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Current advances on biological production of fumaric acid
Journal Pre-proof Current advances on biological production of fumaric acid Feng Guo, Min Wu, Zhongxue Dai, Shangjie Zhang, Wenming Zhang, Weiliang Dong, Jie Zhou, Min Jiang, Fengxue Xin
PII:
S1369-703X(19)30336-5
DOI:
https://doi.org/10.1016/j.bej.2019.107397
Reference:
BEJ 107397
To appear in:
Biochemical Engineering Journal
Received Date:
19 June 2019
Revised Date:
16 August 2019
Accepted Date:
4 October 2019
Please cite this article as: Guo F, Wu M, Dai Z, Zhang S, Zhang W, Dong W, Zhou J, Jiang M, Xin F, Current advances on biological production of fumaric acid, Biochemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.bej.2019.107397
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Current advances on biological production of fumaric acid Feng Guoa, Min Wua, Zhongxue Daia, Shangjie Zhanga, Wenming Zhanga,b, Weiliang Donga,b, Jie Zhoua,b, Min Jianga,b*, Fengxue Xina,b* a
State Key Laboratory of Materials-Oriented Chemical Engineering,
Nanjing Tech University, Nanjing, 211800, P.R. China b
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College of Biotechnology and Pharmaceutical Engineering,
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),
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Nanjing Tech University, Nanjing, 211800, P.R. China
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*Corresponding authors at:
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State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South
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Road 30#, Nanjing 211800, P. R. China.
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E-mail addresses: [email protected] (M. Jiang), [email protected] (F. X. Xin)
This review provides an overview of metabolic pathways for fumaric acid
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production.
Different fumaric acid producers and strain improvement strategies involved were introduced.
Alternative substrates for fumaric acid production and their fermentation 1
performances were shown.
The optimization of fermentation conditions was discussed.
Abstract Fumaric acid is an industrially important platform chemical, which has been widely
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used in food, chemicals, agriculture and pharmaceutical industries. Due to the rising price of raw materials in traditional petrochemical method and demand for sustainable
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development, fermentative fumaric acid production has attracted great attention. In this
context, various strains with enhanced fumaric acid production or elimination of by-
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products were developed through mutagenesis or metabolic engineering. In addition,
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renewable organic wastes together with the fermentation process optimization have also
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been widely investigated. Accordingly, this review will comprehensively summarize the achievements of fermentative fumaric acid production in recent years and major
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obstacles occurring in industrialization to provide insights and perspective for future
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microbial fermentation of fumaric acid. Keywords: Fumaric acid; Strain improvement; Process development; Waste resources
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1. Introduction
Fumaric acid（trans-1,2-ethylenedicarboxylic acid）is a four-carbon dicarboxylic acid, which has been widely applied in food, chemicals, agriculture and pharmaceutical industries. Due to its multifunctional structure including a carbon-carbon double bond
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and two carboxylic acid groups, fumaric acid can easily be esterified and polymerized [1], resulting in the main application as chemical feedstock for the production of paper
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resins, unsaturated polyester resins, alkyd resins, plasticizers, and miscellaneous
industrial products [2]. Moreover, fumaric acid has been widely used as food acidulant
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and beverage ingredient due to its nontoxic nature and unique flavor [3]. Owning to its
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diverse application, fumaric acid has been identified as one of the top ten building block
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chemicals [1]. The worldwide demand for fumaric acid was 225.2 kt in 2012,which is expected to be over 300 kt in 2020 [4].
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Currently, fumaric acid is mainly synthesized through the isomerization of maleic
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acid under the catalysis of mineral acids, peroxy compounds, or thiourea (Fig. 1) [1]. This petrochemical method is the most widely applied process for fumaric acid production due to the high production yield. However, the isomerization process usually occurs at high temperatures, leading to the formation of by-products and huge energy 3
consumption as well [5]. Alternatively, microbial fermentation process has attracted great interests owning to its sustainability and environmentally friendly properties. Microbial fermentation for fumaric acid production has shown promising prospects, and many works have been carried out in recent years including strain isolation and improvement, substrate selection, process optimization, and downstream
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separation. However, reviews regarding fumaric acid production are incredibly insufficient and most of them only involved one of these aspects like strain
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improvement [6] or separation strategies [7]. Accordingly, this review is to provide a
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comprehensive investigation involving the achievements in recent years and obstacles
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restricting fermentation production. The fermentation performance of the most prominent fumaric acid-producing strains and the metabolic engineering involved will
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be introduced first. The alternative substrates, especially renewable waste biomass,
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together with various methods of fermentation optimization will be discussed later.
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Finally, future prospects will also be analyzed.
2. Metabolic pathways and transportation mechanism for fumaric acid production
As illustrated in Fig. 2, fumaric acid is synthesized through three metabolic 4
pathways. The first one is the reductive branch of tricarboxylic acid (TCA) cycle that begins with the carboxylation of pyruvic acid. This pathway was considered as a major contributor for fumaric acid accumulation, in which a maximal theoretical yield of 2 mol/mol glucose can be obtained via CO2 fixation [8]. With the participation of ATP and CO2, pyruvic acid is converted to oxaloacetic acid by pyruvate carboxylase (PYC),
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which is exclusively located in the cytoplasm [1, 9]. Subsequently, oxaloacetic acid is converted to fumaric acid by the action of malate dehydrogenase (MDH) and fumarase
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(FUM) with the participation of NADH. Formerly, FUM was generally considered to
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catalyze both reversible reactions, while recent studies disclosed that its activity of
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catalyzing fumaric acid to L-malic acid could be completely inhibited by 2 mM fumaric acid [10]. This unique enzymatic property probably explains the mechanism of fumaric
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acid accumulation in Rhizopus. oryzae and suggests that expression of FUM from R.
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oryzae could enhance fumaric acid generation. Moreover, nitrogen limitation was also
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revealed to attach a great significance on the activity of cytosolic FUM. When the urea concentration was decreased from 2.0 to 0.1 g/L, the cytosolic FUM activity was rapidly increased by 300%, and the fumaric acid production was increased from 14.4 to 40.3 g/L [11]. The second pathway is the oxidative TCA cycle, which also plays an important 5
role in the synthesis of fumaric acid [12, 13]. In this pathway, pyruvic acid is converted to acetyl-CoA by the pyruvate dehydrogenase system first, and acetyl-CoA subsequently enters TCA cycle. Fumaric acid is synthesized by the catalysis of succinate dehydrogenase (SDH) from succinic acid, and the theoretical yield is limited to 1mol/mol glucose due to the release of CO2. However, in most cases, fumaric acid
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synthesized in this pathway is hard to accumulate, and easily to be converted to L-malic
acid via mitochondrial FUM. In prokaryotes, fumaric acid reductase (FRD) catalyzes
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the conversion of fumaric acid to succinic acid. Since that, mitochondrial FUM and
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FRD were commonly deleted and fumaric acid accumulation in the oxidative route was
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enhanced consequently [14-16]. Moreover, the deletion of some regulators such as DNA-binding response regulator (arcA) or transcriptional repressor of lac operon (lacI)
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was also proved to be beneficial of strengthening the oxidative pathway [14, 17].
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Anyhow, for wild type strains, fumaric acid produced in this way is mainly utilized for
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biomass synthesis and cannot accumulate during the growth stage [8]. Glyoxylate route is also suggested as a potential pathway for fumaric acid
production [18]. Isocitric acid formed in TCA cycle is decomposed into succinic acid and glyoxylic acid under the catalysis of isocitrate lyase. Subsequently, glyoxylic acid is combined with acetyl-CoA and L-malic acid is synthesized under the catalysis of 6
malate synthetase [18-20]. Although the theoretical yield (1mol/mol glucose) is relatively low compared to the reductive TCA cycle, glyoxylate bypass possesses great potential due to the relatively short metabolic pathway. However, since the key enzyme in glyoxylate pathway is strongly repressed in high glucose media, this pathway is difficult to be activated when glucose is used as the substrate [21]. This phenomenon
(PEP), an intermediate formed from glucose [17, 21, 22].
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may be attributed to the inhibition of isocitrate lyase caused by phosphoenolpyruvate
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In addition to these three approaches, amino acid and fatty acid metabolism may
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also be involved in fumaric acid production. Recent metabolic profiling has revealed
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that higher levels of carbon (EMP and TCA) and amino acid metabolism were observed in high fumaric acid -yielding strain [23]. Compared to original strain, 4-aminobutyric
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acid and 5-aminolevulinic acid production was increased by 10.33- and 7.22-fold,
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respectively. These findings provide a new perspective that regulating the metabolism
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of amino acids and fatty acids may also be a viable option to increase fumaric acid production. Recently, a number of studies regarding fumaric acid production by engineering the urea and purine nucleotide cycle confirmed this assumption and suggested that the relationship between carbon metabolism and nitrogen metabolism might have a significant impact on fumaric acid accumulation [24]. 7
Regulation of transportation system is very critical for efficient organic acids secretion. The transportation of dicarboxylic acids including L-malic acid, succinic acid and fumaric acid has been discussed in yeasts, especially Saccharomyces. cerevisiae [25-27]. However, few systematic reviews on transportation mechanism of fumaric acid in fungi were reported so far. It was proposed that fumaric acid and L-malic acid share
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similar import systems due to their competitive relationship with the dicarboxylate
proton symporter. Moreover, fumaric acid entered cells most likely through passive
gene
Spmae1
encoding
the
C4-dicarboxylic
acids
transporter
in
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that
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diffusion in the undissociated form in S. cerevisiae [28]. Recent studies have proved
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Schizosaccharomyces pombe is able to effectively export L-malic acid, fumaric acid, and succinic acid [29]. As for intracellular transport, gene acr1 encoding succinate-
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fumarate transporter (SFC) in S. cerevisiae was discovered to transport the
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mitochondrial fumaric acid into cytoplasm [30], which has been confirmed in S.
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cerevisiae and Torulopsis glabrata [24, 31].
3. Fumaric acid production using wild type and mutagenized strains
3.1 Wild type strain
Some
bacteria
including
Zymomonas
Mobilis,
Bacillus
macerans, 8
Thermoanaerobacter ethanolicus and Erwinia chrysanthemi et al. [32] and yeasts including Scheffersomyces stipitis, Brettanomyces or Brett, Pachysolen tannophilus, and Candida utilis et al. [33] have been identified as fumaric acid producers. However, fungi are still the major producers, especially filamentous Mucoralean fungi, including Rhizopus nigricans [12, 21, 34], R. arrhizus [12, 13, 35-37], R. oryzae [38, 39], and R.
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formosa [1, 34]. Other Mucoralean fungi, such as Cunninghamella [40] and Cirnella
spp., and non-Mucoralean fungi such as Penicillum griseofulvum, Aspergillus glaucus,
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and Caldariomycels fumago were also identified as fumaric acid producers [41].
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During these strains, Rhizopus sp. were the ideal fumaric acid producers, as the
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highest fumaric acid production was obtained by Rhizopus sp. R. arrhizus was once thought to be the most suitable strain for fumaric acid production, and the highest
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production of 121.0 g/L was obtained in stirred tank though the yield is not so high
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(0.37 g/g) [42]. However, the requirement for rich nutrients of R. arrhizus burdened the
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bioprocess cost. Alternatively, R. oryzae with lower nutritional requirements has become the main producer and higher yield and productivity could be obtained as well. As reported, a fairly high productivity of 4.25 g/L/h with a yield of 0.91 g/g was achieved from R. oryzae ATCC 20344 in a rotary biofilm contactor (RBC) combined with downstream separation devices [43, 44]. 9
3.2 Mutagenized strain
Although Rhizopus sp. has been identified as an ideal fumaric acid producer, the production capacity still cannot meet the requirements for commercialization. In this case, random mutagenesis was considered as a promising way to obtain high-yield
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fumaric acid producer. In the last few decades, physical mutagenesis like UV or γ-ray mutagenesis and chemical mutagenesis using multiple alkylating agent, nitroso
compound and LiCl etc. were both widely applied (Table 1). Meanwhile, some novel
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screening methods were also designed. For example, after 3 minutes treatment using
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UV radiation and 4% LiCl, high-yield fumaric acid mutants were screened from agar
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plates containing bromocresol green, in which fumaric acid production capacity could
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be evaluated according to the size of discoloration ring formed by the infiltration of fumaric acid. Based on this selection method, one high-yield fumaric acid mutant R.
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oryzae ZJU11 was isolated after UV irradiation [45]. From an optimized glucose
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concentration of 85 g/L, 57.4 g/L of fumaric acid was produced in flasks, which was 2.05 times higher than that of parent strain. In addition to the enhancement of fumaric acid production, mutants with reduced by-products were also screened. Ethanol is the main by-product during fumaric acid fermentation process, which reduces the
10
conversion of carbon flux to fumaric acid. R. oryzae ME-F01, a mutant with low ethanol production was isolated by allyl alcohol resistance selection method after mutagenesis with UV coupled with nitrosoguanidine (NTG). Compared to the parent, the fumaric acid concentration of the mutant was increased by 21.1%, while ethanol production was decreased by 83.7% [46].
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Directed evolution is a more advanced strategy as it exerts selection pressure
according to specific needs and purposes. Recently, it was also used to screen ideal
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producers, especially the mutants with a capability to utilize other carbon sources. For
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instance, Deng et al. isolated a series of R. oryzae mutants which was able to utilize
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starch directly, and 39.8 g/L of fumaric acid was obtained from cornstarch [47]. Since starch utilization capability could be characterized by the activity of glucoamylase,
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glucose analogue of 2-deoxyglucose (2-DG) was used as evolutionary pressure in this
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study and mutants with high glucoamylase activity were effectively screened. Take
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another instance, R. arrhizus RH-7-13, a xylose-utilizing strain which was selected via continuously increasing the xylose concentration in medium [48]. With 80 g/L of initial xylose, R. arrhizus RH-7-13 could accumulate up to 28.5 g/L of fumaric acid, while parent strain accumulated 13.2 g/L of fumaric acid under similar conditions.
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4. Fumaric acid production using metabolically engineered strains
Recently, metabolic engineering has been developed rapidly, which can help metabolically construct strains with higher fumaric acid production, less by-product formation or more efficient substrate utilization. Strategies to improve fumaric acid
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production through metabolic engineering can be summarized as follows: (1) overexpression of rate limited enzymes; (2) introduction of new metabolic pathways;
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(3) knocking out of genes responsible for by-products generation; (4) energy metabolism balance and cofactor generation; (5) elimination of inhibitor effects to
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enhance synthesis pathways. (6) enhancement or inhibition of related transporters.
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on different strains.
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Advances in terms of these aspects were comprehensively summarized as below based
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4.1 Filamentous fungi
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As the most mainstream fumaric acid producer, R. oryzae has been metabolically engineered to enhance fumaric acid production. Overexpression of endogenous PYC and PPC was considered to increase the carbon flux towards oxaloacetic acid in R. oryzae M16 [15]. Compared to the initial strain, fumaric acid yield of recombinant overexpressing PPC was increased by 26% with yield of 0.78 g/g glucose, but the one 12
overexpressing PYC was significantly affected on growth and the yield was extremely low (<0.05 g/g). Additionally, native FUM in R. oryzae was considered to promote the conversion of L-malic acid to fumaric acid and thereby was overexpressed in R. oryzae M16 [49]. Nevertheless, fumaric acid production was not improved whilst L-malic acid yield was doubled, suggesting the opposite effect of the assumption.
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Besides Rhizopus sp., thermophilic filamentous fungi have shown promising
potential for organic acid production. Recently, Myceliophthora thermophila was
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successfully engineered for fumaric acid production using the CRISPR/Cas9 system
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[50]. After introducing the gene Ckfum originating from C. krusei into M. thermophila,
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fumaric acid titer was increased by triple. To further enhance fumaric acid accumulation, cytoplasmic fr1and fr2 together with mitochondrial fumarase (Mtfum) were deleted.
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Morover, mitochondrial malate carrier (MOC) was deleted to enhance L-malic acid
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synthesis as it was the precursor of fumaric acid. Finally, overexpression of Mtsfc
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encoding C4-dicarboxylate acid transporter led to 17.0 g/L of fumaric acid in fed-batch fermentation.
4.2 Bacteria
As the model bacterium, E. coli has been comprehensively investigated for 13
fumaric acid production through metabolic engineering (Table. 2). In prokaryotes, the enhancement of genes expression involved in oxidative TCA cycle was a common strategy. . Besides these enzymes like FUM and FRD, some regulators have been proved play significant roles in the oxidative pathway. For instance, arcA is a DNAbinding response regulator and its deletion was discovered to intensify the Krebs cycle
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[18]. E. coli with arcA deleted exhibited a better growth capacity, and fumaric acid production was increased by 1.5-fold [18]. As a precursor of glycolytic pathway,
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Oxaloacetic acid plays an important role in organic acid accumulation. Thus, enhancing
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oxaloacetic acid synthesis was a feasible strategy in organic acid production, which has
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been practiced in L-malic acid and succinic acid production by overexpressing PPC or PYC [51-54]. Based on in silico flux response analysis, homologous PPC was
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overexpressed in E. coli CWF41 using the strong tac promoter [17]. Compared to
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control strain E. coli CWF4, the enzyme activity of PPC was enhanced by 4.7 times,
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and fumaric acid production was increased by 2.8 times [14, 17]. Another common way to increase fumaric acid accumulation in E. coli is to enhance the carbon flux in glyoxylate shunt. Thereby, deletion of iclR encoding isocitrate lyase repressor in E. coli was also proved
effective. Furthermore, the deletion of lactate dehydrogenase (LDH)
or pyruvate formate-lyase (PFL) catalyzing lactic acid synthesis increased the flux of 14
pyruvic acid into the oxidative TCA cycle and glyoxylate shunt [55, 56]. Reduction of the main by-products like L-malic acid and succinic acid might be the commonest strategy to enhance fumaric acid accumulation, which was applied in nearly all engineered E. coli [14, 17, 18]. For instance, after deletion of fumABC, frdABCD and iclR in E. coli JM109 (DE3), a strain could hardly accumulate fumaric
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acid, the recombinant was able to produce 2.12 mmol/L of fumaric acid [18]. Moreover, deletion of other by-products such as acetic acid, lactic acid and aspartic acid could also
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improve fumaric acid production. Acetyl-CoA synthase (acs) is one of key enzymes
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responsible for acetic acid secretion and assimilation [57]. To reduce acetic acid
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accumulation, acs was overexpressed in strain E. coli ABCDIA-ptsG--mdh--glk-galpppc-acs and the fumaric acid yield was up to 1.5 times higher than the parent with the
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4.3 Yeasts
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combined strategies [14].
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Compared to E. coli, yeasts are considered as potential industrial candidates for organic acid production owning to their higher acid resistance [58, 59]. However, one major limit for fumaric acid production in yeasts is that the original fumarase affinity to fumaric acid is 17 times higher than that to L-malic acid [60]. The cytosolic fumarase 15
in yeasts mainly catalyzes the conversion of fumaric acid to L-malic acid rather than the reverse reaction, which results in the inability to accumulate large amounts of fumaric acid in cytosol [7]. In this case, the reductive route of fumaric acid biosynthetic pathway was introduced into S. cerevisiae [61]. The resulting strain with overexpression of RoMDH, RoFUM1 (from R. oryzae) and endogenous PYC was able to produce 3.2
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g/L fumaric acid. Inspired by this study, fumaric acid production by simultaneous use
of oxidative and reductive routes was carried out. The oxidative pathway was first
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introduced into S. cerevisiae by the deletion of FUM1 and the resulting strain S.
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cerevisiae FMME004 accumulated 0.3 g/L of fumaric acid [62]. However, fumaric acid
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production was decreased when heterologous genes involved in the reductive pathway were introduced [61], which might attribute to the diversion of carbon flow into TCA
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cycle as the activity of PDC was decreased. With the supplementation of 32 μg/L of
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biotin and an optimal carbon/nitrogen ratio, 5.6 g/L of fumaric acid was obtained in S.
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cerevisiae FMME004-6 finally, in which RoPYC gene was further overexpressed. In addition to S. cerevisiae, other yeasts such as T. glabrata and Scheffersomyces
stipitis have also been explored for fumaric acid production. Recent studies have revealed that four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA) and FUM1 are involved in fumaric acid 16
biosynthesis in T. glabrata [24]. Manipulation of their expression levels is able to regulate the urea and purine nucleotide cycles, and thereby regulate the carbon and nitrogen metabolism. For example, by controlling the expression of ASL at high level and ADSL at low level, 5.6 g/L of fumaric acid was obtained. Moreover, further overexpression of Spmae1 could improve fumaric titer to 8.8 g/L. S. stipitis is an
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excellent xylose-utilizing yeast. Recently, the reductive pathway from R. oryzae FM19 was introduced into S. stipitis [16]. To enhance the reductive pathway activity, FUM
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was deleted to block fumaric acid conversion in the Krebs cycle. Furthermore, the
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heterologous transporter YMAE1 of SpMAE1 was overexpressed, and 4.6 g/L fumaric
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acid was obtained from 50 g/L xylose by the recombinant S. stipitis. Despite these achievements, most engineering strategies focused on the regulation
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of single pathway currently, while the overall metabolism balance was often ignored.
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For instance, after the simultaneous introduction of oxidative and reductive routes, S.
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cerevisiae FMME004-6 produced only 5.6 g/L of fumaric acid. This low titer could attribute to the imbalanced distribution of carbon flux. Therefore, a number of synthetic biology strategies have been adopted to optimize metabolism balance or engineer the transportation efficiency of intermediate metabolites. Recent progress mainly focused on mitochondrial engineering [63], scaffold engineering [64], cofactor engineering [64] 17
and modular assembly [31]. Mitochondrial engineering has been used to construct the oxidative pathway for fumaric acid production in C. glabrata, a classical αketoglutarate producer [63]. This oxidative pathway, containing α-ketoglutarate dehydrogenase complex (KGD), succinyl-CoA synthetase (SUCLG) and succinate dehydrogenase (SDH) were constructed and optimized using the KGD2-SUCLG2 and
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SDH1 expression cassettes. Moreover, SFC1 and SpMAE1 were overexpressed to
enhance the secretion of fumaric acid. The resulting strain with the overexpression of
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ASL gene was able to accumulate 15.8 g/L of fumaric acid from 100 g/L glucose.
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On the other hand, the reductive pathway was also introduced into C. glabrata
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lately. Since there are additional pathways consuming or transporting intermediate metabolites in the reductive TCA pathway, spatial modulation and cofactor engineering
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were performed to enhance the efficiency of conversion from pyruvic acid to fumaric
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acid [64]. By the means of synthesizing DNA scaffolds, RNA scaffolds and protein
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scaffolds [68], structural synthetic biotechnology was developed to improve the pathway transportation efficiency, which has been applied for the production of many chemicals in recent years, such as butyrate [69], hydrogen [70] and L‐ threonine [71]. As for fumaric acid, DNA scaffolds have been recently used in S. cerevisiae and C. glabrata, which both achieved a significant increase in terms of chemicals production 18
[31, 64]. In C. glabrata, three zinc finger proteins ADB1, ADB2 and ADB3 were fused with RoPYC, RoMDH and RoFUM, respectively. DNA scaffold with different ratios of binding sequences resulted in different fumaric acid concentrations while these values were all higher than the one of control without DNA scaffold. Among these, the highest production of 11.2 g/L was obtained from T.G‐ 4G‐ S(1:1:2), which was about 2.8 times
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higher than the control value. Based on this, cofactor engineering was taken to enhance
ATP and NADH generation. Cofactor regeneration has been proved to be an effective
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way to maintain the cellular redox balance by altering the intracellular cofactor pool
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and a number of enzymes have been discovered to directly affect the ratio of
[72],
mitochondrial
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NADH/NAD+ or ATP/ADP, such as cytoplasmic H2O-forming NADH oxidase (NOX) alternative
oxidase
(AOX)
[73],
phosphoenolpyruvate
na
carboxykinase (PCK) [74], and formate dehydrogenase (FDH) [75]. Therefore, PCK
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was overexpressed to generate ATP to motivate the RoPYC‐ catalyzed carboxylation
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of pyruvic acid to oxaloacetic acid, and FDH was overexpressed to generate NADH which could be used to drive the RoMDH‐ catalyzed reduction of oxaloacetic acid to malic acid. After optimizing overexpression of PCK and FDH, the strain T.G‐ 4G‐ S(1:1:2)‐ P(M)‐ F(H) led to a 63.7% increase in fumaric acid production, compared with that of strain T.G‐ 4G‐ S(1:1:2) and the final concentration in a 5-L batch bioreactor was 19
up to 21.6 g/L. Metabolic balance of S. cerevisiae can also be optimized by modular pathway engineering [31]. The fumaric acid biosynthetic pathway was divided into three modules (Fig. 3): (1) PMFM module: reduction module containing RoPYC, RoMDH, RoFUM1 and SpMAE1; (2) KSSS module: oxidation module containing KGD2,
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SUCLG2, SDH1 and SFC1; (3) RPSF module: byproduct module including sRNA-
RHR2, sRNA-PDC6 and DNA scaffold. RNA switch is a type of gene regulatory
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element, which is able to dynamically regulate gene expression via transcription,
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splicing, RNA interference and translation [76]. sRNA-RHR2 and sRNA-PDC6 can be
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designed to regulate GPP (glycerol-1-phosphatephos-phohydrolase1) and PDC activities, respectively [77]. Glycerol and ethanol production were reduced
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consequently. PMFM and KSSS modules with various strengths were assembled and
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optimized, and the highest concentration of 20.5 g/L was obtained by regulating the
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expression strength of KGD2-SUCLG2 at high level and SDH1 at medium level, which showed 49.7% increase compared with the control strain. Taken together, it could be speculated that combined regulation of metabolic pathways could break down the metabolic constraints to achieve the balance of the biological system and thereby increase the production, which cannot be evaded by individual regulation [78]. In 20
addition, fumaric acid production by the final strain with DNA-guided scaffolds was increased by 40.0% with 33.1 g/L compared with that of no DNA scaffold, which might due to the increased local intermediates concentration and the avoidance of feedback inhibition [79-81].
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5. Fermentation Substrates
Substrates cost accounts for about 30- 40% of the total cost in industrial organic
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acids production [82]. Although glucose is the most preferred carbon source for fumaric
fermentation preferences of these carbon sources are
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evaluated [83]. However, the
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acid production, other carbon sources like glycerol, xylose and sucrose were also
generally inferior to those from glucose, as additional pathways are needed to link these
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substrates with glycolysis and TCA cycles. With the rising economic and environmental
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requirements, some renewable raw materials rich in starch or lignocellulose are also
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considered to be promising alternative substrates.
5.1 Xylose
As a widely available pentose in lignocellulose waste, xylose has been explored for fumaric acid production in early 1989 by immobilized R. arrhizus, although the 21
highest productivity was only 0.09 g/L/h [84]. Commonly, high xylose concentration inhibits the growth of R. arrhizus [48]. Therefore, a strain RH 7-13-9#, which can adapt a high xylose concentration was selected through a selection medium. By efficient immobilization fermentation, 45.3 g/L fumaric acid was obtained, and the conversion of xylose reached 73%, which is close to the theoretic yield (77%) [85]. Furthermore,
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co-fermentation of a mixture of glucose and xylose was also investigated. With the
optimum glucose/xylose ratio of 75/25 (w/w), 46.8 g/L fumaric acid was obtained from
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100 g/L of total sugars, which was higher than the production in fermentation of glucose
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and xylose separately [86]. In addition, metabolic engineering has enabled other strains
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to efficiently utilize xylose for fumaric acid production. For instance, 4.7 g/L of fumaric acid was produced from 50 g/L of xylose by S. stipitis PSYPMFfS after the introduction
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of xylose metabolic pathway [16].
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5.2 Glycerol and crude glycerol
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Glycerol is a versatile raw material which has been widely used in the cosmetics and automotive industry. Glycerol has been attempted as carbon source together with other monosaccharides to support fumaric acid fermentation in R. oryzae [87]. As reported, 17 strains/isolates of Rhizopus sp. were tested to produce fumaric acid with 22
40 g/L glycerol as the sole carbon source while the highest titer was only 6.1 g/L at 192 h. In contrast, the titer was much higher when monosaccharide was used as a carbon source, among which the titer of 19.8 g/L from xylose was the highest one. In this case, a co-fermentation strategy was adopted and the production was improved when glycerol was used as co-substrate with monosaccharides such as xylose, fructose, galactose, and
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mannose, except the one with glucose. The highest titer of 28.0 g/L was obtained with
a yield of 0.90 g/g from 40 g/L xylose and 20 g/L glycerol. Additionally, crude glycerol
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is a major by-product in biodiesel industry and its production was increased
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dramatically annually. However, crude glycerol is not a suitable feedstock for many
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microorganisms including R. oryzae [88]. R. arrhizus RH-07-13 is a high-yield fumaric acid producer that has also been investigated for crude glycerol utilization. However,
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the production was only 4.4 g/L using 80 g/L crude glycerol as the sole carbon source
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[89]. In this case, a strategy for co-fermentation of crude glycerol and glucose was
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developed that increased production to 22.8 g/L using the mixed substrate of 40 g/L of crude glycerol and 40 of g/L glucose. Compared with glucose fermentation, the production cost was reduced by approximately 14%, which suggested that the co‑ utilization strategy could be more advantageous rather than sole glucose.
23
5.3 Starchy materials
Starch-rich materials, such as beet molasses [90], corn starch [91], potato flour, cassava flour and sweet potato [92] have been widely used for fumaric acid production. Four kinds of raw starchy materials, including starch, cassava powder, corn powder and
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degermed corn powder with different carbon/nitrogen ratio were compared for fumaric acid production. Among these four substrates, degermed corn powder with nitrogen
concentration of 0.25 g/L was the most suitable substrate. For cassava powder and corn
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powder, although biomass was increased, fumaric acid production was decreased with
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the increase of nitrogen content. When 100 g/L of degermed corn powder was used as
lP
substrate, 35.5 g/L of fumaric acid was obtained via an optimized simultaneous
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saccharification and fermentation (SSF) process [91].
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5.4 Lignocellulosic substrates
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Lignocellulose is the most widely distributed substance in the world, which consists of glucose-rich cellulose, xylose-rich hemicellulose and lignin. It has been considered as an excellent feedstock for bulk chemicals production, such as 2,3-2,3butanediol, succinic acid and fumaric acid [93, 94]. In this context, fumaric acid production from lignocellulose wastes like cassava bagasse [95], corn straw [96] and 24
pulp and paper solid waste (PPSW) [97] etc. was investigated in recent years. Most microorganisms are very weak at using xylose when glucose exists as called carbon catabolite repression. To improve the utilization of xylose in lignocellulose, a novel two-stage corn straw utilization strategy was designed, in which fungi were first cultivated in hydrolysate containing 30 g/L of xylose. After the pre-culture process,
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fumaric acid fermentation was initiated in the hydrolysate containing 80 g/L of glucose. Up to 27.8 g/L of fumaric acid with a productivity of 0.33 g/L/h was finally obtained
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[96].
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PPSW, lignocellulose-rich industrial waste has become the 3rd largest industrial
lP
polluter due to the escalating demand for paper-based products. Recently, PPSW was considered to be an alternative feedstock for fumaric acid fermentation under
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submerged and solid-state fermentation techniques. Fumaric acid production was
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decreased when the particle size of PPSW was increased, and the highest production of 23.5 g/L with productivity of 0.49 g/L/h was achieved at 48 h in submerged
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fermentation when the particle size was in the range of 1.7 mm < x ≤ 3.35 mm. As for solid state fermentation, 41.5 g/kg dry weight fumaric acid was obtained with 75 μm < x ≤ 300 μm particle size after 21 days [97]. In recent years, studies on the production of fumaric acid from lignocellulosic 25
materials continue to be published and some of these results were comparable to those observed with the control medium using pure glucose and xylose [98]. However, pretreatment of raw materials has become a major obstacle to the restriction of renewable biomass fermentation. Currently, the pretreatment process of lignocellulose is still costly and the conversion rate is not so high. Moreover, compounds like
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furaldehydes, phenolic compounds and various minerals could affect the metabolism of
strains, which should be studied further [99]. Anyhow, lignocellulose is still highly
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anticipated to become an important raw material with the development of pretreatment
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process and optimization of solid-state fermentation strategy. In addition, some high
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value-added products are available from lignocellulosic. For instance, chitosan/chitin is a material with a vast market foreground that could be recovered as a by-product from
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lignocellulosic materials. In the two-stage corn straw utilization process introduced
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above, hemicellulose hydrolysate obtained from corn straw pretreated with dilute
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sulphuric acid can be used for biomass and chitin/chitosan synthesis for fungal [96].
26
6. Optimization of fermentation conditions
6.1 Morphology control
Morphology control is the major challenge to maintain the fumaric acid yield and productivity for fungi [100-102]. The main types of fungal morphology include clumps,
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filaments and pellets [101]. Especially, pellets can be further subdivided into aggregated and non-aggregated [103]. The morphology of filaments is conducive to some enzymes
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production, while this will increase the viscosity of fermentation broth, leading to the
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oxygen transfer restriction. Alternatively, pellet will alleviate the problem of high
lP
viscosity and support recycling in subsequent fermentation batches as well, which has been considered as the most favorable morphology for industrial fumaric acid
na
production. In contrast, cells with clump morphology were thought to be detrimental for fumaric acid production, since both biomass and products are difficult to accumulate
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because of the severe internal oxygen limitation. Up to now, the morphology control of
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Rhizopus sp. is still largely based on empirical data. For R. arrhizus, dispersed mycelia were proved to be much more effective than pellets for fumaric acid production [4]. When 25 g/L of glucose and 0.04 g/L of ammonium sulphate were fed, 19.7 g/L of fumaric acid was obtained from R. arrhizus NRRL 2582 with dispersed mycelia, the 27
yield and productivity were 0.84 g/g and 0.18 g/L/h, respectively. In contrast, only 7.0 g/L of fumaric acid was produced from the pellets under similar conditions, together with the yield of 0.29 g/g and the productivity of 0.10 g/L/h. However, as for R. delemar NRRL 1526, the opposite effect was reported [104]. For all this, a consensus is that adequate oxygen transfer is the key for morphology control. Moreover, enough space
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between cells is needed for efficient fumaric acid production since it is important for fumaric acid secretion and the mass transfer between cells and medium [104].
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Various factors have been found to affect the fungi morphology, such as the
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composition of the medium [105-107], trace metals [39, 108, 109], spore concentration
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[110], pH [108, 109, 111], stirring rate [112, 113] and temperature [101]. For example, low inoculation concentration of R. oryzae will help form pellets, while high inoculation
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concentration is conducive to form filaments [110]. More interestingly, different species
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of fungi exhibit different morphology even under the same conditions. For instance,
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high C:N ratio would promote mycelial growth in A. niger [114], but pellets in R. arrhizus [115]. Even for the same fungus, there are still various controversies about its morphology control. As for Rhizopus, the absence of Zn2+ would prevent the formation of pellets [39], while other study indicated that metal ions tend to inhibit pellets formation [109]. Besides, studies showed that pellets would form at pH 2.6-3.36 and 28
large mycelial clumps would form at pH 4-5 for Rhizopus [111]. A proposed strategy to controlling the fungus morphology of is cell immobilization, which will enhance the cells stability and thereby improve fumaric acid yield and productivity [116, 117]. Compared with the conventional submerged fermentation, this technology is characterized by constant or intermittent contact with
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the liquid medium rather than being immersed in fermentation broth at all times, which
allows a high cells concentration and low substrate consumption for biomass generation
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[118, 119]. The application of immobilized cells could also alleviate the problem of
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oxygen transfer and provides a strategy for continuous production of fumaric acid. In
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recent years, various materials such as cork, expanded polystyrene, expanded clay, agar gel cubes and polypropylene tube [120] have been applied as immobilizing materials.
na
For example, in a rotary biofilm contactor bioreactor, R. oryzae ATCC 20344 cells were
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immobilized on the surfaces of plastic discs, in which a high fumaric acid productivity
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of 3.78 g/L/h was achieved [43, 121]. However, one prominent dilemma is that materials used as immobilization matrix are usually weak in mechanical properties, making industrial production difficult.
29
6.2 Neutralizing agents
In the bio-process of fumaric acid production, neutralizing agents are commonly used to control pH [104, 110], and CaCO3 is the most widely used neutralizing agent. The unique advantage is that CaCO3 can release CO2, which can be fixed by PYC and
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thereby reinforces the reductive TCA cycle. However, insoluble calcium fumarate will be produced when CaCO3 was added, which will increase the viscosity of the fermentation broth. Another challenge is that sulfuric acid will be used in subsequent
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stages to release fumaric acid, resulting in insoluble CaSO4 produced, which is a
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compound with narrow industrial application. Accordingly, various other neutralizing
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agents such as Na2CO3, NaHCO3, Ca(OH)2 and (NH4)2CO3 have been studied as
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substitutes for CaCO3 [35, 37, 111]. With addition of Na2CO3 or NaHCO3, heating is no longer needed to recover the fermentation product due to the higher solubility of sodium
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fumarate, thereby downstream processing costs could be reduced. Besides, sodium
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fumarate could facilitate separation and recycling of fungal cell biomass in succeeding batch fermentation steps [35, 111]. However, Na+ in the medium tends to make adverse effect on cell metabolism, and the low solubility of sodium fumarate would cause product inhibition, which limits the utilization of Na2CO3. As for Ca(OH)2 and
30
NaHCO3, these neutralizing agents were reported to increase the accumulation of byproducts like L-malic acid and ethanol. (NH4)2CO3 was also studied as an alternative neutralizing agent under phosphorus limitation [37, 122], and the highest 46.0 g/L of fumaric acid was obtained by R. arrhizus NRRL 1526 although the fermentation time
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was prolonged to 108 h [37].
6.3 Fermentation parameters
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Fermentation parameters such as temperature, metal ion, dissolved oxygen and pH
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are important to industrial fermentation process. Temperature not only effects various reaction rates in fermentation, but also indirectly effects product synthesis by altering
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the physical properties of fermentation broth, including dissolved oxygen, the mass
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transfer rate of the matrix, and the nutrients decomposition and absorption rate. Up to now, due to the strain specificity and the wide range of optimal growth temperatures
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(usually 30-37℃ for fungal), the influence of temperature on fumaric acid production
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has not been deeply studied [104]. Metal ions like Mg2+, Fe2+, Mn2+ and Zn2+, together with vitamins like biotin and
riboflavin, act as cofactors and activators involved in metabolic processes. Especially, Mg2+ and Mn2+ have been proposed to assist the conversion of isocitric acid to α31
ketoglutaric acid, and thereby lead to the fumaric acid accumulation [41]. Biotin, as an activator of PYC, has been widely added to broth to promote the synthesis of organic acids [123]. However, for industrial-scale production, it is almost impossible to add metal salts and vitamins due to cost constraints. Alternatively, some cheap nutrient-rich raw materials like corn steep liquor are usually used as substitutes.
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Dissolved oxygen (DO), as a necessity for the growth of aerobic microorganisms
is generally the controlling factor due to the extremely low oxygen solubility of in water.
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Despite the essential role for growth and metabolism of microorganisms, high DO level
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during fermentation does not necessarily favor the production of fumaric acid, as
lP
excessive DO will stimulate TCA cycle and lead to increased cell growth and energy expenditure. Moreover, too high aeration or agitation rates results in low level of
na
dissolved CO2, which exerts negative influences for CO2 fixation, and thereby reduces
ur
the fumaric acid production. In this case, an improved fermentation strategy was
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patented by DuPont to improve the fumaric acid yield and productivity, in which DO was controlled at a high level (80%-100%) during the cell growth phase, but a low level (30%-80%) during acid production phase [124]. With this strategy, high fumaric acid production of 130.0 g/L was obtained in 142 h, but by-products such as L-malic acid and succinic acid were increased concomitantly. 32
pH is of great importance during fermentation for its multifunctional function, such as effecting enzyme activities involved in various metabolic reactions and regulating the transport of fumaric acid. Fumaric acid production by Rhizopus sp. usually requires pH level of around 6. However, low pH of 3.0 resulted in a sharp decrease of fumaric acid production (9.4 g/L), compared with 30.2 g/L at pH of 5.0 [125]. It might be
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explained that low pH level due to the accumulation of fumaric acid leads free acids to enter cells by passive diffusion, thereby inhibiting the biosynthesis of fumaric acid [37].
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Furthermore, low pH level leads to the decrease of cell membrane permeability, which
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further causes the accumulation of intracellular undissociated fumaric acid and thereby
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leads to the inhibition of fumaric acid [26]. In addition to reducing fumaric acid production, low level of pH also leads to high concentrations of by-products including
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ethanol and glycerol. For these reasons, some strategies to produce fumaric acid at low
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pH using acid-tolerant producers like yeasts or in situ product removal techniques [43,
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126, 127] have been adopted. For example, by releasing OH− from a polyvinyl pyridine adsorbent into the broth, an integrated system of simultaneous fermentation-adsorption was able to produce 85 g/L of fumaric acid from 100 g/L of glucose in 20 h under repetitive fed-batch cycles [43]. The accumulation of fumaric acid, like many other organic acids has been proved 33
to be greatly affected by C:N ratio in the medium. Generally, high C:N ratio is beneficial for fumaric acid overproduction. Microbial growth and proliferation are aborted due to the depletion of a limited nitrogen source, and thereby the pressure caused by excess carbon forces the microorganisms to direct carbon to the accumulation of organic acids. Compared to organic nitrogen sources like yeast extract, inorganic nitrogen sources like
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urea and ammonium salts might be more suitable for fumaric acid production [82]. However, excessive C:N ratio leads to substrate inhibition and reduces cell productivity,
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which might be caused by the inhibition of α-ketoglutarate dehydrogenase enzyme
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complex at high glucose concentration [128]. Some results from the recent continuous
lP
fermentations clearly indicated how nitrogen addition influences the time-dependent product distribution of R. oryzae. As proposed, ethanol production terminated when
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fumaric acid production reached its production window, in which the volumetric
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productivity was approximately 0.3 g/L/h. Therefore, an appropriate continuous
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nitrogen level control could be highly beneficial to fumaric acid accumulation compared to simply limiting the nitrogen at the onset [120]. Moreover, phosphorus source was proposed to play a similar role, as nitrogen source and high phosphorus levels were indicated to reduce carbon flux in the direction of organic acids [128, 129]. Thus, phosphorus source has been considered to control the mycelial growth of R. 34
arrhizus NRRL 1526 instead of nitrogen source. With (NH4)2CO3 as neutralizing agent, the maximum ammonium fumarate productivity of 0.46 kg/m3/h was achieved when 0.1 kg phosphate/m3 was used [37].
7. Conclusions and future prospects
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Fumaric acid, as a valuable dicarboxylic organic acid has been widely applied in food, medicine and construction industries, which is also recognized as one of the “top
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12” chemical building blocks. The earliest attempt to produce fumaric acid by microbial
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fermentation can be traced back to the early 20th century. With the continuous expansion
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of fumaric acid application and the rising prices of fossil raw materials, fumaric acid production by microbial fermentation is exhibiting its potential, although it still remains
na
less economically competitive compared with the traditional petrochemical method. In
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the last few decades, various wild types and mutants were characterized for fumaric acid production, in which R. oryzae was considered to be the most advantageous
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producer. With the development of metabolic engineering, a number of other producers (especially yeasts) have been investigated for fumaric acid production. Furthermore, the rapid development of synthetic biology in recent years has provided new perspectives for metabolic engineering. 35
The cost of substrate is important for the feasibility of industrial fumaric acid production. Although glucose is the most wildly used substrate, many alternatives such as glycerol and xylose have been attempted. To reducing the cost of production, renewable biomaterials are considered to be promising, especially some feedstocks rich in starch or lignocellulose. One of the major dilemmas in fumaric acid fermentation is
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to maintain suitable fermentation conditions. Therefore, a lot of fermentation regulations like morphology, pH and neutralizing agents have been extensively studied.
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Based on the above aspects, the metabolic engineering for fumaric acid production
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in the future should focus on global regulation rather than single genes or pathways.
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Some novel technologies like mitochondrial engineering, scaffold engineering, and cofactor engineering will be instructive and bring guidance for subsequent process
na
optimization. The utilization of waste biomass or co-substrate fermentation may be a
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more competitive strategy which can potentially improve the feasibility of the
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bioproduction process. Besides, the strategy of process optimization should not only focus on the laboratory-scale production increase, but also the feasibility of industrialscale fermentation, which will help to improve the economic competitiveness of the fermentation process. Moreover, the development of fumaric acid derivatives such as L-malic acid and L-aspartic acid, which are 1.5-2 times more valuable than fumaric 36
acid will also increase the value of fumaric acid production.
Acknowledgements
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This work was supported by the National Key Research and Development Program of China (2018YFA0902200), Jiangsu Province Natural Science Foundation for Youths (BK20170993, BK20170997), the Jiangsu Synergetic Innovation Center for Advanced the
National
Natural
Science
Foundation
of
China
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Bio-Manufacture,
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(No. 21978130,No. 21706125, No. 21727818), Jiangsu Key Lab of Biomass-based
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Green Fuels and Chemicals Foundation (JSBEM201908), and Project of State Key
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Laboratory of Materials-Oriented Chemical Engineering (ZK201601).
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Abbreviations
TCA, tricarboxylic acid cycle; PYC, pyruvate carboxylase; MDH, malate
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dehydrogenase; FUM, fumarase; FUMR, fumarase from R. oryzae; SDH, succinate dehydrogenase; FRD, fumaric acid reductase; SFC, succinate-fumarate transporter; arcA, DNA-binding response regulator; iclR, isocitrate lyase repressor; lacI, transcriptional repressor of the lac operon; PEP, phosphoenolpyruvate; PPC, 37
phosphoenolpyruvate carboxylase; Ckfum, fumarase from Candida krusei; Mtfum, mitochondrial fumarase; MOC, mitochondrial malate carrier; LDH, lactate dehydrogenase; PFL, pyruvate formate-lyase; ptsG, The PEP dependent glucosespecific phosphotransferase system; aspA, aspartase; aceBA, glyoxylate shunt operon; argininosuccinate
fumarylacetoacetase;
lyase;
SpMAE,
ADSL,
adenylosuccinate
C4-dicarboxylic
acids
lyase; transporter
FAA, in
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ASL,
Schizosaccharomyces pombe; NOX, NADH oxidase; AOX, mitochondrial alternative
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oxidase; PCK, phosphoenolpyruvate carboxykinase; FDH, formate dehydrogenase;
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KGD, α-ketoglutarate dehydrogenase complex; SUCLG, succinyl-CoA synthetase;
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GPP, glycerol-1-phosphatephos-phohydrolase1; SSF, simultaneous saccharification
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(2010) 761-769.
Jo
ur
na
Fig. 1. The main methods for fumaric acid synthesis.
47
ro of
-p
Fig. 2. Metabolic pathways of fumaric acid synthesis. A: Metabolic pathways in
eukaryocyte; B: Metabolic pathways in prokaryote. Abbreviations: PYC, pyruvate
re
carboxylase; MDH, malate dehydrogenase; FUM, fumarase; FUMR, fumarase from R.
lP
oryzae; SDH, succinate dehydrogenase; FRD, fumaric acid reductase; SFC, succinate-
repressor; lacI,
na
fumarate transporter; arcA, DNA-binding response regulator; iclR, isocitrate lyase transcriptional repressor of the lac operon; PEP, phosphoenolpyruvate;
ur
PPC, phosphoenolpyruvate carboxylase; Ckfum, fumarase from Candida krusei;
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Mtfum, mitochondrial fumarase; MOC, mitochondrial malate carrier; LDH, lactate dehydrogenase; PFL, pyruvate formate-lyase; ptsG, The PEP dependent glucosespecific phosphotransferase system; aspA, aspartase; aceBA, glyoxylate shunt operon; ASL,
argininosuccinate
lyase;
ADSL,
adenylosuccinate
lyase;
FAA, 48
fumarylacetoacetase;
SpMAE,
C4-dicarboxylic
acids
transporter
in
Schizosaccharomyces pombe; KGD, α-ketoglutarate dehydrogenase complex; SUCLG, succinyl-CoA synthetase;
SDH,
succinate
dehydrogenase;
GPP,
glycerol-1-
lP
re
-p
ro of
phosphatephos-phohydrolase1;
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Fig. 3. The fumaric acid biosynthetic pathway involved in modular assembly. The engineered targets are shown in yellow, green and blue, and its related target genes in
ur
red. PMFM module, reduction module; KSSS module, oxidation; RPSF module,
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byproduct module.
49
ro of -p
re
Table 1. Production of fumaric acids in terms of performance and mutagenic strategies for Rhizopus. Mutation
Culture Substrate
Titer
Yield
Productivity
(g/L)
(g/g)
(g/L/h)
57.4
\
\
[40]
lP
Strain strategy
method
Reference
Shake
R. oryzae UV
Glucose
flask
R. oryzae UV ZJU11 R. oryzae
na
ZJU11
Batch
41.1
0.48
0.37
[40]
Glucose
Batch
52.7
\
\
[46]
44.1
0.44
0.53
[47]
32.2
0.32
0.44
[47]
ur
UV, NTG
Glucose
ME-UN-8
Cornstarch
nitrogen ion
(100
DG-3
implantation.
total
Jo
R. oryzae
Shake g/L flask （SSF）
sugar) Raw corn Shake
R. oryzae
nitrogen ion
powder
DG-3
implantation.
(100
flask g/L
（SSF）
total 50
sugar) R. oryzae
Shake
UV, γ-rays
Glucose
RUR709 R. oryzae
26.2
0.32
0.22
[130]
flask UV, γ-rays
Glucose
Batch
32.1
0.42
0.32
[130]
Glucose
Batch
49.4
0.56
0.29
[23]
RUR709 R. oryzae
Femtosecond
FM19
laser
Engineering Strain
Culture
Titer
Yield
Productivity
method
(g/L)
(g/g)
(g/L/h)
<0.05
\
Substrate strategy
R.
ro of
Table 2. Production of fumaric acids in terms of performance and engineering strategies. Reference
oryzae Expression of pyc
Glucose
Batch
0.7
Expression of ppc
Glucose
Batch
Expression of fumR2
Glucose
Batch
R.
oryzae
ppc oryzae
fumR2 Expression
of
Spmae1, Ckfum and
thermophila
Mtsfc; Deletion of
lP
M.
25.0
0.78
0.26
[15]
21.5
0.65
\
[49]
17.0
0.24
\
[50]
28.2
0.39
0.45
[17]
1.7
\
\
[14]
re
R.
[15]
-p
pyc
Fed-
Glucose
batch
SG515
Mtfrd1,
Mtfrd2,
na
Mtfum and moc
Expression of ppc; Deletion E.
coil
of
iclR,
ur
fumC, fumA, fumB,
Fed-
Glucose
CWF812
batch
arcA, ptsG, aspA and
Jo
lacI
E.
coil
Expression of glk,
ABCDIA-
galP, ppc and acs;
ptsG--
Deletion of fumA,
15
mdh--glk-
fumB,
glucose
galp-ppc-
frdABCD, iclR, arcA,
acs
mdh and ptsG
fumC,
g/L
Shake flask
51
Expression of ppc; E.
Fed-
coil Deletion of fumB,
Glycerol
EF02
41.5
0.88
0.51
[22]
3.2
0.05
0.03
[61]
5.6
0.11
0.06
[62]
8.8
0.15
batch fumAC and aspA
S. cerevisiae
Expression
of
FMME-001
Romdh, Rofum1 and
Shake ↑PYC2 + ↑
Glucose flask
pyc2
RoMDH
cerevisiae
Romdh and Rofum1;
Shake Glucose
FMME004-
Deletion of thi2 and
6
fum1
T. glabrata-
Expression of ASL,
ASL(H)-
ADSL and Spmae1;
flask
Shake Glucose
ADSL(L)-
Deletion of ura3 and
SpMAE1
arg8
S.
stipitis
flask
of
Ymae1; Deletion of
Shake Xylose
PSYPMFfS
ura3, leu2, Psfum1
re
Expression
ro of
Expression of Ropyc,
0.12
[24]
-p
S.
4.7
0.10
\
[16]
15.8
0.15
0.21
[63]
21.6
0.22
0.30
[64]
33.1
0.33
\
[31]
flask
lP
and Psfum2 Expression of kgd2, C. glabrata-
SUCLG2,
sdh1,
KS(H)-S(M)–
Spmae1, sfc1 and
Shake
Glucose
na
flask
A-2 S
ASL; Deletion of ura3 and arg8
of
ur
Expression
ADB1-RoPYC-
AsPCK-SpMAE1
T.G‐ 4G‐
and ADB2-RoMDH-
S(1:1:2)‐
ScFDH1-ADB3-
P(M)‐ F(H)
RoFUM; Deletion of
Jo
C. glabrata
ura3
Glucose
and
Batch
arg8;
Scaffold (1:1:2) S.
Expression
of
Shake Glucose
cerevisiae
RoMDH-SDH1,
flask 52
TGFA091-
RoPYC-KGD2-
16
SUCLG2 and SFC1SpMAE1,；Deletion of thi2, fum1, ura3, leu2, trp1 and his3; RNA switches sRNA-RHR2
of and
sRNA-PDC6; DNA
Jo
ur
na
lP
re
-p
ro of
Scaffold (1:2)
53
PII:
S1369-703X(19)30336-5
DOI:
https://doi.org/10.1016/j.bej.2019.107397
Reference:
BEJ 107397
To appear in:
Biochemical Engineering Journal
Received Date:
19 June 2019
Revised Date:
16 August 2019
Accepted Date:
4 October 2019
Please cite this article as: Guo F, Wu M, Dai Z, Zhang S, Zhang W, Dong W, Zhou J, Jiang M, Xin F, Current advances on biological production of fumaric acid, Biochemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.bej.2019.107397
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Current advances on biological production of fumaric acid Feng Guoa, Min Wua, Zhongxue Daia, Shangjie Zhanga, Wenming Zhanga,b, Weiliang Donga,b, Jie Zhoua,b, Min Jianga,b*, Fengxue Xina,b* a
State Key Laboratory of Materials-Oriented Chemical Engineering,
Nanjing Tech University, Nanjing, 211800, P.R. China b
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College of Biotechnology and Pharmaceutical Engineering,
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),
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Nanjing Tech University, Nanjing, 211800, P.R. China
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*Corresponding authors at:
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State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South
na
Road 30#, Nanjing 211800, P. R. China.
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E-mail addresses: [email protected] (M. Jiang), [email protected] (F. X. Xin)
This review provides an overview of metabolic pathways for fumaric acid
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production.
Different fumaric acid producers and strain improvement strategies involved were introduced.
Alternative substrates for fumaric acid production and their fermentation 1
performances were shown.
The optimization of fermentation conditions was discussed.
Abstract Fumaric acid is an industrially important platform chemical, which has been widely
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used in food, chemicals, agriculture and pharmaceutical industries. Due to the rising price of raw materials in traditional petrochemical method and demand for sustainable
-p
development, fermentative fumaric acid production has attracted great attention. In this
context, various strains with enhanced fumaric acid production or elimination of by-
re
products were developed through mutagenesis or metabolic engineering. In addition,
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renewable organic wastes together with the fermentation process optimization have also
na
been widely investigated. Accordingly, this review will comprehensively summarize the achievements of fermentative fumaric acid production in recent years and major
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obstacles occurring in industrialization to provide insights and perspective for future
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microbial fermentation of fumaric acid. Keywords: Fumaric acid; Strain improvement; Process development; Waste resources
2
1. Introduction
Fumaric acid（trans-1,2-ethylenedicarboxylic acid）is a four-carbon dicarboxylic acid, which has been widely applied in food, chemicals, agriculture and pharmaceutical industries. Due to its multifunctional structure including a carbon-carbon double bond
ro of
and two carboxylic acid groups, fumaric acid can easily be esterified and polymerized [1], resulting in the main application as chemical feedstock for the production of paper
-p
resins, unsaturated polyester resins, alkyd resins, plasticizers, and miscellaneous
industrial products [2]. Moreover, fumaric acid has been widely used as food acidulant
re
and beverage ingredient due to its nontoxic nature and unique flavor [3]. Owning to its
lP
diverse application, fumaric acid has been identified as one of the top ten building block
na
chemicals [1]. The worldwide demand for fumaric acid was 225.2 kt in 2012,which is expected to be over 300 kt in 2020 [4].
ur
Currently, fumaric acid is mainly synthesized through the isomerization of maleic
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acid under the catalysis of mineral acids, peroxy compounds, or thiourea (Fig. 1) [1]. This petrochemical method is the most widely applied process for fumaric acid production due to the high production yield. However, the isomerization process usually occurs at high temperatures, leading to the formation of by-products and huge energy 3
consumption as well [5]. Alternatively, microbial fermentation process has attracted great interests owning to its sustainability and environmentally friendly properties. Microbial fermentation for fumaric acid production has shown promising prospects, and many works have been carried out in recent years including strain isolation and improvement, substrate selection, process optimization, and downstream
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separation. However, reviews regarding fumaric acid production are incredibly insufficient and most of them only involved one of these aspects like strain
-p
improvement [6] or separation strategies [7]. Accordingly, this review is to provide a
re
comprehensive investigation involving the achievements in recent years and obstacles
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restricting fermentation production. The fermentation performance of the most prominent fumaric acid-producing strains and the metabolic engineering involved will
na
be introduced first. The alternative substrates, especially renewable waste biomass,
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together with various methods of fermentation optimization will be discussed later.
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Finally, future prospects will also be analyzed.
2. Metabolic pathways and transportation mechanism for fumaric acid production
As illustrated in Fig. 2, fumaric acid is synthesized through three metabolic 4
pathways. The first one is the reductive branch of tricarboxylic acid (TCA) cycle that begins with the carboxylation of pyruvic acid. This pathway was considered as a major contributor for fumaric acid accumulation, in which a maximal theoretical yield of 2 mol/mol glucose can be obtained via CO2 fixation [8]. With the participation of ATP and CO2, pyruvic acid is converted to oxaloacetic acid by pyruvate carboxylase (PYC),
ro of
which is exclusively located in the cytoplasm [1, 9]. Subsequently, oxaloacetic acid is converted to fumaric acid by the action of malate dehydrogenase (MDH) and fumarase
-p
(FUM) with the participation of NADH. Formerly, FUM was generally considered to
re
catalyze both reversible reactions, while recent studies disclosed that its activity of
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catalyzing fumaric acid to L-malic acid could be completely inhibited by 2 mM fumaric acid [10]. This unique enzymatic property probably explains the mechanism of fumaric
na
acid accumulation in Rhizopus. oryzae and suggests that expression of FUM from R.
ur
oryzae could enhance fumaric acid generation. Moreover, nitrogen limitation was also
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revealed to attach a great significance on the activity of cytosolic FUM. When the urea concentration was decreased from 2.0 to 0.1 g/L, the cytosolic FUM activity was rapidly increased by 300%, and the fumaric acid production was increased from 14.4 to 40.3 g/L [11]. The second pathway is the oxidative TCA cycle, which also plays an important 5
role in the synthesis of fumaric acid [12, 13]. In this pathway, pyruvic acid is converted to acetyl-CoA by the pyruvate dehydrogenase system first, and acetyl-CoA subsequently enters TCA cycle. Fumaric acid is synthesized by the catalysis of succinate dehydrogenase (SDH) from succinic acid, and the theoretical yield is limited to 1mol/mol glucose due to the release of CO2. However, in most cases, fumaric acid
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synthesized in this pathway is hard to accumulate, and easily to be converted to L-malic
acid via mitochondrial FUM. In prokaryotes, fumaric acid reductase (FRD) catalyzes
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the conversion of fumaric acid to succinic acid. Since that, mitochondrial FUM and
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FRD were commonly deleted and fumaric acid accumulation in the oxidative route was
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enhanced consequently [14-16]. Moreover, the deletion of some regulators such as DNA-binding response regulator (arcA) or transcriptional repressor of lac operon (lacI)
na
was also proved to be beneficial of strengthening the oxidative pathway [14, 17].
ur
Anyhow, for wild type strains, fumaric acid produced in this way is mainly utilized for
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biomass synthesis and cannot accumulate during the growth stage [8]. Glyoxylate route is also suggested as a potential pathway for fumaric acid
production [18]. Isocitric acid formed in TCA cycle is decomposed into succinic acid and glyoxylic acid under the catalysis of isocitrate lyase. Subsequently, glyoxylic acid is combined with acetyl-CoA and L-malic acid is synthesized under the catalysis of 6
malate synthetase [18-20]. Although the theoretical yield (1mol/mol glucose) is relatively low compared to the reductive TCA cycle, glyoxylate bypass possesses great potential due to the relatively short metabolic pathway. However, since the key enzyme in glyoxylate pathway is strongly repressed in high glucose media, this pathway is difficult to be activated when glucose is used as the substrate [21]. This phenomenon
(PEP), an intermediate formed from glucose [17, 21, 22].
ro of
may be attributed to the inhibition of isocitrate lyase caused by phosphoenolpyruvate
-p
In addition to these three approaches, amino acid and fatty acid metabolism may
re
also be involved in fumaric acid production. Recent metabolic profiling has revealed
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that higher levels of carbon (EMP and TCA) and amino acid metabolism were observed in high fumaric acid -yielding strain [23]. Compared to original strain, 4-aminobutyric
na
acid and 5-aminolevulinic acid production was increased by 10.33- and 7.22-fold,
ur
respectively. These findings provide a new perspective that regulating the metabolism
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of amino acids and fatty acids may also be a viable option to increase fumaric acid production. Recently, a number of studies regarding fumaric acid production by engineering the urea and purine nucleotide cycle confirmed this assumption and suggested that the relationship between carbon metabolism and nitrogen metabolism might have a significant impact on fumaric acid accumulation [24]. 7
Regulation of transportation system is very critical for efficient organic acids secretion. The transportation of dicarboxylic acids including L-malic acid, succinic acid and fumaric acid has been discussed in yeasts, especially Saccharomyces. cerevisiae [25-27]. However, few systematic reviews on transportation mechanism of fumaric acid in fungi were reported so far. It was proposed that fumaric acid and L-malic acid share
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similar import systems due to their competitive relationship with the dicarboxylate
proton symporter. Moreover, fumaric acid entered cells most likely through passive
gene
Spmae1
encoding
the
C4-dicarboxylic
acids
transporter
in
re
that
-p
diffusion in the undissociated form in S. cerevisiae [28]. Recent studies have proved
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Schizosaccharomyces pombe is able to effectively export L-malic acid, fumaric acid, and succinic acid [29]. As for intracellular transport, gene acr1 encoding succinate-
na
fumarate transporter (SFC) in S. cerevisiae was discovered to transport the
ur
mitochondrial fumaric acid into cytoplasm [30], which has been confirmed in S.
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cerevisiae and Torulopsis glabrata [24, 31].
3. Fumaric acid production using wild type and mutagenized strains
3.1 Wild type strain
Some
bacteria
including
Zymomonas
Mobilis,
Bacillus
macerans, 8
Thermoanaerobacter ethanolicus and Erwinia chrysanthemi et al. [32] and yeasts including Scheffersomyces stipitis, Brettanomyces or Brett, Pachysolen tannophilus, and Candida utilis et al. [33] have been identified as fumaric acid producers. However, fungi are still the major producers, especially filamentous Mucoralean fungi, including Rhizopus nigricans [12, 21, 34], R. arrhizus [12, 13, 35-37], R. oryzae [38, 39], and R.
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formosa [1, 34]. Other Mucoralean fungi, such as Cunninghamella [40] and Cirnella
spp., and non-Mucoralean fungi such as Penicillum griseofulvum, Aspergillus glaucus,
-p
and Caldariomycels fumago were also identified as fumaric acid producers [41].
re
During these strains, Rhizopus sp. were the ideal fumaric acid producers, as the
lP
highest fumaric acid production was obtained by Rhizopus sp. R. arrhizus was once thought to be the most suitable strain for fumaric acid production, and the highest
na
production of 121.0 g/L was obtained in stirred tank though the yield is not so high
ur
(0.37 g/g) [42]. However, the requirement for rich nutrients of R. arrhizus burdened the
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bioprocess cost. Alternatively, R. oryzae with lower nutritional requirements has become the main producer and higher yield and productivity could be obtained as well. As reported, a fairly high productivity of 4.25 g/L/h with a yield of 0.91 g/g was achieved from R. oryzae ATCC 20344 in a rotary biofilm contactor (RBC) combined with downstream separation devices [43, 44]. 9
3.2 Mutagenized strain
Although Rhizopus sp. has been identified as an ideal fumaric acid producer, the production capacity still cannot meet the requirements for commercialization. In this case, random mutagenesis was considered as a promising way to obtain high-yield
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fumaric acid producer. In the last few decades, physical mutagenesis like UV or γ-ray mutagenesis and chemical mutagenesis using multiple alkylating agent, nitroso
compound and LiCl etc. were both widely applied (Table 1). Meanwhile, some novel
-p
screening methods were also designed. For example, after 3 minutes treatment using
re
UV radiation and 4% LiCl, high-yield fumaric acid mutants were screened from agar
lP
plates containing bromocresol green, in which fumaric acid production capacity could
na
be evaluated according to the size of discoloration ring formed by the infiltration of fumaric acid. Based on this selection method, one high-yield fumaric acid mutant R.
ur
oryzae ZJU11 was isolated after UV irradiation [45]. From an optimized glucose
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concentration of 85 g/L, 57.4 g/L of fumaric acid was produced in flasks, which was 2.05 times higher than that of parent strain. In addition to the enhancement of fumaric acid production, mutants with reduced by-products were also screened. Ethanol is the main by-product during fumaric acid fermentation process, which reduces the
10
conversion of carbon flux to fumaric acid. R. oryzae ME-F01, a mutant with low ethanol production was isolated by allyl alcohol resistance selection method after mutagenesis with UV coupled with nitrosoguanidine (NTG). Compared to the parent, the fumaric acid concentration of the mutant was increased by 21.1%, while ethanol production was decreased by 83.7% [46].
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Directed evolution is a more advanced strategy as it exerts selection pressure
according to specific needs and purposes. Recently, it was also used to screen ideal
-p
producers, especially the mutants with a capability to utilize other carbon sources. For
re
instance, Deng et al. isolated a series of R. oryzae mutants which was able to utilize
lP
starch directly, and 39.8 g/L of fumaric acid was obtained from cornstarch [47]. Since starch utilization capability could be characterized by the activity of glucoamylase,
na
glucose analogue of 2-deoxyglucose (2-DG) was used as evolutionary pressure in this
ur
study and mutants with high glucoamylase activity were effectively screened. Take
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another instance, R. arrhizus RH-7-13, a xylose-utilizing strain which was selected via continuously increasing the xylose concentration in medium [48]. With 80 g/L of initial xylose, R. arrhizus RH-7-13 could accumulate up to 28.5 g/L of fumaric acid, while parent strain accumulated 13.2 g/L of fumaric acid under similar conditions.
11
4. Fumaric acid production using metabolically engineered strains
Recently, metabolic engineering has been developed rapidly, which can help metabolically construct strains with higher fumaric acid production, less by-product formation or more efficient substrate utilization. Strategies to improve fumaric acid
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production through metabolic engineering can be summarized as follows: (1) overexpression of rate limited enzymes; (2) introduction of new metabolic pathways;
-p
(3) knocking out of genes responsible for by-products generation; (4) energy metabolism balance and cofactor generation; (5) elimination of inhibitor effects to
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enhance synthesis pathways. (6) enhancement or inhibition of related transporters.
na
on different strains.
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Advances in terms of these aspects were comprehensively summarized as below based
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4.1 Filamentous fungi
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As the most mainstream fumaric acid producer, R. oryzae has been metabolically engineered to enhance fumaric acid production. Overexpression of endogenous PYC and PPC was considered to increase the carbon flux towards oxaloacetic acid in R. oryzae M16 [15]. Compared to the initial strain, fumaric acid yield of recombinant overexpressing PPC was increased by 26% with yield of 0.78 g/g glucose, but the one 12
overexpressing PYC was significantly affected on growth and the yield was extremely low (<0.05 g/g). Additionally, native FUM in R. oryzae was considered to promote the conversion of L-malic acid to fumaric acid and thereby was overexpressed in R. oryzae M16 [49]. Nevertheless, fumaric acid production was not improved whilst L-malic acid yield was doubled, suggesting the opposite effect of the assumption.
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Besides Rhizopus sp., thermophilic filamentous fungi have shown promising
potential for organic acid production. Recently, Myceliophthora thermophila was
-p
successfully engineered for fumaric acid production using the CRISPR/Cas9 system
re
[50]. After introducing the gene Ckfum originating from C. krusei into M. thermophila,
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fumaric acid titer was increased by triple. To further enhance fumaric acid accumulation, cytoplasmic fr1and fr2 together with mitochondrial fumarase (Mtfum) were deleted.
na
Morover, mitochondrial malate carrier (MOC) was deleted to enhance L-malic acid
ur
synthesis as it was the precursor of fumaric acid. Finally, overexpression of Mtsfc
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encoding C4-dicarboxylate acid transporter led to 17.0 g/L of fumaric acid in fed-batch fermentation.
4.2 Bacteria
As the model bacterium, E. coli has been comprehensively investigated for 13
fumaric acid production through metabolic engineering (Table. 2). In prokaryotes, the enhancement of genes expression involved in oxidative TCA cycle was a common strategy. . Besides these enzymes like FUM and FRD, some regulators have been proved play significant roles in the oxidative pathway. For instance, arcA is a DNAbinding response regulator and its deletion was discovered to intensify the Krebs cycle
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[18]. E. coli with arcA deleted exhibited a better growth capacity, and fumaric acid production was increased by 1.5-fold [18]. As a precursor of glycolytic pathway,
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Oxaloacetic acid plays an important role in organic acid accumulation. Thus, enhancing
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oxaloacetic acid synthesis was a feasible strategy in organic acid production, which has
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been practiced in L-malic acid and succinic acid production by overexpressing PPC or PYC [51-54]. Based on in silico flux response analysis, homologous PPC was
na
overexpressed in E. coli CWF41 using the strong tac promoter [17]. Compared to
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control strain E. coli CWF4, the enzyme activity of PPC was enhanced by 4.7 times,
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and fumaric acid production was increased by 2.8 times [14, 17]. Another common way to increase fumaric acid accumulation in E. coli is to enhance the carbon flux in glyoxylate shunt. Thereby, deletion of iclR encoding isocitrate lyase repressor in E. coli was also proved
effective. Furthermore, the deletion of lactate dehydrogenase (LDH)
or pyruvate formate-lyase (PFL) catalyzing lactic acid synthesis increased the flux of 14
pyruvic acid into the oxidative TCA cycle and glyoxylate shunt [55, 56]. Reduction of the main by-products like L-malic acid and succinic acid might be the commonest strategy to enhance fumaric acid accumulation, which was applied in nearly all engineered E. coli [14, 17, 18]. For instance, after deletion of fumABC, frdABCD and iclR in E. coli JM109 (DE3), a strain could hardly accumulate fumaric
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acid, the recombinant was able to produce 2.12 mmol/L of fumaric acid [18]. Moreover, deletion of other by-products such as acetic acid, lactic acid and aspartic acid could also
-p
improve fumaric acid production. Acetyl-CoA synthase (acs) is one of key enzymes
re
responsible for acetic acid secretion and assimilation [57]. To reduce acetic acid
lP
accumulation, acs was overexpressed in strain E. coli ABCDIA-ptsG--mdh--glk-galpppc-acs and the fumaric acid yield was up to 1.5 times higher than the parent with the
ur
4.3 Yeasts
na
combined strategies [14].
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Compared to E. coli, yeasts are considered as potential industrial candidates for organic acid production owning to their higher acid resistance [58, 59]. However, one major limit for fumaric acid production in yeasts is that the original fumarase affinity to fumaric acid is 17 times higher than that to L-malic acid [60]. The cytosolic fumarase 15
in yeasts mainly catalyzes the conversion of fumaric acid to L-malic acid rather than the reverse reaction, which results in the inability to accumulate large amounts of fumaric acid in cytosol [7]. In this case, the reductive route of fumaric acid biosynthetic pathway was introduced into S. cerevisiae [61]. The resulting strain with overexpression of RoMDH, RoFUM1 (from R. oryzae) and endogenous PYC was able to produce 3.2
ro of
g/L fumaric acid. Inspired by this study, fumaric acid production by simultaneous use
of oxidative and reductive routes was carried out. The oxidative pathway was first
-p
introduced into S. cerevisiae by the deletion of FUM1 and the resulting strain S.
re
cerevisiae FMME004 accumulated 0.3 g/L of fumaric acid [62]. However, fumaric acid
lP
production was decreased when heterologous genes involved in the reductive pathway were introduced [61], which might attribute to the diversion of carbon flow into TCA
na
cycle as the activity of PDC was decreased. With the supplementation of 32 μg/L of
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biotin and an optimal carbon/nitrogen ratio, 5.6 g/L of fumaric acid was obtained in S.
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cerevisiae FMME004-6 finally, in which RoPYC gene was further overexpressed. In addition to S. cerevisiae, other yeasts such as T. glabrata and Scheffersomyces
stipitis have also been explored for fumaric acid production. Recent studies have revealed that four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA) and FUM1 are involved in fumaric acid 16
biosynthesis in T. glabrata [24]. Manipulation of their expression levels is able to regulate the urea and purine nucleotide cycles, and thereby regulate the carbon and nitrogen metabolism. For example, by controlling the expression of ASL at high level and ADSL at low level, 5.6 g/L of fumaric acid was obtained. Moreover, further overexpression of Spmae1 could improve fumaric titer to 8.8 g/L. S. stipitis is an
ro of
excellent xylose-utilizing yeast. Recently, the reductive pathway from R. oryzae FM19 was introduced into S. stipitis [16]. To enhance the reductive pathway activity, FUM
-p
was deleted to block fumaric acid conversion in the Krebs cycle. Furthermore, the
re
heterologous transporter YMAE1 of SpMAE1 was overexpressed, and 4.6 g/L fumaric
lP
acid was obtained from 50 g/L xylose by the recombinant S. stipitis. Despite these achievements, most engineering strategies focused on the regulation
na
of single pathway currently, while the overall metabolism balance was often ignored.
ur
For instance, after the simultaneous introduction of oxidative and reductive routes, S.
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cerevisiae FMME004-6 produced only 5.6 g/L of fumaric acid. This low titer could attribute to the imbalanced distribution of carbon flux. Therefore, a number of synthetic biology strategies have been adopted to optimize metabolism balance or engineer the transportation efficiency of intermediate metabolites. Recent progress mainly focused on mitochondrial engineering [63], scaffold engineering [64], cofactor engineering [64] 17
and modular assembly [31]. Mitochondrial engineering has been used to construct the oxidative pathway for fumaric acid production in C. glabrata, a classical αketoglutarate producer [63]. This oxidative pathway, containing α-ketoglutarate dehydrogenase complex (KGD), succinyl-CoA synthetase (SUCLG) and succinate dehydrogenase (SDH) were constructed and optimized using the KGD2-SUCLG2 and
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SDH1 expression cassettes. Moreover, SFC1 and SpMAE1 were overexpressed to
enhance the secretion of fumaric acid. The resulting strain with the overexpression of
-p
ASL gene was able to accumulate 15.8 g/L of fumaric acid from 100 g/L glucose.
re
On the other hand, the reductive pathway was also introduced into C. glabrata
lP
lately. Since there are additional pathways consuming or transporting intermediate metabolites in the reductive TCA pathway, spatial modulation and cofactor engineering
na
were performed to enhance the efficiency of conversion from pyruvic acid to fumaric
ur
acid [64]. By the means of synthesizing DNA scaffolds, RNA scaffolds and protein
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scaffolds [68], structural synthetic biotechnology was developed to improve the pathway transportation efficiency, which has been applied for the production of many chemicals in recent years, such as butyrate [69], hydrogen [70] and L‐ threonine [71]. As for fumaric acid, DNA scaffolds have been recently used in S. cerevisiae and C. glabrata, which both achieved a significant increase in terms of chemicals production 18
[31, 64]. In C. glabrata, three zinc finger proteins ADB1, ADB2 and ADB3 were fused with RoPYC, RoMDH and RoFUM, respectively. DNA scaffold with different ratios of binding sequences resulted in different fumaric acid concentrations while these values were all higher than the one of control without DNA scaffold. Among these, the highest production of 11.2 g/L was obtained from T.G‐ 4G‐ S(1:1:2), which was about 2.8 times
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higher than the control value. Based on this, cofactor engineering was taken to enhance
ATP and NADH generation. Cofactor regeneration has been proved to be an effective
-p
way to maintain the cellular redox balance by altering the intracellular cofactor pool
re
and a number of enzymes have been discovered to directly affect the ratio of
[72],
mitochondrial
lP
NADH/NAD+ or ATP/ADP, such as cytoplasmic H2O-forming NADH oxidase (NOX) alternative
oxidase
(AOX)
[73],
phosphoenolpyruvate
na
carboxykinase (PCK) [74], and formate dehydrogenase (FDH) [75]. Therefore, PCK
ur
was overexpressed to generate ATP to motivate the RoPYC‐ catalyzed carboxylation
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of pyruvic acid to oxaloacetic acid, and FDH was overexpressed to generate NADH which could be used to drive the RoMDH‐ catalyzed reduction of oxaloacetic acid to malic acid. After optimizing overexpression of PCK and FDH, the strain T.G‐ 4G‐ S(1:1:2)‐ P(M)‐ F(H) led to a 63.7% increase in fumaric acid production, compared with that of strain T.G‐ 4G‐ S(1:1:2) and the final concentration in a 5-L batch bioreactor was 19
up to 21.6 g/L. Metabolic balance of S. cerevisiae can also be optimized by modular pathway engineering [31]. The fumaric acid biosynthetic pathway was divided into three modules (Fig. 3): (1) PMFM module: reduction module containing RoPYC, RoMDH, RoFUM1 and SpMAE1; (2) KSSS module: oxidation module containing KGD2,
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SUCLG2, SDH1 and SFC1; (3) RPSF module: byproduct module including sRNA-
RHR2, sRNA-PDC6 and DNA scaffold. RNA switch is a type of gene regulatory
-p
element, which is able to dynamically regulate gene expression via transcription,
re
splicing, RNA interference and translation [76]. sRNA-RHR2 and sRNA-PDC6 can be
lP
designed to regulate GPP (glycerol-1-phosphatephos-phohydrolase1) and PDC activities, respectively [77]. Glycerol and ethanol production were reduced
na
consequently. PMFM and KSSS modules with various strengths were assembled and
ur
optimized, and the highest concentration of 20.5 g/L was obtained by regulating the
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expression strength of KGD2-SUCLG2 at high level and SDH1 at medium level, which showed 49.7% increase compared with the control strain. Taken together, it could be speculated that combined regulation of metabolic pathways could break down the metabolic constraints to achieve the balance of the biological system and thereby increase the production, which cannot be evaded by individual regulation [78]. In 20
addition, fumaric acid production by the final strain with DNA-guided scaffolds was increased by 40.0% with 33.1 g/L compared with that of no DNA scaffold, which might due to the increased local intermediates concentration and the avoidance of feedback inhibition [79-81].
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5. Fermentation Substrates
Substrates cost accounts for about 30- 40% of the total cost in industrial organic
-p
acids production [82]. Although glucose is the most preferred carbon source for fumaric
fermentation preferences of these carbon sources are
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evaluated [83]. However, the
re
acid production, other carbon sources like glycerol, xylose and sucrose were also
generally inferior to those from glucose, as additional pathways are needed to link these
na
substrates with glycolysis and TCA cycles. With the rising economic and environmental
ur
requirements, some renewable raw materials rich in starch or lignocellulose are also
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considered to be promising alternative substrates.
5.1 Xylose
As a widely available pentose in lignocellulose waste, xylose has been explored for fumaric acid production in early 1989 by immobilized R. arrhizus, although the 21
highest productivity was only 0.09 g/L/h [84]. Commonly, high xylose concentration inhibits the growth of R. arrhizus [48]. Therefore, a strain RH 7-13-9#, which can adapt a high xylose concentration was selected through a selection medium. By efficient immobilization fermentation, 45.3 g/L fumaric acid was obtained, and the conversion of xylose reached 73%, which is close to the theoretic yield (77%) [85]. Furthermore,
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co-fermentation of a mixture of glucose and xylose was also investigated. With the
optimum glucose/xylose ratio of 75/25 (w/w), 46.8 g/L fumaric acid was obtained from
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100 g/L of total sugars, which was higher than the production in fermentation of glucose
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and xylose separately [86]. In addition, metabolic engineering has enabled other strains
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to efficiently utilize xylose for fumaric acid production. For instance, 4.7 g/L of fumaric acid was produced from 50 g/L of xylose by S. stipitis PSYPMFfS after the introduction
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of xylose metabolic pathway [16].
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5.2 Glycerol and crude glycerol
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Glycerol is a versatile raw material which has been widely used in the cosmetics and automotive industry. Glycerol has been attempted as carbon source together with other monosaccharides to support fumaric acid fermentation in R. oryzae [87]. As reported, 17 strains/isolates of Rhizopus sp. were tested to produce fumaric acid with 22
40 g/L glycerol as the sole carbon source while the highest titer was only 6.1 g/L at 192 h. In contrast, the titer was much higher when monosaccharide was used as a carbon source, among which the titer of 19.8 g/L from xylose was the highest one. In this case, a co-fermentation strategy was adopted and the production was improved when glycerol was used as co-substrate with monosaccharides such as xylose, fructose, galactose, and
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mannose, except the one with glucose. The highest titer of 28.0 g/L was obtained with
a yield of 0.90 g/g from 40 g/L xylose and 20 g/L glycerol. Additionally, crude glycerol
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is a major by-product in biodiesel industry and its production was increased
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dramatically annually. However, crude glycerol is not a suitable feedstock for many
lP
microorganisms including R. oryzae [88]. R. arrhizus RH-07-13 is a high-yield fumaric acid producer that has also been investigated for crude glycerol utilization. However,
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the production was only 4.4 g/L using 80 g/L crude glycerol as the sole carbon source
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[89]. In this case, a strategy for co-fermentation of crude glycerol and glucose was
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developed that increased production to 22.8 g/L using the mixed substrate of 40 g/L of crude glycerol and 40 of g/L glucose. Compared with glucose fermentation, the production cost was reduced by approximately 14%, which suggested that the co‑ utilization strategy could be more advantageous rather than sole glucose.
23
5.3 Starchy materials
Starch-rich materials, such as beet molasses [90], corn starch [91], potato flour, cassava flour and sweet potato [92] have been widely used for fumaric acid production. Four kinds of raw starchy materials, including starch, cassava powder, corn powder and
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degermed corn powder with different carbon/nitrogen ratio were compared for fumaric acid production. Among these four substrates, degermed corn powder with nitrogen
concentration of 0.25 g/L was the most suitable substrate. For cassava powder and corn
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powder, although biomass was increased, fumaric acid production was decreased with
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the increase of nitrogen content. When 100 g/L of degermed corn powder was used as
lP
substrate, 35.5 g/L of fumaric acid was obtained via an optimized simultaneous
na
saccharification and fermentation (SSF) process [91].
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5.4 Lignocellulosic substrates
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Lignocellulose is the most widely distributed substance in the world, which consists of glucose-rich cellulose, xylose-rich hemicellulose and lignin. It has been considered as an excellent feedstock for bulk chemicals production, such as 2,3-2,3butanediol, succinic acid and fumaric acid [93, 94]. In this context, fumaric acid production from lignocellulose wastes like cassava bagasse [95], corn straw [96] and 24
pulp and paper solid waste (PPSW) [97] etc. was investigated in recent years. Most microorganisms are very weak at using xylose when glucose exists as called carbon catabolite repression. To improve the utilization of xylose in lignocellulose, a novel two-stage corn straw utilization strategy was designed, in which fungi were first cultivated in hydrolysate containing 30 g/L of xylose. After the pre-culture process,
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fumaric acid fermentation was initiated in the hydrolysate containing 80 g/L of glucose. Up to 27.8 g/L of fumaric acid with a productivity of 0.33 g/L/h was finally obtained
-p
[96].
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PPSW, lignocellulose-rich industrial waste has become the 3rd largest industrial
lP
polluter due to the escalating demand for paper-based products. Recently, PPSW was considered to be an alternative feedstock for fumaric acid fermentation under
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submerged and solid-state fermentation techniques. Fumaric acid production was
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decreased when the particle size of PPSW was increased, and the highest production of 23.5 g/L with productivity of 0.49 g/L/h was achieved at 48 h in submerged
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fermentation when the particle size was in the range of 1.7 mm < x ≤ 3.35 mm. As for solid state fermentation, 41.5 g/kg dry weight fumaric acid was obtained with 75 μm < x ≤ 300 μm particle size after 21 days [97]. In recent years, studies on the production of fumaric acid from lignocellulosic 25
materials continue to be published and some of these results were comparable to those observed with the control medium using pure glucose and xylose [98]. However, pretreatment of raw materials has become a major obstacle to the restriction of renewable biomass fermentation. Currently, the pretreatment process of lignocellulose is still costly and the conversion rate is not so high. Moreover, compounds like
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furaldehydes, phenolic compounds and various minerals could affect the metabolism of
strains, which should be studied further [99]. Anyhow, lignocellulose is still highly
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anticipated to become an important raw material with the development of pretreatment
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process and optimization of solid-state fermentation strategy. In addition, some high
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value-added products are available from lignocellulosic. For instance, chitosan/chitin is a material with a vast market foreground that could be recovered as a by-product from
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lignocellulosic materials. In the two-stage corn straw utilization process introduced
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above, hemicellulose hydrolysate obtained from corn straw pretreated with dilute
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sulphuric acid can be used for biomass and chitin/chitosan synthesis for fungal [96].
26
6. Optimization of fermentation conditions
6.1 Morphology control
Morphology control is the major challenge to maintain the fumaric acid yield and productivity for fungi [100-102]. The main types of fungal morphology include clumps,
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filaments and pellets [101]. Especially, pellets can be further subdivided into aggregated and non-aggregated [103]. The morphology of filaments is conducive to some enzymes
-p
production, while this will increase the viscosity of fermentation broth, leading to the
re
oxygen transfer restriction. Alternatively, pellet will alleviate the problem of high
lP
viscosity and support recycling in subsequent fermentation batches as well, which has been considered as the most favorable morphology for industrial fumaric acid
na
production. In contrast, cells with clump morphology were thought to be detrimental for fumaric acid production, since both biomass and products are difficult to accumulate
ur
because of the severe internal oxygen limitation. Up to now, the morphology control of
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Rhizopus sp. is still largely based on empirical data. For R. arrhizus, dispersed mycelia were proved to be much more effective than pellets for fumaric acid production [4]. When 25 g/L of glucose and 0.04 g/L of ammonium sulphate were fed, 19.7 g/L of fumaric acid was obtained from R. arrhizus NRRL 2582 with dispersed mycelia, the 27
yield and productivity were 0.84 g/g and 0.18 g/L/h, respectively. In contrast, only 7.0 g/L of fumaric acid was produced from the pellets under similar conditions, together with the yield of 0.29 g/g and the productivity of 0.10 g/L/h. However, as for R. delemar NRRL 1526, the opposite effect was reported [104]. For all this, a consensus is that adequate oxygen transfer is the key for morphology control. Moreover, enough space
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between cells is needed for efficient fumaric acid production since it is important for fumaric acid secretion and the mass transfer between cells and medium [104].
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Various factors have been found to affect the fungi morphology, such as the
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composition of the medium [105-107], trace metals [39, 108, 109], spore concentration
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[110], pH [108, 109, 111], stirring rate [112, 113] and temperature [101]. For example, low inoculation concentration of R. oryzae will help form pellets, while high inoculation
na
concentration is conducive to form filaments [110]. More interestingly, different species
ur
of fungi exhibit different morphology even under the same conditions. For instance,
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high C:N ratio would promote mycelial growth in A. niger [114], but pellets in R. arrhizus [115]. Even for the same fungus, there are still various controversies about its morphology control. As for Rhizopus, the absence of Zn2+ would prevent the formation of pellets [39], while other study indicated that metal ions tend to inhibit pellets formation [109]. Besides, studies showed that pellets would form at pH 2.6-3.36 and 28
large mycelial clumps would form at pH 4-5 for Rhizopus [111]. A proposed strategy to controlling the fungus morphology of is cell immobilization, which will enhance the cells stability and thereby improve fumaric acid yield and productivity [116, 117]. Compared with the conventional submerged fermentation, this technology is characterized by constant or intermittent contact with
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the liquid medium rather than being immersed in fermentation broth at all times, which
allows a high cells concentration and low substrate consumption for biomass generation
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[118, 119]. The application of immobilized cells could also alleviate the problem of
re
oxygen transfer and provides a strategy for continuous production of fumaric acid. In
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recent years, various materials such as cork, expanded polystyrene, expanded clay, agar gel cubes and polypropylene tube [120] have been applied as immobilizing materials.
na
For example, in a rotary biofilm contactor bioreactor, R. oryzae ATCC 20344 cells were
ur
immobilized on the surfaces of plastic discs, in which a high fumaric acid productivity
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of 3.78 g/L/h was achieved [43, 121]. However, one prominent dilemma is that materials used as immobilization matrix are usually weak in mechanical properties, making industrial production difficult.
29
6.2 Neutralizing agents
In the bio-process of fumaric acid production, neutralizing agents are commonly used to control pH [104, 110], and CaCO3 is the most widely used neutralizing agent. The unique advantage is that CaCO3 can release CO2, which can be fixed by PYC and
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thereby reinforces the reductive TCA cycle. However, insoluble calcium fumarate will be produced when CaCO3 was added, which will increase the viscosity of the fermentation broth. Another challenge is that sulfuric acid will be used in subsequent
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stages to release fumaric acid, resulting in insoluble CaSO4 produced, which is a
re
compound with narrow industrial application. Accordingly, various other neutralizing
lP
agents such as Na2CO3, NaHCO3, Ca(OH)2 and (NH4)2CO3 have been studied as
na
substitutes for CaCO3 [35, 37, 111]. With addition of Na2CO3 or NaHCO3, heating is no longer needed to recover the fermentation product due to the higher solubility of sodium
ur
fumarate, thereby downstream processing costs could be reduced. Besides, sodium
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fumarate could facilitate separation and recycling of fungal cell biomass in succeeding batch fermentation steps [35, 111]. However, Na+ in the medium tends to make adverse effect on cell metabolism, and the low solubility of sodium fumarate would cause product inhibition, which limits the utilization of Na2CO3. As for Ca(OH)2 and
30
NaHCO3, these neutralizing agents were reported to increase the accumulation of byproducts like L-malic acid and ethanol. (NH4)2CO3 was also studied as an alternative neutralizing agent under phosphorus limitation [37, 122], and the highest 46.0 g/L of fumaric acid was obtained by R. arrhizus NRRL 1526 although the fermentation time
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was prolonged to 108 h [37].
6.3 Fermentation parameters
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Fermentation parameters such as temperature, metal ion, dissolved oxygen and pH
re
are important to industrial fermentation process. Temperature not only effects various reaction rates in fermentation, but also indirectly effects product synthesis by altering
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the physical properties of fermentation broth, including dissolved oxygen, the mass
na
transfer rate of the matrix, and the nutrients decomposition and absorption rate. Up to now, due to the strain specificity and the wide range of optimal growth temperatures
ur
(usually 30-37℃ for fungal), the influence of temperature on fumaric acid production
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has not been deeply studied [104]. Metal ions like Mg2+, Fe2+, Mn2+ and Zn2+, together with vitamins like biotin and
riboflavin, act as cofactors and activators involved in metabolic processes. Especially, Mg2+ and Mn2+ have been proposed to assist the conversion of isocitric acid to α31
ketoglutaric acid, and thereby lead to the fumaric acid accumulation [41]. Biotin, as an activator of PYC, has been widely added to broth to promote the synthesis of organic acids [123]. However, for industrial-scale production, it is almost impossible to add metal salts and vitamins due to cost constraints. Alternatively, some cheap nutrient-rich raw materials like corn steep liquor are usually used as substitutes.
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Dissolved oxygen (DO), as a necessity for the growth of aerobic microorganisms
is generally the controlling factor due to the extremely low oxygen solubility of in water.
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Despite the essential role for growth and metabolism of microorganisms, high DO level
re
during fermentation does not necessarily favor the production of fumaric acid, as
lP
excessive DO will stimulate TCA cycle and lead to increased cell growth and energy expenditure. Moreover, too high aeration or agitation rates results in low level of
na
dissolved CO2, which exerts negative influences for CO2 fixation, and thereby reduces
ur
the fumaric acid production. In this case, an improved fermentation strategy was
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patented by DuPont to improve the fumaric acid yield and productivity, in which DO was controlled at a high level (80%-100%) during the cell growth phase, but a low level (30%-80%) during acid production phase [124]. With this strategy, high fumaric acid production of 130.0 g/L was obtained in 142 h, but by-products such as L-malic acid and succinic acid were increased concomitantly. 32
pH is of great importance during fermentation for its multifunctional function, such as effecting enzyme activities involved in various metabolic reactions and regulating the transport of fumaric acid. Fumaric acid production by Rhizopus sp. usually requires pH level of around 6. However, low pH of 3.0 resulted in a sharp decrease of fumaric acid production (9.4 g/L), compared with 30.2 g/L at pH of 5.0 [125]. It might be
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explained that low pH level due to the accumulation of fumaric acid leads free acids to enter cells by passive diffusion, thereby inhibiting the biosynthesis of fumaric acid [37].
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Furthermore, low pH level leads to the decrease of cell membrane permeability, which
re
further causes the accumulation of intracellular undissociated fumaric acid and thereby
lP
leads to the inhibition of fumaric acid [26]. In addition to reducing fumaric acid production, low level of pH also leads to high concentrations of by-products including
na
ethanol and glycerol. For these reasons, some strategies to produce fumaric acid at low
ur
pH using acid-tolerant producers like yeasts or in situ product removal techniques [43,
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126, 127] have been adopted. For example, by releasing OH− from a polyvinyl pyridine adsorbent into the broth, an integrated system of simultaneous fermentation-adsorption was able to produce 85 g/L of fumaric acid from 100 g/L of glucose in 20 h under repetitive fed-batch cycles [43]. The accumulation of fumaric acid, like many other organic acids has been proved 33
to be greatly affected by C:N ratio in the medium. Generally, high C:N ratio is beneficial for fumaric acid overproduction. Microbial growth and proliferation are aborted due to the depletion of a limited nitrogen source, and thereby the pressure caused by excess carbon forces the microorganisms to direct carbon to the accumulation of organic acids. Compared to organic nitrogen sources like yeast extract, inorganic nitrogen sources like
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urea and ammonium salts might be more suitable for fumaric acid production [82]. However, excessive C:N ratio leads to substrate inhibition and reduces cell productivity,
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which might be caused by the inhibition of α-ketoglutarate dehydrogenase enzyme
re
complex at high glucose concentration [128]. Some results from the recent continuous
lP
fermentations clearly indicated how nitrogen addition influences the time-dependent product distribution of R. oryzae. As proposed, ethanol production terminated when
na
fumaric acid production reached its production window, in which the volumetric
ur
productivity was approximately 0.3 g/L/h. Therefore, an appropriate continuous
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nitrogen level control could be highly beneficial to fumaric acid accumulation compared to simply limiting the nitrogen at the onset [120]. Moreover, phosphorus source was proposed to play a similar role, as nitrogen source and high phosphorus levels were indicated to reduce carbon flux in the direction of organic acids [128, 129]. Thus, phosphorus source has been considered to control the mycelial growth of R. 34
arrhizus NRRL 1526 instead of nitrogen source. With (NH4)2CO3 as neutralizing agent, the maximum ammonium fumarate productivity of 0.46 kg/m3/h was achieved when 0.1 kg phosphate/m3 was used [37].
7. Conclusions and future prospects
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Fumaric acid, as a valuable dicarboxylic organic acid has been widely applied in food, medicine and construction industries, which is also recognized as one of the “top
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12” chemical building blocks. The earliest attempt to produce fumaric acid by microbial
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fermentation can be traced back to the early 20th century. With the continuous expansion
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of fumaric acid application and the rising prices of fossil raw materials, fumaric acid production by microbial fermentation is exhibiting its potential, although it still remains
na
less economically competitive compared with the traditional petrochemical method. In
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the last few decades, various wild types and mutants were characterized for fumaric acid production, in which R. oryzae was considered to be the most advantageous
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producer. With the development of metabolic engineering, a number of other producers (especially yeasts) have been investigated for fumaric acid production. Furthermore, the rapid development of synthetic biology in recent years has provided new perspectives for metabolic engineering. 35
The cost of substrate is important for the feasibility of industrial fumaric acid production. Although glucose is the most wildly used substrate, many alternatives such as glycerol and xylose have been attempted. To reducing the cost of production, renewable biomaterials are considered to be promising, especially some feedstocks rich in starch or lignocellulose. One of the major dilemmas in fumaric acid fermentation is
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to maintain suitable fermentation conditions. Therefore, a lot of fermentation regulations like morphology, pH and neutralizing agents have been extensively studied.
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Based on the above aspects, the metabolic engineering for fumaric acid production
re
in the future should focus on global regulation rather than single genes or pathways.
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Some novel technologies like mitochondrial engineering, scaffold engineering, and cofactor engineering will be instructive and bring guidance for subsequent process
na
optimization. The utilization of waste biomass or co-substrate fermentation may be a
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more competitive strategy which can potentially improve the feasibility of the
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bioproduction process. Besides, the strategy of process optimization should not only focus on the laboratory-scale production increase, but also the feasibility of industrialscale fermentation, which will help to improve the economic competitiveness of the fermentation process. Moreover, the development of fumaric acid derivatives such as L-malic acid and L-aspartic acid, which are 1.5-2 times more valuable than fumaric 36
acid will also increase the value of fumaric acid production.
Acknowledgements
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This work was supported by the National Key Research and Development Program of China (2018YFA0902200), Jiangsu Province Natural Science Foundation for Youths (BK20170993, BK20170997), the Jiangsu Synergetic Innovation Center for Advanced the
National
Natural
Science
Foundation
of
China
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Bio-Manufacture,
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(No. 21978130,No. 21706125, No. 21727818), Jiangsu Key Lab of Biomass-based
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Green Fuels and Chemicals Foundation (JSBEM201908), and Project of State Key
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Laboratory of Materials-Oriented Chemical Engineering (ZK201601).
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Abbreviations
TCA, tricarboxylic acid cycle; PYC, pyruvate carboxylase; MDH, malate
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dehydrogenase; FUM, fumarase; FUMR, fumarase from R. oryzae; SDH, succinate dehydrogenase; FRD, fumaric acid reductase; SFC, succinate-fumarate transporter; arcA, DNA-binding response regulator; iclR, isocitrate lyase repressor; lacI, transcriptional repressor of the lac operon; PEP, phosphoenolpyruvate; PPC, 37
phosphoenolpyruvate carboxylase; Ckfum, fumarase from Candida krusei; Mtfum, mitochondrial fumarase; MOC, mitochondrial malate carrier; LDH, lactate dehydrogenase; PFL, pyruvate formate-lyase; ptsG, The PEP dependent glucosespecific phosphotransferase system; aspA, aspartase; aceBA, glyoxylate shunt operon; argininosuccinate
fumarylacetoacetase;
lyase;
SpMAE,
ADSL,
adenylosuccinate
C4-dicarboxylic
acids
lyase; transporter
FAA, in
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ASL,
Schizosaccharomyces pombe; NOX, NADH oxidase; AOX, mitochondrial alternative
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oxidase; PCK, phosphoenolpyruvate carboxykinase; FDH, formate dehydrogenase;
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KGD, α-ketoglutarate dehydrogenase complex; SUCLG, succinyl-CoA synthetase;
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GPP, glycerol-1-phosphatephos-phohydrolase1; SSF, simultaneous saccharification
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ur
na
Fig. 1. The main methods for fumaric acid synthesis.
47
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-p
Fig. 2. Metabolic pathways of fumaric acid synthesis. A: Metabolic pathways in
eukaryocyte; B: Metabolic pathways in prokaryote. Abbreviations: PYC, pyruvate
re
carboxylase; MDH, malate dehydrogenase; FUM, fumarase; FUMR, fumarase from R.
lP
oryzae; SDH, succinate dehydrogenase; FRD, fumaric acid reductase; SFC, succinate-
repressor; lacI,
na
fumarate transporter; arcA, DNA-binding response regulator; iclR, isocitrate lyase transcriptional repressor of the lac operon; PEP, phosphoenolpyruvate;
ur
PPC, phosphoenolpyruvate carboxylase; Ckfum, fumarase from Candida krusei;
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Mtfum, mitochondrial fumarase; MOC, mitochondrial malate carrier; LDH, lactate dehydrogenase; PFL, pyruvate formate-lyase; ptsG, The PEP dependent glucosespecific phosphotransferase system; aspA, aspartase; aceBA, glyoxylate shunt operon; ASL,
argininosuccinate
lyase;
ADSL,
adenylosuccinate
lyase;
FAA, 48
fumarylacetoacetase;
SpMAE,
C4-dicarboxylic
acids
transporter
in
Schizosaccharomyces pombe; KGD, α-ketoglutarate dehydrogenase complex; SUCLG, succinyl-CoA synthetase;
SDH,
succinate
dehydrogenase;
GPP,
glycerol-1-
lP
re
-p
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phosphatephos-phohydrolase1;
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Fig. 3. The fumaric acid biosynthetic pathway involved in modular assembly. The engineered targets are shown in yellow, green and blue, and its related target genes in
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red. PMFM module, reduction module; KSSS module, oxidation; RPSF module,
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byproduct module.
49
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Table 1. Production of fumaric acids in terms of performance and mutagenic strategies for Rhizopus. Mutation
Culture Substrate
Titer
Yield
Productivity
(g/L)
(g/g)
(g/L/h)
57.4
\
\
[40]
lP
Strain strategy
method
Reference
Shake
R. oryzae UV
Glucose
flask
R. oryzae UV ZJU11 R. oryzae
na
ZJU11
Batch
41.1
0.48
0.37
[40]
Glucose
Batch
52.7
\
\
[46]
44.1
0.44
0.53
[47]
32.2
0.32
0.44
[47]
ur
UV, NTG
Glucose
ME-UN-8
Cornstarch
nitrogen ion
(100
DG-3
implantation.
total
Jo
R. oryzae
Shake g/L flask （SSF）
sugar) Raw corn Shake
R. oryzae
nitrogen ion
powder
DG-3
implantation.
(100
flask g/L
（SSF）
total 50
sugar) R. oryzae
Shake
UV, γ-rays
Glucose
RUR709 R. oryzae
26.2
0.32
0.22
[130]
flask UV, γ-rays
Glucose
Batch
32.1
0.42
0.32
[130]
Glucose
Batch
49.4
0.56
0.29
[23]
RUR709 R. oryzae
Femtosecond
FM19
laser
Engineering Strain
Culture
Titer
Yield
Productivity
method
(g/L)
(g/g)
(g/L/h)
<0.05
\
Substrate strategy
R.
ro of
Table 2. Production of fumaric acids in terms of performance and engineering strategies. Reference
oryzae Expression of pyc
Glucose
Batch
0.7
Expression of ppc
Glucose
Batch
Expression of fumR2
Glucose
Batch
R.
oryzae
ppc oryzae
fumR2 Expression
of
Spmae1, Ckfum and
thermophila
Mtsfc; Deletion of
lP
M.
25.0
0.78
0.26
[15]
21.5
0.65
\
[49]
17.0
0.24
\
[50]
28.2
0.39
0.45
[17]
1.7
\
\
[14]
re
R.
[15]
-p
pyc
Fed-
Glucose
batch
SG515
Mtfrd1,
Mtfrd2,
na
Mtfum and moc
Expression of ppc; Deletion E.
coil
of
iclR,
ur
fumC, fumA, fumB,
Fed-
Glucose
CWF812
batch
arcA, ptsG, aspA and
Jo
lacI
E.
coil
Expression of glk,
ABCDIA-
galP, ppc and acs;
ptsG--
Deletion of fumA,
15
mdh--glk-
fumB,
glucose
galp-ppc-
frdABCD, iclR, arcA,
acs
mdh and ptsG
fumC,
g/L
Shake flask
51
Expression of ppc; E.
Fed-
coil Deletion of fumB,
Glycerol
EF02
41.5
0.88
0.51
[22]
3.2
0.05
0.03
[61]
5.6
0.11
0.06
[62]
8.8
0.15
batch fumAC and aspA
S. cerevisiae
Expression
of
FMME-001
Romdh, Rofum1 and
Shake ↑PYC2 + ↑
Glucose flask
pyc2
RoMDH
cerevisiae
Romdh and Rofum1;
Shake Glucose
FMME004-
Deletion of thi2 and
6
fum1
T. glabrata-
Expression of ASL,
ASL(H)-
ADSL and Spmae1;
flask
Shake Glucose
ADSL(L)-
Deletion of ura3 and
SpMAE1
arg8
S.
stipitis
flask
of
Ymae1; Deletion of
Shake Xylose
PSYPMFfS
ura3, leu2, Psfum1
re
Expression
ro of
Expression of Ropyc,
0.12
[24]
-p
S.
4.7
0.10
\
[16]
15.8
0.15
0.21
[63]
21.6
0.22
0.30
[64]
33.1
0.33
\
[31]
flask
lP
and Psfum2 Expression of kgd2, C. glabrata-
SUCLG2,
sdh1,
KS(H)-S(M)–
Spmae1, sfc1 and
Shake
Glucose
na
flask
A-2 S
ASL; Deletion of ura3 and arg8
of
ur
Expression
ADB1-RoPYC-
AsPCK-SpMAE1
T.G‐ 4G‐
and ADB2-RoMDH-
S(1:1:2)‐
ScFDH1-ADB3-
P(M)‐ F(H)
RoFUM; Deletion of
Jo
C. glabrata
ura3
Glucose
and
Batch
arg8;
Scaffold (1:1:2) S.
Expression
of
Shake Glucose
cerevisiae
RoMDH-SDH1,
flask 52
TGFA091-
RoPYC-KGD2-
16
SUCLG2 and SFC1SpMAE1,；Deletion of thi2, fum1, ura3, leu2, trp1 and his3; RNA switches sRNA-RHR2
of and
sRNA-PDC6; DNA
Jo
ur
na
lP
re
-p
ro of
Scaffold (1:2)
53