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Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH
Author’s Accepted Manuscript Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH Zhiyong Cui, Cuijuan Gao, Jiaojiao Li, Jin Hou, Carol Sze Ki Lin, Qingsheng Qi www.elsevier.com/locate/ymben
PII: DOI: Reference:
S1096-7176(17)30099-X http://dx.doi.org/10.1016/j.ymben.2017.06.007 YMBEN1260
To appear in: Metabolic Engineering Received date: 5 April 2017 Revised date: 27 May 2017 Accepted date: 12 June 2017 Cite this article as: Zhiyong Cui, Cuijuan Gao, Jiaojiao Li, Jin Hou, Carol Sze Ki Lin and Qingsheng Qi, Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2017.06.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. 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.
Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH Zhiyong Cui a, Cuijuan Gao a, b, Jiaojiao Li a, Jin Hou a, Carol Sze Ki Lin c, Qingsheng Qi a* a
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, China b
c
School of Life Science, Linyi University, Linyi, 276000, China
School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
*
Corresponding author. Tel.: +86 531 88365628. [email protected]
Abstract Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.
Keywords
Yarrowia lipolytica, Succinic acid production, Acetic acid overflow, Acetate: succinate
CoA-transferase
1. Introduction Along with the petrochemical resource consumption and environmental pollution, microbial synthesis of value-added industrial products has received more and more attention as a sustainable process (Liang and Qi, 2014). Among all the platform chemicals, succinic acid has wide range of applications in many fields like food, chemical and agricultural, and it can be used as a precursor material to synthesize γ-butyrolactone, 1, 4-butanedioic acid, tetrahydrofuran and other value-added chemical products (Mckinlay et al., 2007; Werpy, 2004). In microbial cells, succinic acid is one of the intermediate metabolites of tricarboxylic acid (TCA) cycle, and it can be fermentatively produced from biomass resources (Vuoristo et al., 2015; Yin et al., 2015). Current succinate-production bacteria primarily include Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens (Borges and Pereira, 2010; Lee et al., 2000), genetically engineered Escherichia coli and Corynebacterium glutamicum (Li et al., 2013; Wang et al., 2016; Zhang et al., 2016). Although succinate production from these bacteria could achieve high yields and titers, the fermentation process normally require anaerobic condition with addition of alkali to maintain neutral pH condition. The addition of alkali increases the probability of bacterial contamination during the cultivation and it leads to more complex downstream industrial processing (DSP) steps (Beauprez et al., 2010; Jansen and Gulik, 2014). In contrast to prokaryotes, yeast is highly tolerant of low pH condition, makes it an attractive industrial host. As an eukaryotic model organism, Saccharomyces cerevisiae can tolerate a variety of environmental stresses, including low pH condition, which makes it
possesses a promising potential in industrial application (Raab and Lang, 2011; Rezaei et al., 2015). However, until now only a relative low succinic acid titer and yield were achieved in engineered S. cerevisiae (Raab et al., 2010; Yan et al., 2014). While Yarrowia lipolytica is a unconventional and strictly aerobic yeast, it relays on the complete TCA cycle and electron transport chain to sustain growth (Liu et al., 2015; Nicaud, 2012). Y. lipolytica can accumulate a large number of organic acids, such as citric acid, isocitric acid and α-ketoglutaric acid and it was widely studied due to its intrinsic properties (Gonçalves et al., 2014; Rywin´Ska et al., 2013; Zhu and Jackson, 2015). The whole genome sequence of Y. lipolytica model strain Po1f has been released (Liu and Alper, 2014). Genetic tools including CRISPR-Cas9 mediated gene editing (Schwartz et al., 2016), DNA assembler (Gao et al., 2014) and Golden Gate Assmbly system (Celińska et al., 2017) also were developed in Y. lipolytica by different research groups. Nowadays, we can carry out a variety of rational metabolic rewriting for production of high value-added chemicals other than traditional products such as lipid, citric acid and polyol in this strain (Gao et al., 2017; Li et al., 2016). Engineered Y. lipolytica had been reported to accumulate succinic acid by deleting the encoding gene or replacing the promoter of the succinate dehydrogenase (Jost et al., 2015; Yuzbashev et al., 2010). Through directional evolution, Yuzbashev et al. (2016) constructed a recombinant Y. lipolytica that can produce 40.5 g/L succinic acid with a yield of 0.36 g/g. In the meantime, we also engineered a succinate-production strain PGC01003 by knocking out the SDH5 subunit of succinate dehydrogenase in wild type strain Po1f. Although this strain has achieved very high succinate titer after long time fermentation at neutral pH from glycerol and it demonstrated the robustness of the species, there are still many problems that need to be solved (Gao
et al., 2016). The PGC01003 produced much acetate during the fermentation process, which affected the cell growth and succinate production yield. In addition, the high succinate titer was achieved by adjusting the pH during the fermentation. When the strain was cultivated at low pH, the resultant succinic acid titer (5.2 g/L) was relatively low. In this study, to achieve efficient production of succinic acid at low pH, the key factors such as Ylpdc and Ylach that would lead to acetate formation in PGC01003 were first identified. Then the strain was metabolically engineered by deleting the key gene that caused acetic acid overflow and overexpressing the genes that can improve the formation of succinic acid through reductive carboxylation and oxidative TCA pathway (Figure 1).
Figure 1 Pathways related to succinate production in Y. lipolytica. SDH, succinate dehydrogenase; ACH, acetyl-CoA hydrolase; PDC, pyruvate decarboxylase; ACS, acetyl-CoA
synthase; PYC, pyruvate carboxylase; PCK, phosphoenolpyruvate carboxykinase; CIT, citrate synthase; ACO, aconitase; KGDH, α-ketoglutarate dehydrogenase; SCS, succinyl-CoA synthase; ICL, isocitrate lyase; MLS, malate synthase; ACL, ATP citrate lyase.
2. Materials and methods 2.1. Strains, media, and growth conditions DH5α was grown in Luria-Bertani broth (LB) containing ampicillin (50 mg/L) or kanamycin (10 mg/L) for routine subcloning and plasmid propagation. Y. lipolytica PGC01003, a succinate production engineered strain derived from Po1f (ATCC MYA-2613), was constructed previously (Gao et al., 2016). PGC01003 was used for further metabolic engineering and its derived strains were presented in Table S1.
YNBG-ura, YNBG-casamino acids and YNBG-hyg medium containing 6.7 g/L yeast nitrogen base (with ammonium sulfate, Solarbo and without amino acids), 20 g/L glycerol and 2 g/L casamino acids, 0.5 g/L urea or hygromycin, respectively, were used for transformants screening. The yeast strains were cultivated in YPG medium containing 20 g/L glycerol, 10 g/L yeast extract and 20 g/L tryptone. The cultivation medium for shake flasks and bioreactor fermentation is comprised of 10 g/L yeast extract, 20 g/L tryptone and 20-100 g/L of glycerol as indicated in the text. All media were sterilized at 121 °C for 20 min, and then were inoculated under sterile conditions. 2.2. Plasmids construction and strain engineering
The primers used in this study are listed in Table S2. Yeast genomes were extracted by
TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Transformation of Y. lipolytica was performed using the the Lithium Acetate Method (Chen et al., 1997), and the transformants were selected in YNBG, YNBG-ura, YNBG-casamino acids or YNBG-hyg plates. The Y. lipolytica Ylpdc and Ylach genes were deleted through homologous recombination using LEU2 as the selection marker. The deletion cassettes comprise 1000 bp upper homologous arm, LEU2 marker and 1000 bp down homologous arm were ligated with pBluescript SK (-) vector by Gibson Assembly Cloning Kit (New England Biolabs (NEB), England) (Gibson et al., 2009). The resulting plasmids pPUT-PDC and pPUT-ACH were used for amplification of deletion cassettes. The PCR products were transformed into strain PGC01003 for deletion of Ylpdc and Ylach, respectively.
The LEU2 and the previously used URA3 auxotrophic markers for gene deletion were flanked with LoxP sites to allow the retrieval of the markers. The plasmid pUB4-Cre carried cre gene encoding for Cre recombinase which mediates recombination between two Lox sites was transformed into PGC11505 and selected on YNBG-hyg plates (Guo et al., 2014). The strain named PGC52 without LEU2 and URA3 markers were screened and selected. The integration expression plasmid pINA1312 or pINA1269 were kindly provided by Professor Catherine Madzak (Institut National de la Recherche Agronomique, AgroParisTech, France) was used for overexpressing SeACSL641P and YlACS in PGC01003 and YlACH, ScPCK, YlPCK, ScPYC, YlPYC, YlCIT, YlACO, YlICL and YlMLS in PGC52 (Madzak et al., 2004). The codon optimized SeacsL641P (Starai et al., 2005) was synthesized (Genscript, Nanjing, China), and the target fragment was amplified using primers oACS-F and oACS-R. The others desired genes were PCR amplified
from the genomic DNA of Y. lipolytica W29 or Saccharomyces cerevisiae EBY100 with corresponding primers (Table 2). The DNA fragments flanked with appropriate homologous arm were assembled with the BamHI and KpnI digested pINA1312 or pINA1269, respectively. The resulting plasmids were then linearized by NotI, and introduced into Y. lipolytica PGC01003 or PGC52. The transformations were selected on YNBG-casamino acids plates. For co-overexpression of Ylicl and Ylmls or Ylkgdh and Ylscs2 (encoding gene for succinyl-CoA synthetase beta subunit), Ylicl-Ylmls and Ylkgdh-Ylscs2 were fused by a widely used GGGS linker (Chen, 2015) and ligated into the plasmid pINA1269. The resulting plasmids were linearized and transformed into PGC62. 2.3 YlACH activity assay The Y. lipolytica strains were cultured overnight to OD600 ≈ 1. The 50 mL culture broth were harvested and suspended in 800 μL extracting buffer (50 mM Tris-HCl, 20% glycerol pH 8.0). The cells were broken using 0.4 – 0.6 mm glass beads (Sigma-Aldrich, St. Louis, MO) for 10 min prior to centrifugation. The supernatant was obtained then used for further analysis. CoA-transferase activities were determined according to previously reported study (Fleck and Brock, 2009). In general, the release of CoASH was detected with 5, 5′-dithiobis- (2-nitrobenzoic acid) at 412 nm, taking a millimolar extinction coefficient of 13. 6 mM-1 cm-1 (Srere, 1963). Succinyl-CoA is significantly instable under alkaline pH condition. Therefore, 50 mM succinate were used as substrates for determining the CoA-transferase or hydrolase activity at pH 8.0 and the control was the one without any succinate. 2.4. Shake flask fermentation
The fermentation were carried out in 250 mL shake flasks with 50 mL YPG medium, and
cultivated at 28 °C and 220 rpm. Samples were taken every 12 hours for measuring optical density, residual glycerol and organic acids. In acetate overflow analysis, the initial glycerol concentration was 20 g/L, the concentrations of succinic acid and acetate were quantified after 48 h when glycerol was completely consumed. For succinic acid production, the initial concentration of glycerol was 40 g/L and 1-2 mL glycerol stock (500 g/L) was fed at 24 h and 72 h. All the fermentation conducted in shake flask had not added any alkali to maintain neutral pH condition.
2.5. Fed-batch fermentation in a bioreactor
The strains were first inoculated into 5 mL 2% YPG medium and incubated at 28 °C and 220 rpm for 24 h. Culture (1 mL) was inoculated into 50 mL 2% YPG medium in 250 mL shake flasks as seed culture at 28 °C and 220 rpm. Seed culture (50 mL) was then inoculated into 1.0 L fermentation medium to start fed-batch fermentation.
Fed-batch fermentation without any pH control was carried out in 2.5 L fermenter (B. Braun Melsungen AG, Melsungen, Germany), at 28°C, 600 rpm and 2 vvm. The YPG with 30-100 g/L glycerol was used as the initial batch medium, and 30-100 mL glycerol was fed from 750 g/L stock when the glycerol concentration dropped below 10 g/L.
2.6. Substrate and product analysis
Succinic acid, glycerol and by-products concentrations were determined by HPLC equipped with an Aminex HPX-87H column (Bio-Rad, Inc., Hercules, CA) and a refractive index detector. The pH changes were monitored by on-line pH analyzer of fermenter. All samples were passed through 0.22 μm filters before loading. The analysis was performed using 5 mM H2SO4 as mobile phase at 0.6 mL/min, and the column temperature was 65 °C.
3. Results
3.1 Identification of the key factor involved in acetic acid overflow in SDH negative Y. lipolytica
Previously, we constructed a succinate-production strain PGC01003 by inactivating the succinate dehydrogenase. Although this strain produced 160 g/L succinate from glycerol after 400 hours fed-batch cultivation, the yield and productivity were relatively low (Gao et al., 2016). Moreover, the pH needed to be maintained at neutral condition by adding alkaline reagent throughout the fermentation process, which would increase the cost of downstream processing. Compared with the wild type Y. lipolytica, PGC01003 accumulated high level of acetate. As a main by-product formed in both aerobic and anaerobic condition, acetate is responsible for the regulation of central metabolism and multiple physiological features (Wolfe et al., 2005). Excessive amounts of acetic acid not only inhibited the cell growth but also led to loss of carbon source. Therefore, further work needs to be done to eliminate acetate formation in PGC01003. However, it was still unclear which pathway is responsible for acetic acid formation.
Figure 2 Strategies to diminish acetic acid accumulation in SDH negative Y. lipolytica strain PGC01003. (A) Effect of the different genetic modifications on PGC01003 strain fermentation
profiles including succinic acid and acetic acid production. (B) The cell growth of PGC01003 can be partly recovered by deletion of the Ylach. The initial concentration of glycerol was 20 g/L. After 48 h of culture, the OD600, succinic acid and acetic acid concentrations were determined. In general, acetic acid is generated through pyruvate decarboxylase (PDC) bypass. Additional NADH can be obtained from NAD+ through PDC-bypass, which does benefit to cell growth, for interrupted TCA cycle. However, we found that inactivation of Ylpdc which encodes pyruvate decarboxylase (PDC) and catalyzes the first reaction of PDC-bypass, did not reduce the acetic acid formation as expected (Figure 2a). Reutilization of acetic acid is another way that maybe affect the acetic acid accumulation in the medium. Therefore, by overexpressing the acetyl-CoA synthetase (ACS), which may consume the acetic acid accumulated in the medium. Two sources of ACS, one endogenous derived from Y. lipolytica and the other from Salmonella enteric SeACSL641P (Starai et al., 2005), were expressed in Y. lipolytica. As summarized in Figure 2a, overexpression of YlACS did not result in an increase of succinic acid production and the acetic acid accumulation barely changed. Strain PGC11401 with expression of SeACSL641P yielded as high as 6.3 g/L succinic acid, while acetic acid accumulation decreased to 4.7 g/L.
By digging out metabolic pathways and genes from literatures, gene ach1 attracted our attention. The encoding product of ach1 from Saccharomyces cerevisiae originally named acetyl-CoA hydrolase, but recent study showed that it also has a CoA-transferase enzyme activity and maybe involve in exchanging acetyl unit between mitochondria and cytosol (Yun, 2015). To evaluate the role of YlACH in Y. lipolytica, the Ylach gene was knocked-out in PGC01003, generating strain PGC11505. Surprisingly, the acetic acid accumulation significantly reduced from 7.5 g/L to 0.2 g/L (Figure 2a). Moreover, the succinic acid titer increased to 7.0 g/L in shake flasks (increased by
27.3%), and the cell growth also restored comparing with the parent strain PGC01003 (Figure 2b). To further confirm the restored cell growth was caused by acetic acid reduction, we directly added different concentration of acetic acid to the medium and found that cell growth and succinic acid production of PGC11505 were again impaired (Figure S1). Obviously, acetic acid accumulation is harmful to cell growth and succinic acid production, deletion of Ylach in SDH negative Y. lipolytica can relieve this adverse impact. These experiments demonstrated that high level of acetic acid accumulation in SDH negative Y. lipolytica mainly generated from acetyl-CoA, but not pyruvate as it usually observed in other species (König, 1998).
Table 1 Activity measurement of PG52 and PGC52 YlACH.
Hydrolase (U/mg)
CoA transferase (U/mg)
PGC52
n.d.
n.d.
PGC52 YlACH
0.03±0.01
1.89±0.13
The n.d. stand for not detected. One unit is defined as the amount of enzyme producing 1 μmol of succinyl-CoA per minute (succinyl-CoA is not stable in the condition of pH 8.0).
To confirm the function of YlACH in Y. lipolytica，the Ylach gene was expressed under the control of strong promoter hp4d in PGC52 (the strain derived from PGC11505, and the URA3 and LEU2 selection markers have been recycled) and the enzyme activity was analyzed. As shown in Table 1, YlACH showed high acetate: succinate CoA-transferase activity rather than hydrolase activity. The inactivation of the acetyl-CoA transferase blocked the formation of acetic acid from acetyl-CoA, which would subsequently reduce the reverse reaction of succinic acid to succinyl-CoA. The reduction of acetic acid concentration also restored the cell growth, therefore, with an improved
succinic acid production.
3.2 Regulation of by-products formation through reductive carboxylation
Deletion of Ylach improved succinic acid production and restored the cell growth. However, we found that there was a dramatic increase in pyruvate formation at the end of fermentation (from 0.4 g/L to 6.0 g/L, data not shown). In addition, erythritol and mannitol accumulated as well. The formation of the by-products, including pyruvate and its upstream metabolites, indicated that there was a metabolic flux repression from acetyl-CoA, which is a direct downstream metabolite of pyruvate.
In order to channel pyruvate to succinic acid production, a series of gene overexpression were investigated, including the genes encoding the phosphoenolpyruvate carboxykinase from S. cerevisiae (ScPCK, named PGC62) and Y. lipolytica (YlPCK, named PGC73), pyruvate carboxylase from S. cerevisiae (ScPYC, named PGC82) and Y. lipolytica (YlPYC, named PGC91), Y. lipolytica endogenous citrate synthase (YlCIT, named PGC104), aconitase (YlACO, named PGC112), isocitrate lyase (YlICL, named PGC125) and malate synthase (YlMLS, named PGC131) (Table 2).
Table 2 Fermentation profiles of Y. lipolytica PGC52 and its derivative strains in shake flasks Engineered
Succinic
Pyruvate
Erythritol
Mannitol
Malate
Succinic acid
strain
acid
(g/L)
(g/L)
(g/L)
(g/L)
yield
(g/L)
(g/g glycerol)
PGC52
12.05±0.22
5.47±1.98
5.88±1.58
2.0±0.15
n.d.
0.29
PGC62
30.15±0.73
n.d.
n.d.
2.22±0.65
2.3±0.28
0.5
PGC73
13.99±0.16
n.d.
n.d.
3.19±0.02
0.39±0.14
0.39
PGC82
15.72±0.11
n.d.
5.05±1.41
3.72±0.27
2.54±0.34
0.36
PGC91
26.16±1.41
n.d.
4.47±0.7
3.82±0.11
5.89±0.2
0.41
PGC104
17.03±0.44
5.68±0.56
4.3±0.22
1.55±0.27
n.d.
0.34
PGC112
15.1±0.16
4.14±0.3
6.32±0.61
2.56±0.46
n.d.
0.29
PGC125
18.51±0.9
8.48±1.05
4.95±1.28
4.04±0.07
n.d.
0.27
PGC131
22.54±0.08
5.78±0.85
5.26±0.79
4.53±0.63
n.d.
0.36
The n.d. stand for not detected. The initial concentration of glycerol was 40 g/L. After 96 h of culture, the succinic acid and by-products concentration were determined by HPLC.
These recombinant strains were evaluated and all of these genes overexpression enhanced succinic acid production. These cultivations all shown highly acidic pH (a value range of 2.5 – 3.5) at the end of fermentation. Among them, overexpression of YlCIT, YlACO, YlICL and YlMLS, increased succinic acid titer by 41.3%, 25.3%, 53.6% and 87.1%, respectively. Nevertheless, the pyruvate production was not significantly reduced. Overexpression of PYC or PCK, both reductive carboxylation enzymes from S. cerevisiae and Y. lipolytica, eliminated the pyruvate accumulation completely. Among these four engineered strains, overexpression of ScPCK and YlPYC enhanced succinic acid titer significantly, PGC62 with ScPCK overexpression showed a maximum succinic acid titer (30.2 g/L) and yield (0.5 g/g), which enhanced by 150.2% and 72.4% compared with control, respectively. The PGC91 with YlPYC overexpression also increased succinic acid titer and yield by 115.7% and 41.4%, respectively. Interestingly, both overexpression ScPCK and YlPYC resulted in a considerable malate accumulation, confirming that excess oxaloacetate generated from phosphoenolpyruvate or pyruvate carboxylation. These results revealed that enhanced pyruvate and phosphoenolpyruvate carboxylation can overcome the problem of by-products accumulation caused
by Ylach deletion.
3.3 Simultaneous overexpression of reductive carboxylation and oxidative TCA pathway
As shown above, overexpression ScPCK in PGC52 presented the highest improvement on succinic acid production. The resultant succinic acid concentration was 1.5-fold higher than that obtained from PGC52. To further increase the metabolic efficiency, the key genes involved in glyoxylate bypass and oxidative TCA were investigated.
Figure 3 Combinatorial effect of different genetic modifications in Y. lipolytica on succinic acid titer. The initial concentration of glycerol was 40 g/L. After 96 h of culture, the succinic acid concentration of engineered strains were determined.
Three key enzymes, YlACL, YlICL and YlMLS, involved with glyoxylate bypass, were first
attempted in a ScPCK overexpression strain. Unexpectedly, the enhancement of the metabolic flow of glyoxylate bypass had no obvious improvement to succinic acid production (Figure 3). Chen (2016) demonstrated that overexpression of α-ketoglutarate dehydrogenase and succinyl-CoA synthase beta subunit was beneficial to fumarate production. In this study, it was found that overexpression of YlSCS2 can further improve the succinic acid production to 37.0 g/L, which was 24% higher than the strain expressing with only ScPCK. On the contrary, succinic acid production was reduced by 19% in YlKGDH overexpressing strain PGC194. In addition, the YlCIT overexpressing strain PGC143 produced 31.4 g/L succinic acid, which was similar to its parent strain.
3.4 Fed-batch fermentation of succinic acid at low pH value
Figure 4 Fed-batch fermentation profile of engineered Y. lipolytica strains PGC01003 (A), PGC11505 (B) and PGC202 (C). Open circle stands for OD600; open square stands for succinic acid concentration; open triangle stands for glycerol concentration; open diamand stands for acetic acid concentration; dashed line stands for the pH of culture medium.
Comparing with the strain only with SDH inactivation, PGC202 has largely improved succinic acid production and reduced acetic acid accumulation. It showed excellent characteristics for
industrial application. To evaluate its capability, fed-batch fermentation was then performed without pH control. Three engineered strains, PGC01003, PGC11505 and PGC202 were compared. As shown in Figure 4, pH of all the three strains were kept at a value around of 3.0 – 4.3 at the end of fermentation. Original strain PGC01003 presented poor capacity for succinic acid synthesis without adding alkaline. The succinic acid titer only reached to 5.4 g/L with 10.6 g/L acetic acid accumulation during fermentation. Meanwhile, both PGC11505 and PGC202 accumulated a small amount of acetic acid (around 0.5 – 1.4 g/L), and they grew well in the acidic environment. These results demonstrated that elimination of acetic acid is critical to cell growth and succinic acid production at low pH. The succinic acid titer of PGC11505 was 45.6 g/L, with a yield of 0.2 g/g glycerol. The highest succinic acid production of 67.5 g/L was obtained by PGC202 using defined medium containing 181.3 g/L of glycerol, with a yield of 0.37 g/g glycerol, and productivity of 0.49 g/L/h.
Figure 5 Fermentation performance of Y. lipolytica PGC202 in fed-batch fermentation with different initial glycerol concentration. (A) Comparison of SA production and by-product accumulation by PGC202 in batch fermentation at various initial concentration of glycerol. (B) The fermentation results of the PGC202 strain with the 50 g/L initial glycerol. Open circle stands for OD600; open square stands for succinic acid concentration; open triangle stands for glycerol
concentration; dashed line stands for the pH of culture medium; open diamand stands for mannitol concentration.
Through analysis of fermentation products, large number of by-products, such as mannitol and arabitol were accumulated by PGC202. However, these kinds of by-products were not detected in shake flasks fermentation. Mannitol, similar to erythritol and arabitol, is a storage for carbon source as well as reducing force, which can be enhanced by using fermentation medium with high glycerol concentration (Juszczyk et al., 2013; Tomaszewska et al., 2012). These studies reported that the formation of by-products would lead to reduction of succinic acid yield, were mainly caused by highly initial glycerol concentration. Therefore, the initial glycerol concentration was set from 30 to 70 g/L to reduce by-products formation. As shown in Figure 5, mannitol titers were positively correlated with the initial glycerol concentration. Initial glycerol of 70 g/L led to high resultant succinic acid concentration (93.3 g/L) with high productivity (0.7 g/L/h), while about 19.9 g/L mannitol was formed. Although PGC202 accumulated only 11.0 g/L mannitol when the initial glycerol concentration was 30 g/L, the highest succinic acid titer of 110.7 g/L was achieved in 138 h with a final pH of 3.4 and a yield of 0.53 g/g glycerol, which is equivalent to 81.8% of theoretical yield when medium containing 50 g/L initial glycerol was used. This is the highest fermentative succinic acid titer and yield achieved by Y. lipolytica at natural low pH reported to date, using defined medium.
4. Discussion
Because of low processing cost and environmental friendliness, microbial synthesis of succinic acid from cheap substrates has been drawed increasing attentions in both academic research and
industrial manufacture (Cao et al., 2013). At present, bacteria have an obvious advantage in succinic acid production. Except for yield and productivity, the downstream separation and purification steps are also a determined factor affecting the cost of succinic acid production. The succinate obtained from the bacterial fermentation need to be converted to free acid form (rather than the salt form), which cost about 60% to 70% of the total production cost (Cok et al., 2013; Zeikus et al., 1999). Yeast can carry out low pH fermentation, directly convert the substrate into succinic acid, which largely reduce the cost of downstream processing. Compare to bacteria, low-pH yeast would be more suitable as a succinic acid production host if the succinic acid production efficiency of yeast could improve further. However, the succinic acid titer and yield of S. cerevisiae still could not meet the need of industrial application today after many years of research efforts. As a unconventional yeast, Y. lipolytica can utilise many unspecific substrates such as alkanes, ethanol and glycerol (Ledesma-Amaro and Nicaud, 2016). As the primary by-product from the production of biodiesel, glycerol is one of the low-cost feedstock. With the rise of biodiesel production scale, glycerol was regarded as the second top feedstock in succinic acid production (Tan et al., 2014). Several studies have demonstrated that Y. lipolytica as an efficient industrial production host in succinic acid production (Gao et al., 2016; Yuzbashev et al., 2016). Herein, Y. lipolytica central metabolic pathway was engineered to improve succinic acid production through elimination of acetic acid overflow and by-products formation, succinic acid production of strain PGC202 reached 110.7 g/L with a maximum yield of 0.53 g/g of glycerol and a productivity of 0.8 g/L/h in fed-batch fermentation.
The main by-product of the parent strain PGC01003 is acetic acid, and the acetic acid overflow should be due to the imbalance of metabolic flux between glycolysis and TCA cycle. Excessive acetic acid generated during the fermentation process not only inhibited the cell growth, but it also
affected the global metabolism of cells (Bernal et al., 2016), and ultimately reduced the production of succinic acid. However, interference of pyruvate decarboxylation bypass, which is the primary acetic acid formation pathway, would not lead to reduction of acetic acid accumulation in SDH negative Y. lipolytica. As an alternative option, YlACH was inactivated to evaluate the role of YlACH in acetic acid overflow. Surprisingly, the acetic acid accumulation was significantly reduced, and succinic acid production was improved at the same time. YlACH exhibited high amino acid sequence identity to the acetyl-CoA hydrolase (ScACH) from S. cerevisiae, and acetate: succinate CoA-transferase (TvASCT) from Trichomonas vaginalis. In view of acetyl-CoA hydrolysis is an energy consuming process, these two enzymes showed high activity for CoA transferase, and only a minor acetyl-CoA hydrolase activity (Fleck and Brock, 2009; van Grinsven et al., 2008). The results of enzymatic activity determination also demonstrated that YlACH has high specific activity for the acetate: succinate CoA-transferase other than acetyl-CoA hydrolase. From the information above, the absence of succinate dehydrogenase in PGC01003 may cause TCA cycle obstruction, and this resulted excessive acetyl-CoA transfer into acetic acid by CoA transferase.
Although the deletion of Ylach eliminated acetic acid overflow, the engineered strain PGC11505 produced some other by-products such as pyruvate, erythritol and mannitol. It indicated that the additional deletion of Ylach in the PGC01003 caused the accumulation of acetyl-CoA, in which the feedback inhibited the pyruvate dehydrogenase and led to pyruvate and its upstream metabolites formation. Therefore, the distribution of metabolic flux was reconstructed to relieve the metabolic repression and by-products formation. As expected, the succinic acid production of all the engineered strains were improved, and the overexpression of reductive carboxylation achieved the higher succinic acid titers and yields. In Y. lipolytica, the reductive carboxylation represents the
committed step in many organic acids biosynthesis, the pyruvate carboxylase from different species were commonly used (Wang et al., 2015; Yin et al., 2012). Results from this study indicated that phosphoenolpyruvate carboxykinase was more conducive to succinic acid production than pyruvate carboxylase. Actually, the reaction catalyzed by PCK is accompanied by ATP generation, which may be useful to SDH negative strain (Kim et al., 2004; Tan et al., 2013). The overexpression of reductive carboxylation also led to considerable malate accumulation, which enlightened us that reductive carboxylation plays an important role in the regulation of redox state, since this pathway consumes NADH.
5. Conclusions
This study provides a rational engineering strategy for the enhancement of succinic acid production in Y. lipolytica. Through a series of analysis and well-designed experiments, the CoA-transferase encoded by Ylach was identified being responsible for acetic acid overflow in Y. lipolytica mutant during fermentation. Deletion of Ylach significantly reduced acetic acid formation. By further strengthening the reductive carboxylation and oxidative TCA, an engineered strain PGC202 was obtained, which can produce 110.7 g/L succinic acid in fed-batch cultivation using glycerol without pH control. This is the highest fermentative succinic acid titer achieved in yeast at low pH condition.
Results from this study successfully demonstrated that an innovative feedstock
valorization solution using an engineered Y. lipolytica strain based on renewable and low-cost feedstocks, which could contribute to the future bio-based economy.
Acknowledgments
This work was supported by the key research and development plan of Shandong Province 2016ZDJS07A19 and 2016GSF121019.
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Highlights 1. The first time revealed that acetic acid overflow is caused by acetate: succinate CoA-transferase
in SDH negative Y. lipolytica. 2. Succinic acid titer achieved in this study is the highest level producted at low pH by yeast. 3. Yarrowia lipolytica could be developed as an efficient succinic acid production strain with great industrial prospects.
PII: DOI: Reference:
S1096-7176(17)30099-X http://dx.doi.org/10.1016/j.ymben.2017.06.007 YMBEN1260
To appear in: Metabolic Engineering Received date: 5 April 2017 Revised date: 27 May 2017 Accepted date: 12 June 2017 Cite this article as: Zhiyong Cui, Cuijuan Gao, Jiaojiao Li, Jin Hou, Carol Sze Ki Lin and Qingsheng Qi, Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2017.06.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. 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.
Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH Zhiyong Cui a, Cuijuan Gao a, b, Jiaojiao Li a, Jin Hou a, Carol Sze Ki Lin c, Qingsheng Qi a* a
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, China b
c
School of Life Science, Linyi University, Linyi, 276000, China
School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
*
Corresponding author. Tel.: +86 531 88365628. [email protected]
Abstract Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.
Keywords
Yarrowia lipolytica, Succinic acid production, Acetic acid overflow, Acetate: succinate
CoA-transferase
1. Introduction Along with the petrochemical resource consumption and environmental pollution, microbial synthesis of value-added industrial products has received more and more attention as a sustainable process (Liang and Qi, 2014). Among all the platform chemicals, succinic acid has wide range of applications in many fields like food, chemical and agricultural, and it can be used as a precursor material to synthesize γ-butyrolactone, 1, 4-butanedioic acid, tetrahydrofuran and other value-added chemical products (Mckinlay et al., 2007; Werpy, 2004). In microbial cells, succinic acid is one of the intermediate metabolites of tricarboxylic acid (TCA) cycle, and it can be fermentatively produced from biomass resources (Vuoristo et al., 2015; Yin et al., 2015). Current succinate-production bacteria primarily include Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens (Borges and Pereira, 2010; Lee et al., 2000), genetically engineered Escherichia coli and Corynebacterium glutamicum (Li et al., 2013; Wang et al., 2016; Zhang et al., 2016). Although succinate production from these bacteria could achieve high yields and titers, the fermentation process normally require anaerobic condition with addition of alkali to maintain neutral pH condition. The addition of alkali increases the probability of bacterial contamination during the cultivation and it leads to more complex downstream industrial processing (DSP) steps (Beauprez et al., 2010; Jansen and Gulik, 2014). In contrast to prokaryotes, yeast is highly tolerant of low pH condition, makes it an attractive industrial host. As an eukaryotic model organism, Saccharomyces cerevisiae can tolerate a variety of environmental stresses, including low pH condition, which makes it
possesses a promising potential in industrial application (Raab and Lang, 2011; Rezaei et al., 2015). However, until now only a relative low succinic acid titer and yield were achieved in engineered S. cerevisiae (Raab et al., 2010; Yan et al., 2014). While Yarrowia lipolytica is a unconventional and strictly aerobic yeast, it relays on the complete TCA cycle and electron transport chain to sustain growth (Liu et al., 2015; Nicaud, 2012). Y. lipolytica can accumulate a large number of organic acids, such as citric acid, isocitric acid and α-ketoglutaric acid and it was widely studied due to its intrinsic properties (Gonçalves et al., 2014; Rywin´Ska et al., 2013; Zhu and Jackson, 2015). The whole genome sequence of Y. lipolytica model strain Po1f has been released (Liu and Alper, 2014). Genetic tools including CRISPR-Cas9 mediated gene editing (Schwartz et al., 2016), DNA assembler (Gao et al., 2014) and Golden Gate Assmbly system (Celińska et al., 2017) also were developed in Y. lipolytica by different research groups. Nowadays, we can carry out a variety of rational metabolic rewriting for production of high value-added chemicals other than traditional products such as lipid, citric acid and polyol in this strain (Gao et al., 2017; Li et al., 2016). Engineered Y. lipolytica had been reported to accumulate succinic acid by deleting the encoding gene or replacing the promoter of the succinate dehydrogenase (Jost et al., 2015; Yuzbashev et al., 2010). Through directional evolution, Yuzbashev et al. (2016) constructed a recombinant Y. lipolytica that can produce 40.5 g/L succinic acid with a yield of 0.36 g/g. In the meantime, we also engineered a succinate-production strain PGC01003 by knocking out the SDH5 subunit of succinate dehydrogenase in wild type strain Po1f. Although this strain has achieved very high succinate titer after long time fermentation at neutral pH from glycerol and it demonstrated the robustness of the species, there are still many problems that need to be solved (Gao
et al., 2016). The PGC01003 produced much acetate during the fermentation process, which affected the cell growth and succinate production yield. In addition, the high succinate titer was achieved by adjusting the pH during the fermentation. When the strain was cultivated at low pH, the resultant succinic acid titer (5.2 g/L) was relatively low. In this study, to achieve efficient production of succinic acid at low pH, the key factors such as Ylpdc and Ylach that would lead to acetate formation in PGC01003 were first identified. Then the strain was metabolically engineered by deleting the key gene that caused acetic acid overflow and overexpressing the genes that can improve the formation of succinic acid through reductive carboxylation and oxidative TCA pathway (Figure 1).
Figure 1 Pathways related to succinate production in Y. lipolytica. SDH, succinate dehydrogenase; ACH, acetyl-CoA hydrolase; PDC, pyruvate decarboxylase; ACS, acetyl-CoA
synthase; PYC, pyruvate carboxylase; PCK, phosphoenolpyruvate carboxykinase; CIT, citrate synthase; ACO, aconitase; KGDH, α-ketoglutarate dehydrogenase; SCS, succinyl-CoA synthase; ICL, isocitrate lyase; MLS, malate synthase; ACL, ATP citrate lyase.
2. Materials and methods 2.1. Strains, media, and growth conditions DH5α was grown in Luria-Bertani broth (LB) containing ampicillin (50 mg/L) or kanamycin (10 mg/L) for routine subcloning and plasmid propagation. Y. lipolytica PGC01003, a succinate production engineered strain derived from Po1f (ATCC MYA-2613), was constructed previously (Gao et al., 2016). PGC01003 was used for further metabolic engineering and its derived strains were presented in Table S1.
YNBG-ura, YNBG-casamino acids and YNBG-hyg medium containing 6.7 g/L yeast nitrogen base (with ammonium sulfate, Solarbo and without amino acids), 20 g/L glycerol and 2 g/L casamino acids, 0.5 g/L urea or hygromycin, respectively, were used for transformants screening. The yeast strains were cultivated in YPG medium containing 20 g/L glycerol, 10 g/L yeast extract and 20 g/L tryptone. The cultivation medium for shake flasks and bioreactor fermentation is comprised of 10 g/L yeast extract, 20 g/L tryptone and 20-100 g/L of glycerol as indicated in the text. All media were sterilized at 121 °C for 20 min, and then were inoculated under sterile conditions. 2.2. Plasmids construction and strain engineering
The primers used in this study are listed in Table S2. Yeast genomes were extracted by
TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Transformation of Y. lipolytica was performed using the the Lithium Acetate Method (Chen et al., 1997), and the transformants were selected in YNBG, YNBG-ura, YNBG-casamino acids or YNBG-hyg plates. The Y. lipolytica Ylpdc and Ylach genes were deleted through homologous recombination using LEU2 as the selection marker. The deletion cassettes comprise 1000 bp upper homologous arm, LEU2 marker and 1000 bp down homologous arm were ligated with pBluescript SK (-) vector by Gibson Assembly Cloning Kit (New England Biolabs (NEB), England) (Gibson et al., 2009). The resulting plasmids pPUT-PDC and pPUT-ACH were used for amplification of deletion cassettes. The PCR products were transformed into strain PGC01003 for deletion of Ylpdc and Ylach, respectively.
The LEU2 and the previously used URA3 auxotrophic markers for gene deletion were flanked with LoxP sites to allow the retrieval of the markers. The plasmid pUB4-Cre carried cre gene encoding for Cre recombinase which mediates recombination between two Lox sites was transformed into PGC11505 and selected on YNBG-hyg plates (Guo et al., 2014). The strain named PGC52 without LEU2 and URA3 markers were screened and selected. The integration expression plasmid pINA1312 or pINA1269 were kindly provided by Professor Catherine Madzak (Institut National de la Recherche Agronomique, AgroParisTech, France) was used for overexpressing SeACSL641P and YlACS in PGC01003 and YlACH, ScPCK, YlPCK, ScPYC, YlPYC, YlCIT, YlACO, YlICL and YlMLS in PGC52 (Madzak et al., 2004). The codon optimized SeacsL641P (Starai et al., 2005) was synthesized (Genscript, Nanjing, China), and the target fragment was amplified using primers oACS-F and oACS-R. The others desired genes were PCR amplified
from the genomic DNA of Y. lipolytica W29 or Saccharomyces cerevisiae EBY100 with corresponding primers (Table 2). The DNA fragments flanked with appropriate homologous arm were assembled with the BamHI and KpnI digested pINA1312 or pINA1269, respectively. The resulting plasmids were then linearized by NotI, and introduced into Y. lipolytica PGC01003 or PGC52. The transformations were selected on YNBG-casamino acids plates. For co-overexpression of Ylicl and Ylmls or Ylkgdh and Ylscs2 (encoding gene for succinyl-CoA synthetase beta subunit), Ylicl-Ylmls and Ylkgdh-Ylscs2 were fused by a widely used GGGS linker (Chen, 2015) and ligated into the plasmid pINA1269. The resulting plasmids were linearized and transformed into PGC62. 2.3 YlACH activity assay The Y. lipolytica strains were cultured overnight to OD600 ≈ 1. The 50 mL culture broth were harvested and suspended in 800 μL extracting buffer (50 mM Tris-HCl, 20% glycerol pH 8.0). The cells were broken using 0.4 – 0.6 mm glass beads (Sigma-Aldrich, St. Louis, MO) for 10 min prior to centrifugation. The supernatant was obtained then used for further analysis. CoA-transferase activities were determined according to previously reported study (Fleck and Brock, 2009). In general, the release of CoASH was detected with 5, 5′-dithiobis- (2-nitrobenzoic acid) at 412 nm, taking a millimolar extinction coefficient of 13. 6 mM-1 cm-1 (Srere, 1963). Succinyl-CoA is significantly instable under alkaline pH condition. Therefore, 50 mM succinate were used as substrates for determining the CoA-transferase or hydrolase activity at pH 8.0 and the control was the one without any succinate. 2.4. Shake flask fermentation
The fermentation were carried out in 250 mL shake flasks with 50 mL YPG medium, and
cultivated at 28 °C and 220 rpm. Samples were taken every 12 hours for measuring optical density, residual glycerol and organic acids. In acetate overflow analysis, the initial glycerol concentration was 20 g/L, the concentrations of succinic acid and acetate were quantified after 48 h when glycerol was completely consumed. For succinic acid production, the initial concentration of glycerol was 40 g/L and 1-2 mL glycerol stock (500 g/L) was fed at 24 h and 72 h. All the fermentation conducted in shake flask had not added any alkali to maintain neutral pH condition.
2.5. Fed-batch fermentation in a bioreactor
The strains were first inoculated into 5 mL 2% YPG medium and incubated at 28 °C and 220 rpm for 24 h. Culture (1 mL) was inoculated into 50 mL 2% YPG medium in 250 mL shake flasks as seed culture at 28 °C and 220 rpm. Seed culture (50 mL) was then inoculated into 1.0 L fermentation medium to start fed-batch fermentation.
Fed-batch fermentation without any pH control was carried out in 2.5 L fermenter (B. Braun Melsungen AG, Melsungen, Germany), at 28°C, 600 rpm and 2 vvm. The YPG with 30-100 g/L glycerol was used as the initial batch medium, and 30-100 mL glycerol was fed from 750 g/L stock when the glycerol concentration dropped below 10 g/L.
2.6. Substrate and product analysis
Succinic acid, glycerol and by-products concentrations were determined by HPLC equipped with an Aminex HPX-87H column (Bio-Rad, Inc., Hercules, CA) and a refractive index detector. The pH changes were monitored by on-line pH analyzer of fermenter. All samples were passed through 0.22 μm filters before loading. The analysis was performed using 5 mM H2SO4 as mobile phase at 0.6 mL/min, and the column temperature was 65 °C.
3. Results
3.1 Identification of the key factor involved in acetic acid overflow in SDH negative Y. lipolytica
Previously, we constructed a succinate-production strain PGC01003 by inactivating the succinate dehydrogenase. Although this strain produced 160 g/L succinate from glycerol after 400 hours fed-batch cultivation, the yield and productivity were relatively low (Gao et al., 2016). Moreover, the pH needed to be maintained at neutral condition by adding alkaline reagent throughout the fermentation process, which would increase the cost of downstream processing. Compared with the wild type Y. lipolytica, PGC01003 accumulated high level of acetate. As a main by-product formed in both aerobic and anaerobic condition, acetate is responsible for the regulation of central metabolism and multiple physiological features (Wolfe et al., 2005). Excessive amounts of acetic acid not only inhibited the cell growth but also led to loss of carbon source. Therefore, further work needs to be done to eliminate acetate formation in PGC01003. However, it was still unclear which pathway is responsible for acetic acid formation.
Figure 2 Strategies to diminish acetic acid accumulation in SDH negative Y. lipolytica strain PGC01003. (A) Effect of the different genetic modifications on PGC01003 strain fermentation
profiles including succinic acid and acetic acid production. (B) The cell growth of PGC01003 can be partly recovered by deletion of the Ylach. The initial concentration of glycerol was 20 g/L. After 48 h of culture, the OD600, succinic acid and acetic acid concentrations were determined. In general, acetic acid is generated through pyruvate decarboxylase (PDC) bypass. Additional NADH can be obtained from NAD+ through PDC-bypass, which does benefit to cell growth, for interrupted TCA cycle. However, we found that inactivation of Ylpdc which encodes pyruvate decarboxylase (PDC) and catalyzes the first reaction of PDC-bypass, did not reduce the acetic acid formation as expected (Figure 2a). Reutilization of acetic acid is another way that maybe affect the acetic acid accumulation in the medium. Therefore, by overexpressing the acetyl-CoA synthetase (ACS), which may consume the acetic acid accumulated in the medium. Two sources of ACS, one endogenous derived from Y. lipolytica and the other from Salmonella enteric SeACSL641P (Starai et al., 2005), were expressed in Y. lipolytica. As summarized in Figure 2a, overexpression of YlACS did not result in an increase of succinic acid production and the acetic acid accumulation barely changed. Strain PGC11401 with expression of SeACSL641P yielded as high as 6.3 g/L succinic acid, while acetic acid accumulation decreased to 4.7 g/L.
By digging out metabolic pathways and genes from literatures, gene ach1 attracted our attention. The encoding product of ach1 from Saccharomyces cerevisiae originally named acetyl-CoA hydrolase, but recent study showed that it also has a CoA-transferase enzyme activity and maybe involve in exchanging acetyl unit between mitochondria and cytosol (Yun, 2015). To evaluate the role of YlACH in Y. lipolytica, the Ylach gene was knocked-out in PGC01003, generating strain PGC11505. Surprisingly, the acetic acid accumulation significantly reduced from 7.5 g/L to 0.2 g/L (Figure 2a). Moreover, the succinic acid titer increased to 7.0 g/L in shake flasks (increased by
27.3%), and the cell growth also restored comparing with the parent strain PGC01003 (Figure 2b). To further confirm the restored cell growth was caused by acetic acid reduction, we directly added different concentration of acetic acid to the medium and found that cell growth and succinic acid production of PGC11505 were again impaired (Figure S1). Obviously, acetic acid accumulation is harmful to cell growth and succinic acid production, deletion of Ylach in SDH negative Y. lipolytica can relieve this adverse impact. These experiments demonstrated that high level of acetic acid accumulation in SDH negative Y. lipolytica mainly generated from acetyl-CoA, but not pyruvate as it usually observed in other species (König, 1998).
Table 1 Activity measurement of PG52 and PGC52 YlACH.
Hydrolase (U/mg)
CoA transferase (U/mg)
PGC52
n.d.
n.d.
PGC52 YlACH
0.03±0.01
1.89±0.13
The n.d. stand for not detected. One unit is defined as the amount of enzyme producing 1 μmol of succinyl-CoA per minute (succinyl-CoA is not stable in the condition of pH 8.0).
To confirm the function of YlACH in Y. lipolytica，the Ylach gene was expressed under the control of strong promoter hp4d in PGC52 (the strain derived from PGC11505, and the URA3 and LEU2 selection markers have been recycled) and the enzyme activity was analyzed. As shown in Table 1, YlACH showed high acetate: succinate CoA-transferase activity rather than hydrolase activity. The inactivation of the acetyl-CoA transferase blocked the formation of acetic acid from acetyl-CoA, which would subsequently reduce the reverse reaction of succinic acid to succinyl-CoA. The reduction of acetic acid concentration also restored the cell growth, therefore, with an improved
succinic acid production.
3.2 Regulation of by-products formation through reductive carboxylation
Deletion of Ylach improved succinic acid production and restored the cell growth. However, we found that there was a dramatic increase in pyruvate formation at the end of fermentation (from 0.4 g/L to 6.0 g/L, data not shown). In addition, erythritol and mannitol accumulated as well. The formation of the by-products, including pyruvate and its upstream metabolites, indicated that there was a metabolic flux repression from acetyl-CoA, which is a direct downstream metabolite of pyruvate.
In order to channel pyruvate to succinic acid production, a series of gene overexpression were investigated, including the genes encoding the phosphoenolpyruvate carboxykinase from S. cerevisiae (ScPCK, named PGC62) and Y. lipolytica (YlPCK, named PGC73), pyruvate carboxylase from S. cerevisiae (ScPYC, named PGC82) and Y. lipolytica (YlPYC, named PGC91), Y. lipolytica endogenous citrate synthase (YlCIT, named PGC104), aconitase (YlACO, named PGC112), isocitrate lyase (YlICL, named PGC125) and malate synthase (YlMLS, named PGC131) (Table 2).
Table 2 Fermentation profiles of Y. lipolytica PGC52 and its derivative strains in shake flasks Engineered
Succinic
Pyruvate
Erythritol
Mannitol
Malate
Succinic acid
strain
acid
(g/L)
(g/L)
(g/L)
(g/L)
yield
(g/L)
(g/g glycerol)
PGC52
12.05±0.22
5.47±1.98
5.88±1.58
2.0±0.15
n.d.
0.29
PGC62
30.15±0.73
n.d.
n.d.
2.22±0.65
2.3±0.28
0.5
PGC73
13.99±0.16
n.d.
n.d.
3.19±0.02
0.39±0.14
0.39
PGC82
15.72±0.11
n.d.
5.05±1.41
3.72±0.27
2.54±0.34
0.36
PGC91
26.16±1.41
n.d.
4.47±0.7
3.82±0.11
5.89±0.2
0.41
PGC104
17.03±0.44
5.68±0.56
4.3±0.22
1.55±0.27
n.d.
0.34
PGC112
15.1±0.16
4.14±0.3
6.32±0.61
2.56±0.46
n.d.
0.29
PGC125
18.51±0.9
8.48±1.05
4.95±1.28
4.04±0.07
n.d.
0.27
PGC131
22.54±0.08
5.78±0.85
5.26±0.79
4.53±0.63
n.d.
0.36
The n.d. stand for not detected. The initial concentration of glycerol was 40 g/L. After 96 h of culture, the succinic acid and by-products concentration were determined by HPLC.
These recombinant strains were evaluated and all of these genes overexpression enhanced succinic acid production. These cultivations all shown highly acidic pH (a value range of 2.5 – 3.5) at the end of fermentation. Among them, overexpression of YlCIT, YlACO, YlICL and YlMLS, increased succinic acid titer by 41.3%, 25.3%, 53.6% and 87.1%, respectively. Nevertheless, the pyruvate production was not significantly reduced. Overexpression of PYC or PCK, both reductive carboxylation enzymes from S. cerevisiae and Y. lipolytica, eliminated the pyruvate accumulation completely. Among these four engineered strains, overexpression of ScPCK and YlPYC enhanced succinic acid titer significantly, PGC62 with ScPCK overexpression showed a maximum succinic acid titer (30.2 g/L) and yield (0.5 g/g), which enhanced by 150.2% and 72.4% compared with control, respectively. The PGC91 with YlPYC overexpression also increased succinic acid titer and yield by 115.7% and 41.4%, respectively. Interestingly, both overexpression ScPCK and YlPYC resulted in a considerable malate accumulation, confirming that excess oxaloacetate generated from phosphoenolpyruvate or pyruvate carboxylation. These results revealed that enhanced pyruvate and phosphoenolpyruvate carboxylation can overcome the problem of by-products accumulation caused
by Ylach deletion.
3.3 Simultaneous overexpression of reductive carboxylation and oxidative TCA pathway
As shown above, overexpression ScPCK in PGC52 presented the highest improvement on succinic acid production. The resultant succinic acid concentration was 1.5-fold higher than that obtained from PGC52. To further increase the metabolic efficiency, the key genes involved in glyoxylate bypass and oxidative TCA were investigated.
Figure 3 Combinatorial effect of different genetic modifications in Y. lipolytica on succinic acid titer. The initial concentration of glycerol was 40 g/L. After 96 h of culture, the succinic acid concentration of engineered strains were determined.
Three key enzymes, YlACL, YlICL and YlMLS, involved with glyoxylate bypass, were first
attempted in a ScPCK overexpression strain. Unexpectedly, the enhancement of the metabolic flow of glyoxylate bypass had no obvious improvement to succinic acid production (Figure 3). Chen (2016) demonstrated that overexpression of α-ketoglutarate dehydrogenase and succinyl-CoA synthase beta subunit was beneficial to fumarate production. In this study, it was found that overexpression of YlSCS2 can further improve the succinic acid production to 37.0 g/L, which was 24% higher than the strain expressing with only ScPCK. On the contrary, succinic acid production was reduced by 19% in YlKGDH overexpressing strain PGC194. In addition, the YlCIT overexpressing strain PGC143 produced 31.4 g/L succinic acid, which was similar to its parent strain.
3.4 Fed-batch fermentation of succinic acid at low pH value
Figure 4 Fed-batch fermentation profile of engineered Y. lipolytica strains PGC01003 (A), PGC11505 (B) and PGC202 (C). Open circle stands for OD600; open square stands for succinic acid concentration; open triangle stands for glycerol concentration; open diamand stands for acetic acid concentration; dashed line stands for the pH of culture medium.
Comparing with the strain only with SDH inactivation, PGC202 has largely improved succinic acid production and reduced acetic acid accumulation. It showed excellent characteristics for
industrial application. To evaluate its capability, fed-batch fermentation was then performed without pH control. Three engineered strains, PGC01003, PGC11505 and PGC202 were compared. As shown in Figure 4, pH of all the three strains were kept at a value around of 3.0 – 4.3 at the end of fermentation. Original strain PGC01003 presented poor capacity for succinic acid synthesis without adding alkaline. The succinic acid titer only reached to 5.4 g/L with 10.6 g/L acetic acid accumulation during fermentation. Meanwhile, both PGC11505 and PGC202 accumulated a small amount of acetic acid (around 0.5 – 1.4 g/L), and they grew well in the acidic environment. These results demonstrated that elimination of acetic acid is critical to cell growth and succinic acid production at low pH. The succinic acid titer of PGC11505 was 45.6 g/L, with a yield of 0.2 g/g glycerol. The highest succinic acid production of 67.5 g/L was obtained by PGC202 using defined medium containing 181.3 g/L of glycerol, with a yield of 0.37 g/g glycerol, and productivity of 0.49 g/L/h.
Figure 5 Fermentation performance of Y. lipolytica PGC202 in fed-batch fermentation with different initial glycerol concentration. (A) Comparison of SA production and by-product accumulation by PGC202 in batch fermentation at various initial concentration of glycerol. (B) The fermentation results of the PGC202 strain with the 50 g/L initial glycerol. Open circle stands for OD600; open square stands for succinic acid concentration; open triangle stands for glycerol
concentration; dashed line stands for the pH of culture medium; open diamand stands for mannitol concentration.
Through analysis of fermentation products, large number of by-products, such as mannitol and arabitol were accumulated by PGC202. However, these kinds of by-products were not detected in shake flasks fermentation. Mannitol, similar to erythritol and arabitol, is a storage for carbon source as well as reducing force, which can be enhanced by using fermentation medium with high glycerol concentration (Juszczyk et al., 2013; Tomaszewska et al., 2012). These studies reported that the formation of by-products would lead to reduction of succinic acid yield, were mainly caused by highly initial glycerol concentration. Therefore, the initial glycerol concentration was set from 30 to 70 g/L to reduce by-products formation. As shown in Figure 5, mannitol titers were positively correlated with the initial glycerol concentration. Initial glycerol of 70 g/L led to high resultant succinic acid concentration (93.3 g/L) with high productivity (0.7 g/L/h), while about 19.9 g/L mannitol was formed. Although PGC202 accumulated only 11.0 g/L mannitol when the initial glycerol concentration was 30 g/L, the highest succinic acid titer of 110.7 g/L was achieved in 138 h with a final pH of 3.4 and a yield of 0.53 g/g glycerol, which is equivalent to 81.8% of theoretical yield when medium containing 50 g/L initial glycerol was used. This is the highest fermentative succinic acid titer and yield achieved by Y. lipolytica at natural low pH reported to date, using defined medium.
4. Discussion
Because of low processing cost and environmental friendliness, microbial synthesis of succinic acid from cheap substrates has been drawed increasing attentions in both academic research and
industrial manufacture (Cao et al., 2013). At present, bacteria have an obvious advantage in succinic acid production. Except for yield and productivity, the downstream separation and purification steps are also a determined factor affecting the cost of succinic acid production. The succinate obtained from the bacterial fermentation need to be converted to free acid form (rather than the salt form), which cost about 60% to 70% of the total production cost (Cok et al., 2013; Zeikus et al., 1999). Yeast can carry out low pH fermentation, directly convert the substrate into succinic acid, which largely reduce the cost of downstream processing. Compare to bacteria, low-pH yeast would be more suitable as a succinic acid production host if the succinic acid production efficiency of yeast could improve further. However, the succinic acid titer and yield of S. cerevisiae still could not meet the need of industrial application today after many years of research efforts. As a unconventional yeast, Y. lipolytica can utilise many unspecific substrates such as alkanes, ethanol and glycerol (Ledesma-Amaro and Nicaud, 2016). As the primary by-product from the production of biodiesel, glycerol is one of the low-cost feedstock. With the rise of biodiesel production scale, glycerol was regarded as the second top feedstock in succinic acid production (Tan et al., 2014). Several studies have demonstrated that Y. lipolytica as an efficient industrial production host in succinic acid production (Gao et al., 2016; Yuzbashev et al., 2016). Herein, Y. lipolytica central metabolic pathway was engineered to improve succinic acid production through elimination of acetic acid overflow and by-products formation, succinic acid production of strain PGC202 reached 110.7 g/L with a maximum yield of 0.53 g/g of glycerol and a productivity of 0.8 g/L/h in fed-batch fermentation.
The main by-product of the parent strain PGC01003 is acetic acid, and the acetic acid overflow should be due to the imbalance of metabolic flux between glycolysis and TCA cycle. Excessive acetic acid generated during the fermentation process not only inhibited the cell growth, but it also
affected the global metabolism of cells (Bernal et al., 2016), and ultimately reduced the production of succinic acid. However, interference of pyruvate decarboxylation bypass, which is the primary acetic acid formation pathway, would not lead to reduction of acetic acid accumulation in SDH negative Y. lipolytica. As an alternative option, YlACH was inactivated to evaluate the role of YlACH in acetic acid overflow. Surprisingly, the acetic acid accumulation was significantly reduced, and succinic acid production was improved at the same time. YlACH exhibited high amino acid sequence identity to the acetyl-CoA hydrolase (ScACH) from S. cerevisiae, and acetate: succinate CoA-transferase (TvASCT) from Trichomonas vaginalis. In view of acetyl-CoA hydrolysis is an energy consuming process, these two enzymes showed high activity for CoA transferase, and only a minor acetyl-CoA hydrolase activity (Fleck and Brock, 2009; van Grinsven et al., 2008). The results of enzymatic activity determination also demonstrated that YlACH has high specific activity for the acetate: succinate CoA-transferase other than acetyl-CoA hydrolase. From the information above, the absence of succinate dehydrogenase in PGC01003 may cause TCA cycle obstruction, and this resulted excessive acetyl-CoA transfer into acetic acid by CoA transferase.
Although the deletion of Ylach eliminated acetic acid overflow, the engineered strain PGC11505 produced some other by-products such as pyruvate, erythritol and mannitol. It indicated that the additional deletion of Ylach in the PGC01003 caused the accumulation of acetyl-CoA, in which the feedback inhibited the pyruvate dehydrogenase and led to pyruvate and its upstream metabolites formation. Therefore, the distribution of metabolic flux was reconstructed to relieve the metabolic repression and by-products formation. As expected, the succinic acid production of all the engineered strains were improved, and the overexpression of reductive carboxylation achieved the higher succinic acid titers and yields. In Y. lipolytica, the reductive carboxylation represents the
committed step in many organic acids biosynthesis, the pyruvate carboxylase from different species were commonly used (Wang et al., 2015; Yin et al., 2012). Results from this study indicated that phosphoenolpyruvate carboxykinase was more conducive to succinic acid production than pyruvate carboxylase. Actually, the reaction catalyzed by PCK is accompanied by ATP generation, which may be useful to SDH negative strain (Kim et al., 2004; Tan et al., 2013). The overexpression of reductive carboxylation also led to considerable malate accumulation, which enlightened us that reductive carboxylation plays an important role in the regulation of redox state, since this pathway consumes NADH.
5. Conclusions
This study provides a rational engineering strategy for the enhancement of succinic acid production in Y. lipolytica. Through a series of analysis and well-designed experiments, the CoA-transferase encoded by Ylach was identified being responsible for acetic acid overflow in Y. lipolytica mutant during fermentation. Deletion of Ylach significantly reduced acetic acid formation. By further strengthening the reductive carboxylation and oxidative TCA, an engineered strain PGC202 was obtained, which can produce 110.7 g/L succinic acid in fed-batch cultivation using glycerol without pH control. This is the highest fermentative succinic acid titer achieved in yeast at low pH condition.
Results from this study successfully demonstrated that an innovative feedstock
valorization solution using an engineered Y. lipolytica strain based on renewable and low-cost feedstocks, which could contribute to the future bio-based economy.
Acknowledgments
This work was supported by the key research and development plan of Shandong Province 2016ZDJS07A19 and 2016GSF121019.
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Highlights 1. The first time revealed that acetic acid overflow is caused by acetate: succinate CoA-transferase
in SDH negative Y. lipolytica. 2. Succinic acid titer achieved in this study is the highest level producted at low pH by yeast. 3. Yarrowia lipolytica could be developed as an efficient succinic acid production strain with great industrial prospects.