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Making brilliant colors by microorganisms
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ScienceDirect Making brilliant colors by microorganisms Jian Zha1, Xia Wu1 and Mattheos AG Koffas2,3 Anthocyanins, the colorful molecules found in plants, have positive health effects in humans, and are used as food colorants and nutraceuticals. Currently, the industrial supply of anthocyanins largely depends on extraction from plants, a method that lacks robustness and is potentially unsustainable. A promising alternative is biosynthesis by metabolically engineered microbes, which has achieved considerable success. Here, we review recent progress on anthocyanin biosynthesis in engineered microorganisms and the engineering approaches for enhancing anthocyanin production. The de novo anthocyanin production strategies and microbial production of unusual anthocyanins such as deuterated cyanidin 3-O-glucoside and pyranoanthocyanins are also covered. These engineering strategies will provide a guidance to microbial production of anthocyanins. Existing problems and future directions are also discussed. Addresses 1 School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, Shaanxi 710021, China 2 Department of Chemical and Biological Engineering, and Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 3 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA Corresponding author: Koffas, Mattheos AG ([email protected])
Current Opinion in Biotechnology 2020, 61:135–141 This review comes from a themed issue on Food biotechnology
The traditional way of obtaining anthocyanins for industrial use is extraction from plant tissues [7–9], which has several disadvantages, such as seasonal and regional fluctuations in anthocyanin content in plants, and difficulty in quality control, making such methods less robust and difficult to meet demands for higher purity products [10,11]. Recently, production of anthocyanins using genetically engineered tobacco cell suspension culture was developed that allowed the biosynthesis of blue anthocyanins [12]. Another feasible alternative for anthocyanin production is biosynthesis in metabolically engineered microorganisms, which has already shown promising potential in the synthesis of plant-derived value-added compounds, such as terpenoids, alkaloids, and flavonoids [13–15]. Microbes are advantageous over plants in several aspects, such as fast growth and easy cultivation, and the availability of sophisticated genetic tools in metabolic engineering and synthetic biology, thus making microbial production of natural products facile, controllable, and cost-effective [15–18]. Microbial production of typical anthocyanins, such as cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside, has been achieved in Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Lactococcus lactis [16,19,20,21]. Moreover, direct production of anthocyanins from glucose without the feed of expensive precursors has also been accomplished using microorganisms [20,22].
Edited by Mark A Blenner and Jan Peter van Pijkeren
https://doi.org/10.1016/j.copbio.2019.12.020 0958-1669/ã 2019 Elsevier Ltd. All rights reserved.
In this review, we will highlight the recent progress on anthocyanin production in genetically modified microorganisms with a main focus on metabolic engineering strategies and will point out some critical issues in future studies.
Biosynthesis pathway of anthocyanins in plants Introduction Anthocyanins are an important group of water-soluble flavonoid compounds in the plant kingdom (Figure 1). They endow plants with brilliant colors such as red, purple, and blue, which attract insects for pollination [1,2]. In addition, anthocyanins have demonstrated medicinal roles in humans and animals, such as the strong absorption of ultraviolet light and the prevention of cancer, cardiovascular diseases, neurodegenerative diseases, obesity, and diabetes [3–5]. These unique properties have been leveraged to be applied as food colorants, nutraceuticals, cosmetic additives, and so on [1,6]. www.sciencedirect.com
In plants, anthocyanins are synthesized via the general flavonoid pathway, whereby three molecules of malonylCoA and one molecule of 4-coumaroyl-CoA resulting from phenylalanine or tyrosine are condensed by chalcone synthase (CHS) to form chalcones (Figure 2). With the isomerization catalyzed by chalcone isomerase (CHI), chalcones are then transformed to flavanones, which then undergo hydroxylation by flavanone 3-hydroxylase (F3H) to form different dihydroflavonols. These molecules are reduced by dihydroflavonol 4-reductase (DFR) to form leucoanthocyanidins. Oxidation of leucoanthocyanidins by anthocyanidin synthase (ANS) generates the unstable flavylium cations anthocyanidins. Leucoanthocyanidins can also be reduced to flavan-3-ols, such as catechin, Current Opinion in Biotechnology 2020, 61:135–141
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Figure 1
Pelargonidin
Cyanidin
Malvinidin
Peonidin
Petunidin
Delphinidin Current Opinion in Biotechnology
The core structure of anthocyanins and representative anthocyanins.
which can also be oxidized by ANS to produce anthocyanidins, even though this reaction is not considered a part of the anthocyanin canonical pathway. Anthocyanidins are very unstable under neutral pH, and several structural modifications confer improved stability, such as methylation, acylation, and flavonoid glucosyltransferase (FGT)-catalyzed glycosylation. Anthocyanins are transported to and stored in vacuoles after biosynthesis, where they can be stabilized through structural decorations, lowered pH, and co-pigmentation [1].
439 mg/L by feeding 580 mg/L catechin [24,25]. In addition to E. coli, reported anthocyanin production hosts have been extended to S. cerevisiae, S. venezuelae, C. glutamicum, and L. lactis with great success, which, however, still does not meet industrial requirements due to relatively low production titers [16,19,20,21]. As a result, various engineering strategies have been adopted for highly efficient anthocyanin production.
Production of anthocyanins in metabolically engineered microbes
Construction of an anthocyanin-producing strain involves coexpression of multiple plant genes/enzymes. The enzyme orthologs from different plants present distinct kinetic and thermodynamic properties, which can result in very different production levels of the same anthocyanin compound when they are expressed heterologously in microbes. Thus, gene/ enzyme screening and selection from diverse species constitute a common and feasible way of improving the production of anthocyanins and other flavonoids [26,27]. To achieve maximal cyanidin production in E. coli, Yan et al. compared the in vivo activities of ANS from four plants, and the Petunia
The first attempt to synthesize recombinant anthocyanins in engineered microbes was reported in 2005 by Yan et al., in which a recombinant E. coli strain produced 6.0 mg/L of cyanidin 3-O-glucoside and 5.6 mg/L of pelargonidin 3-O-glucoside using naringenin and eriodictyol as the respective precursors [23]. Further engineering and optimization dramatically increased the titers of the synthesized anthocyanins, and the highest titer of cyanidin 3-O-glucoside in E. coli has reached Current Opinion in Biotechnology 2020, 61:135–141
Engineering approaches to improve anthocyanin production
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Anthocyanin production Zha, Wu and Koffas 137
Figure 2
The Shikimate Pathway
Phenylalanine
Cinnamic acid
Tyrosine
p-Coumaric acid
p-Coumaroyl-CoA
Naringenin chalcone
Naringenin
Anthocyanins Current Opinion in Biotechnology
The pathway for anthocyanin biosynthesis in plants. Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, cytochrome P450 reductase; TAL, tyrosine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 3’-hydroxylase; F3’5’H, flavonoid 3’, 5’-hydroxylase; DFR, dihydroflavonol reductase; LAR, leucocyanidin reductase; ANS, anthocyanidin synthase; FGT, flavonoid glucosyltransferase; OMT, O-methyltransferase; ACT, acyltransferases.
hybrida ANS showed the best performance [27]. Similarly, naringenin production using coumaric acid in E. coli was optimized by comparing different orthologs for the pathway genes including 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, and gene combinations of different sources showed drastic variations in naringenin titer, with the highest titer achieved using the three genes from Arabidopsis thaliana, Petunia hybrida, and Cucurbita maxima, respectively [28]. www.sciencedirect.com
The gene/enzyme screening is the very basic step of enhancing anthocyanin production, which does not guarantee the functional expression of these genes/enzymes in the selected microbial hosts, due to the differences in gene expression machinery between plants and microbes. To tackle this problem, various gene modifications can be applied such as fusion engineering and truncation engineering (Figure 3). For example, a SUMO tag was fused Current Opinion in Biotechnology 2020, 61:135–141
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to the N-terminus of flavonoid 3-O-glucosyltransferase to enhance the soluble expression of this enzyme for the production of cyanidin 3-O-glucoside from catechin, which resulted in a 1.1-fold elevation in cyanidin 3-O-glucoside titer [19]. Fusion of F3GT from A. thaliana to the N-terminus of ANS from Petunia hybrida with a pentapeptide linker led to higher production of cyanidin 3-O-glucoside compared with the uncoupled ANS and F3GT using catechin as the precursor, and the underlying mechanism was proposed to be a higher local concentration of the unstable intermediate cyanidin aglycone, its faster transition in the chimeric enzyme system, and its slower degradation [27]. To achieve the functional expression of F3’5’H from Catharanthus roseus, the first four codons (N-terminal membrane anchor region) of F3’5’H were deleted, and the next two codons were replaced with methionine and alanine [29]. The modified F3’5’H fused with a truncated P450 reductase from C. roseus successfully catalyzed the hydroxylation at C3’ and directed the formation of quercetin by feeding coumaric acid. Anthocyanins generally undergo glycosylation for higher stability, and UDP-glucose is one of the most commonly used co-substrates in this process (Figure 3). Since UDPglucose is also involved in many other metabolic pathways such as the biosynthesis of cell wall and glycogen, its global regulation is crucial [6]. The frequently used approaches for improving UDP-glucose availability are overexpression of the biosynthetic genes and partial inhibition of the degradation pathways to increase UDP-glucose supply. Such attempts in E. coli include the upregulation of genes responsible for UDP biosynthesis from orotic acid ( pyrE, pyrF, cmk, ndk), the increased supply of glucose-1-phosphate (glf, glk, pgm), and the condensation of UDP and glucose-1-phosphate (galU1), as well as inhibition of the competitive UDPglucose consumption pathways [25,27,30]. In one case, overexpression of pgm and galU1 along with the coexpression of ANS and 3GT resulted in a 57.8% increase in cyanidin 3-O-glucoside production using catechin as the precursor [27]. S-Adenosyl-L-methionine is also an indispensable cosubstrate for the production of methylated anthocyanins such as peonidin 3-O-glucoside. Through the CRISPR interference-mediated silencing of the transcriptional repressor MetJ, SAM availability was improved and peonidin 3-O-glucoside production was increased by twofold in E. coli [31]. Many target products in metabolically engineered microorganisms are toxic to host strains, thereby inhibiting their high-titer production. In the production of cyanidin 3-O-glucoside, over 70% of the total products are retained inside the cells, which may potentially interfere with cellular metabolism [25,27]. A feasible strategy Current Opinion in Biotechnology 2020, 61:135–141
to continuously synthesize anthocyanins at acceptable levels is to transfer them from cytoplasm to extracellular environments through specific efflux pumps. A relevant E. coli efflux pump YadH has been identified and its overexpression led to 15% more production of cyanidin 3-O-glucoside. Moreover, deletion of another efflux pump TolC, probably responsible for the secretion of the substrate catechin, further promoted the production of cyanidin 3-O-glucoside [24]. Apart from relying on intrinsic transporters in microbial hosts, introduction of transporters from plants is also a possible route to implement the transportation process in engineered microbes. In plants, anthocyanins are transported to and accumulate in vacuoles by specific transporters [32], such as ATP-binding cassette transporter ZmMRP3 and glutathione S-transferase encoded by Bronze-2 [33,34]. Recently, an ATP-binding cassette transporter AtABCC2 from Arabidopsis was reported to direct the co-transport of cyanidin 3-O-glucoside and glutathione to vacuoles [35]. These transporters have not been tested in microbial strains for the efflux of anthocyanins, and their roles in improving anthocyanin production are not clear. Regarding the high-content intracellular accumulation of anthocyanins in current studies, it would be highly interesting to introduce plant-derived transporters to microbes and to investigate their potential ability to enhance anthocyanin production.
Separation of enzyme expression and anthocyanin production using a two-step biocatalysis The unstable nature of anthocyanins at neutral pH is a big challenge for their microbial production. Unlike in plants where naturally synthesized anthocyanins are stored and stabilized in vacuoles [1,36], there is a lack of such an organelle in bacterial cells. In addition, the near neutral pH in the intracellular environment in E. coli and other hosts makes the synthesized anthocyanins rather labile. This issue drives the development of a two-step biocatalysis strategy, which proves effective in elevating anthocyanin production [27]. In the first step, cells are grown in medium at pH 7, where enzyme expression and cosubstrate (such as UDP-glucose) generation are accomplished. In the second step, cells with fully expressed enzymes are transferred to fresh medium adjusted to pH 5 with the supplementation of precursors, where anthocyanins can be synthesized and stabilized. Protective agents such as glutamate can be used to prevent cell lysis under a low pH. Such a two-step procedure separates enzyme expression from anthocyanin production, and balances the pH dependence in primary metabolism (cell growth and enzyme expression) and secondary metabolism (anthocyanin production). This strategy could increase the titer of cyanidin 3-O-glucoside by 15-fold (38.9 mg/L) compared to the traditional single-step production (2.5 mg/L) in E. coli [27]. Apart from pH, other process parameters also play important roles in www.sciencedirect.com
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Figure 3
Orotic acid
Transporters
Glucose Anthocyanins Transporters
Current Opinion in Biotechnology
The strategies applied in microbial biosynthesis of anthocyanins. The modifications of the anthocyanin-producing strains involve pathway enzymes, the supply of UDP-glucose, and the transport of substrates and anthocyanins. Abbreviations: G-6-P, Glucose-6-phosphate; G-1-P, glucose-1-phosphate; UDP-G, UDP-glucose; galU1, glucose-1-phosphate uridylyltransferase gene, pgm, phosphoglucomutase gene; pyrE, orotate phosphoribosyltransferase; pyrF, orotidine-5’-phosphate decarboxylase, cmk, cytidine monophosphate kinase gene; ndk, nucleoside diphosphate kinase gene; glk, glucokinase gene; glf, glucose facilitator diffusion gene.
anthocyanin production, such as induction time-point, substrate feeding, amount of dissolved oxygen, and temperature, which affect the physiological function of cells and thereby affect the stability of expressed enzymes, the supply of co-substrate, and so on [24,26,28]. These process conditions should also be optimized in anthocyanin production. De novo biosynthesis of anthocyanins
The initial studies on microbial production of anthocyanins relied on the supplementation of flavonoid precursors, such as (+)-catechin, something that can potentially increase the production cost and decrease industrial feasibility. Therefore, using cheap carbon sources such as glucose or glycerol for microbial production is of great industrial importance. Recently, de novo production of anthocyanins has been achieved first by using microbial consortia and later by using recombinant S. cerevisiae. Given that the entire pathway starting from glucose is extremely long, the division of the metabolic pathway into multiple strains can alleviate the metabolic burden and improve the overall metabolic performance. This strategy was showcased by splitting the biosynthetic pathway of pelargonidin 3-O-glucoside starting from tyrosine (containing 15 genes) into four E. coli strains including a high-yield tyrosine-producing strain to achieve de novo production of 10 mg/L pelargonidin 3-O-glucoside from 20 g/L glucose [22]. In another study, using a single S. cerevisiae strain as the host, Levisson et al. introduced 13 genes responsible for the biosynthesis of pelargonidin 3-O-glucoside, deleted the genes for the degradation or competitive utilization of phenylalanine (aro10, pdc5, and pdc6), pelargonidin 3-O-glucoside (exg1, and spr1), and coumaroyl-CoA (tsc13), and enhanced the supply of phenylalanine and tyrosine via alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase www.sciencedirect.com
(Aro3, Aro4) [20]. The combinatorial modifications resulted in the production of 0.01 mmol/gCDW pelargonidin and 0.001 mmol/gCDW pelargonidin 3-O-glucoside from glucose in controlled aerobic batch cultures. A similar study was performed and achieved mg/L level production of anthocyanins (delphinidin-3-O-glucoside, cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside) [37]. Although the production levels are very low, the de novo production demonstrates the possibility of microbial production of anthocyanins from cheap carbon sources, and opens up an avenue to efficient biosystems for anthocyanin production.
Production of unusual anthocyanins by bacterial strains Cyanidin 3-O-glucoside, one of the most commonly found anthocyanins in nature, has potential therapeutic roles in medical applications. To facilitate pharmacokinetic study of this compound, the synthesis of a deuterated version of this compound was recently demonstrated for the first time using recombinant E. coli. Specifically, an E. coli strain co-expressing ANS from Petunia hybrida and F3GT from Arabidopsis was grown in medium with D2O as the solvent and deuterated glycerol D8 as the carbon source. The strain produced deuterated cyanidin 3-O-glucoside when feeding normal catechin. This deuterated compound is more stable than the undeuterated one possibly due to the higher stability of the carbon-deuterium bonds than the carbon-hydrogen bonds [38]. Another unusual form of anthocyanins is pyranoanthocyanins, which are mostly found in red wines and are formed by the condensation of anthocyanins and compounds containing a polarizable double bond such as Current Opinion in Biotechnology 2020, 61:135–141
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acetaldehyde, pyruvic acid, hydroxycinnamic acids, and vinylphenols. To produce pyranoanthocyanins by microorganisms, an E. coli strain was engineered to produce 4-vinylphenol by expressing TAL and phenolic acid decarboxylase (PDC). Co-culturing this strain with a cyanidin 3-O-glucoside-producing E. coli strain and by optimizing co-culture parameters, including testing the presence of ethanol, generated 19.5 mg/L pyranocyanidin 3-O-glucoside-4-phenol. Additional expression of HpaBC in 4-vinylphenol-producing E. coli resulted in the production of another pyranoanthocyanin, pyranocyanidin 3-O-glucoside-4-catechol, and optimization of the co-culture conditions elevated its titer to 13 mg/L [39]. Pyranoanthocyanin production was also reported in L. lactis when using catechin to synthesize cyanidin 3O-glucoside. Interestingly, pyranoanthocyanins (including methylpyranocyanidin and methylpyranopeonidin) were generated concomitantly, which might be a spontaneous process given the potential reaction between cyanidin/peonidin (methylated cyanidin by unknown flavonoid methyltransferases in L. lactis) and the L. lactis metabolite acetoacetic acid. The same phenomenon was observed when using gallocatechin as the substrate, in which two new pyranoanthocyanins, that is, methylpyranodelphinidin and methylpyranopetunidin, were produced [21].
Conflict of interest statement
Conclusion Production of colorful anthocyanins by engineered microorganisms is a promising way of supplying these compounds for industrial use. With the continuously emerging techniques in metabolic engineering and synthetic biology, the production titers of some anthocyanins have been significantly increased through multidimensional engineering of pathway enzymes, global regulation of cosubstrate supply, transmembrane transportation of accumulated intracellular anthocyanins, and effective stabilization of newly synthesized anthocyanins. However, many problems still remain to be solved to further improve the production and to increase the commercial competitiveness of the microbial production. For example, the kinetics of the recombinant enzymes, their expression profiles, as well as the stability of the produced molecules have not been thoroughly studied and remain largely unknown. In addition, engineering of transporters for efficient secretion of intracellular anthocyanins into the media also lacks in-depth and mechanistic investigation. These points are all worth studying and can be interesting and useful foci of future exploration. Finally, the production of more complex anthocyanins with different colors (including the sought-after blue anthocyanins) is another important target for future metabolic engineering endeavors. Given the big strides on microbial anthocyanin production in the past years, it is highly expected that engineered microorganisms will become very competitive in supplying anthocyanins in the near future. Current Opinion in Biotechnology 2020, 61:135–141
Nothing declared.
Acknowledgements This work was supported by the startup funds from Shaanxi University of Science and Technology awarded to JZ and XW. The financial support from National Natural Science Foundation for Young Scientists of China (31900114) to XW is also acknowledged.
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Current Opinion in Biotechnology 2020, 61:135–141
ScienceDirect Making brilliant colors by microorganisms Jian Zha1, Xia Wu1 and Mattheos AG Koffas2,3 Anthocyanins, the colorful molecules found in plants, have positive health effects in humans, and are used as food colorants and nutraceuticals. Currently, the industrial supply of anthocyanins largely depends on extraction from plants, a method that lacks robustness and is potentially unsustainable. A promising alternative is biosynthesis by metabolically engineered microbes, which has achieved considerable success. Here, we review recent progress on anthocyanin biosynthesis in engineered microorganisms and the engineering approaches for enhancing anthocyanin production. The de novo anthocyanin production strategies and microbial production of unusual anthocyanins such as deuterated cyanidin 3-O-glucoside and pyranoanthocyanins are also covered. These engineering strategies will provide a guidance to microbial production of anthocyanins. Existing problems and future directions are also discussed. Addresses 1 School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, Shaanxi 710021, China 2 Department of Chemical and Biological Engineering, and Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 3 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA Corresponding author: Koffas, Mattheos AG ([email protected])
Current Opinion in Biotechnology 2020, 61:135–141 This review comes from a themed issue on Food biotechnology
The traditional way of obtaining anthocyanins for industrial use is extraction from plant tissues [7–9], which has several disadvantages, such as seasonal and regional fluctuations in anthocyanin content in plants, and difficulty in quality control, making such methods less robust and difficult to meet demands for higher purity products [10,11]. Recently, production of anthocyanins using genetically engineered tobacco cell suspension culture was developed that allowed the biosynthesis of blue anthocyanins [12]. Another feasible alternative for anthocyanin production is biosynthesis in metabolically engineered microorganisms, which has already shown promising potential in the synthesis of plant-derived value-added compounds, such as terpenoids, alkaloids, and flavonoids [13–15]. Microbes are advantageous over plants in several aspects, such as fast growth and easy cultivation, and the availability of sophisticated genetic tools in metabolic engineering and synthetic biology, thus making microbial production of natural products facile, controllable, and cost-effective [15–18]. Microbial production of typical anthocyanins, such as cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside, has been achieved in Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Lactococcus lactis [16,19,20,21]. Moreover, direct production of anthocyanins from glucose without the feed of expensive precursors has also been accomplished using microorganisms [20,22].
Edited by Mark A Blenner and Jan Peter van Pijkeren
https://doi.org/10.1016/j.copbio.2019.12.020 0958-1669/ã 2019 Elsevier Ltd. All rights reserved.
In this review, we will highlight the recent progress on anthocyanin production in genetically modified microorganisms with a main focus on metabolic engineering strategies and will point out some critical issues in future studies.
Biosynthesis pathway of anthocyanins in plants Introduction Anthocyanins are an important group of water-soluble flavonoid compounds in the plant kingdom (Figure 1). They endow plants with brilliant colors such as red, purple, and blue, which attract insects for pollination [1,2]. In addition, anthocyanins have demonstrated medicinal roles in humans and animals, such as the strong absorption of ultraviolet light and the prevention of cancer, cardiovascular diseases, neurodegenerative diseases, obesity, and diabetes [3–5]. These unique properties have been leveraged to be applied as food colorants, nutraceuticals, cosmetic additives, and so on [1,6]. www.sciencedirect.com
In plants, anthocyanins are synthesized via the general flavonoid pathway, whereby three molecules of malonylCoA and one molecule of 4-coumaroyl-CoA resulting from phenylalanine or tyrosine are condensed by chalcone synthase (CHS) to form chalcones (Figure 2). With the isomerization catalyzed by chalcone isomerase (CHI), chalcones are then transformed to flavanones, which then undergo hydroxylation by flavanone 3-hydroxylase (F3H) to form different dihydroflavonols. These molecules are reduced by dihydroflavonol 4-reductase (DFR) to form leucoanthocyanidins. Oxidation of leucoanthocyanidins by anthocyanidin synthase (ANS) generates the unstable flavylium cations anthocyanidins. Leucoanthocyanidins can also be reduced to flavan-3-ols, such as catechin, Current Opinion in Biotechnology 2020, 61:135–141
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Figure 1
Pelargonidin
Cyanidin
Malvinidin
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The core structure of anthocyanins and representative anthocyanins.
which can also be oxidized by ANS to produce anthocyanidins, even though this reaction is not considered a part of the anthocyanin canonical pathway. Anthocyanidins are very unstable under neutral pH, and several structural modifications confer improved stability, such as methylation, acylation, and flavonoid glucosyltransferase (FGT)-catalyzed glycosylation. Anthocyanins are transported to and stored in vacuoles after biosynthesis, where they can be stabilized through structural decorations, lowered pH, and co-pigmentation [1].
439 mg/L by feeding 580 mg/L catechin [24,25]. In addition to E. coli, reported anthocyanin production hosts have been extended to S. cerevisiae, S. venezuelae, C. glutamicum, and L. lactis with great success, which, however, still does not meet industrial requirements due to relatively low production titers [16,19,20,21]. As a result, various engineering strategies have been adopted for highly efficient anthocyanin production.
Production of anthocyanins in metabolically engineered microbes
Construction of an anthocyanin-producing strain involves coexpression of multiple plant genes/enzymes. The enzyme orthologs from different plants present distinct kinetic and thermodynamic properties, which can result in very different production levels of the same anthocyanin compound when they are expressed heterologously in microbes. Thus, gene/ enzyme screening and selection from diverse species constitute a common and feasible way of improving the production of anthocyanins and other flavonoids [26,27]. To achieve maximal cyanidin production in E. coli, Yan et al. compared the in vivo activities of ANS from four plants, and the Petunia
The first attempt to synthesize recombinant anthocyanins in engineered microbes was reported in 2005 by Yan et al., in which a recombinant E. coli strain produced 6.0 mg/L of cyanidin 3-O-glucoside and 5.6 mg/L of pelargonidin 3-O-glucoside using naringenin and eriodictyol as the respective precursors [23]. Further engineering and optimization dramatically increased the titers of the synthesized anthocyanins, and the highest titer of cyanidin 3-O-glucoside in E. coli has reached Current Opinion in Biotechnology 2020, 61:135–141
Engineering approaches to improve anthocyanin production
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Figure 2
The Shikimate Pathway
Phenylalanine
Cinnamic acid
Tyrosine
p-Coumaric acid
p-Coumaroyl-CoA
Naringenin chalcone
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Anthocyanins Current Opinion in Biotechnology
The pathway for anthocyanin biosynthesis in plants. Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, cytochrome P450 reductase; TAL, tyrosine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 3’-hydroxylase; F3’5’H, flavonoid 3’, 5’-hydroxylase; DFR, dihydroflavonol reductase; LAR, leucocyanidin reductase; ANS, anthocyanidin synthase; FGT, flavonoid glucosyltransferase; OMT, O-methyltransferase; ACT, acyltransferases.
hybrida ANS showed the best performance [27]. Similarly, naringenin production using coumaric acid in E. coli was optimized by comparing different orthologs for the pathway genes including 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, and gene combinations of different sources showed drastic variations in naringenin titer, with the highest titer achieved using the three genes from Arabidopsis thaliana, Petunia hybrida, and Cucurbita maxima, respectively [28]. www.sciencedirect.com
The gene/enzyme screening is the very basic step of enhancing anthocyanin production, which does not guarantee the functional expression of these genes/enzymes in the selected microbial hosts, due to the differences in gene expression machinery between plants and microbes. To tackle this problem, various gene modifications can be applied such as fusion engineering and truncation engineering (Figure 3). For example, a SUMO tag was fused Current Opinion in Biotechnology 2020, 61:135–141
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to the N-terminus of flavonoid 3-O-glucosyltransferase to enhance the soluble expression of this enzyme for the production of cyanidin 3-O-glucoside from catechin, which resulted in a 1.1-fold elevation in cyanidin 3-O-glucoside titer [19]. Fusion of F3GT from A. thaliana to the N-terminus of ANS from Petunia hybrida with a pentapeptide linker led to higher production of cyanidin 3-O-glucoside compared with the uncoupled ANS and F3GT using catechin as the precursor, and the underlying mechanism was proposed to be a higher local concentration of the unstable intermediate cyanidin aglycone, its faster transition in the chimeric enzyme system, and its slower degradation [27]. To achieve the functional expression of F3’5’H from Catharanthus roseus, the first four codons (N-terminal membrane anchor region) of F3’5’H were deleted, and the next two codons were replaced with methionine and alanine [29]. The modified F3’5’H fused with a truncated P450 reductase from C. roseus successfully catalyzed the hydroxylation at C3’ and directed the formation of quercetin by feeding coumaric acid. Anthocyanins generally undergo glycosylation for higher stability, and UDP-glucose is one of the most commonly used co-substrates in this process (Figure 3). Since UDPglucose is also involved in many other metabolic pathways such as the biosynthesis of cell wall and glycogen, its global regulation is crucial [6]. The frequently used approaches for improving UDP-glucose availability are overexpression of the biosynthetic genes and partial inhibition of the degradation pathways to increase UDP-glucose supply. Such attempts in E. coli include the upregulation of genes responsible for UDP biosynthesis from orotic acid ( pyrE, pyrF, cmk, ndk), the increased supply of glucose-1-phosphate (glf, glk, pgm), and the condensation of UDP and glucose-1-phosphate (galU1), as well as inhibition of the competitive UDPglucose consumption pathways [25,27,30]. In one case, overexpression of pgm and galU1 along with the coexpression of ANS and 3GT resulted in a 57.8% increase in cyanidin 3-O-glucoside production using catechin as the precursor [27]. S-Adenosyl-L-methionine is also an indispensable cosubstrate for the production of methylated anthocyanins such as peonidin 3-O-glucoside. Through the CRISPR interference-mediated silencing of the transcriptional repressor MetJ, SAM availability was improved and peonidin 3-O-glucoside production was increased by twofold in E. coli [31]. Many target products in metabolically engineered microorganisms are toxic to host strains, thereby inhibiting their high-titer production. In the production of cyanidin 3-O-glucoside, over 70% of the total products are retained inside the cells, which may potentially interfere with cellular metabolism [25,27]. A feasible strategy Current Opinion in Biotechnology 2020, 61:135–141
to continuously synthesize anthocyanins at acceptable levels is to transfer them from cytoplasm to extracellular environments through specific efflux pumps. A relevant E. coli efflux pump YadH has been identified and its overexpression led to 15% more production of cyanidin 3-O-glucoside. Moreover, deletion of another efflux pump TolC, probably responsible for the secretion of the substrate catechin, further promoted the production of cyanidin 3-O-glucoside [24]. Apart from relying on intrinsic transporters in microbial hosts, introduction of transporters from plants is also a possible route to implement the transportation process in engineered microbes. In plants, anthocyanins are transported to and accumulate in vacuoles by specific transporters [32], such as ATP-binding cassette transporter ZmMRP3 and glutathione S-transferase encoded by Bronze-2 [33,34]. Recently, an ATP-binding cassette transporter AtABCC2 from Arabidopsis was reported to direct the co-transport of cyanidin 3-O-glucoside and glutathione to vacuoles [35]. These transporters have not been tested in microbial strains for the efflux of anthocyanins, and their roles in improving anthocyanin production are not clear. Regarding the high-content intracellular accumulation of anthocyanins in current studies, it would be highly interesting to introduce plant-derived transporters to microbes and to investigate their potential ability to enhance anthocyanin production.
Separation of enzyme expression and anthocyanin production using a two-step biocatalysis The unstable nature of anthocyanins at neutral pH is a big challenge for their microbial production. Unlike in plants where naturally synthesized anthocyanins are stored and stabilized in vacuoles [1,36], there is a lack of such an organelle in bacterial cells. In addition, the near neutral pH in the intracellular environment in E. coli and other hosts makes the synthesized anthocyanins rather labile. This issue drives the development of a two-step biocatalysis strategy, which proves effective in elevating anthocyanin production [27]. In the first step, cells are grown in medium at pH 7, where enzyme expression and cosubstrate (such as UDP-glucose) generation are accomplished. In the second step, cells with fully expressed enzymes are transferred to fresh medium adjusted to pH 5 with the supplementation of precursors, where anthocyanins can be synthesized and stabilized. Protective agents such as glutamate can be used to prevent cell lysis under a low pH. Such a two-step procedure separates enzyme expression from anthocyanin production, and balances the pH dependence in primary metabolism (cell growth and enzyme expression) and secondary metabolism (anthocyanin production). This strategy could increase the titer of cyanidin 3-O-glucoside by 15-fold (38.9 mg/L) compared to the traditional single-step production (2.5 mg/L) in E. coli [27]. Apart from pH, other process parameters also play important roles in www.sciencedirect.com
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Figure 3
Orotic acid
Transporters
Glucose Anthocyanins Transporters
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The strategies applied in microbial biosynthesis of anthocyanins. The modifications of the anthocyanin-producing strains involve pathway enzymes, the supply of UDP-glucose, and the transport of substrates and anthocyanins. Abbreviations: G-6-P, Glucose-6-phosphate; G-1-P, glucose-1-phosphate; UDP-G, UDP-glucose; galU1, glucose-1-phosphate uridylyltransferase gene, pgm, phosphoglucomutase gene; pyrE, orotate phosphoribosyltransferase; pyrF, orotidine-5’-phosphate decarboxylase, cmk, cytidine monophosphate kinase gene; ndk, nucleoside diphosphate kinase gene; glk, glucokinase gene; glf, glucose facilitator diffusion gene.
anthocyanin production, such as induction time-point, substrate feeding, amount of dissolved oxygen, and temperature, which affect the physiological function of cells and thereby affect the stability of expressed enzymes, the supply of co-substrate, and so on [24,26,28]. These process conditions should also be optimized in anthocyanin production. De novo biosynthesis of anthocyanins
The initial studies on microbial production of anthocyanins relied on the supplementation of flavonoid precursors, such as (+)-catechin, something that can potentially increase the production cost and decrease industrial feasibility. Therefore, using cheap carbon sources such as glucose or glycerol for microbial production is of great industrial importance. Recently, de novo production of anthocyanins has been achieved first by using microbial consortia and later by using recombinant S. cerevisiae. Given that the entire pathway starting from glucose is extremely long, the division of the metabolic pathway into multiple strains can alleviate the metabolic burden and improve the overall metabolic performance. This strategy was showcased by splitting the biosynthetic pathway of pelargonidin 3-O-glucoside starting from tyrosine (containing 15 genes) into four E. coli strains including a high-yield tyrosine-producing strain to achieve de novo production of 10 mg/L pelargonidin 3-O-glucoside from 20 g/L glucose [22]. In another study, using a single S. cerevisiae strain as the host, Levisson et al. introduced 13 genes responsible for the biosynthesis of pelargonidin 3-O-glucoside, deleted the genes for the degradation or competitive utilization of phenylalanine (aro10, pdc5, and pdc6), pelargonidin 3-O-glucoside (exg1, and spr1), and coumaroyl-CoA (tsc13), and enhanced the supply of phenylalanine and tyrosine via alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase www.sciencedirect.com
(Aro3, Aro4) [20]. The combinatorial modifications resulted in the production of 0.01 mmol/gCDW pelargonidin and 0.001 mmol/gCDW pelargonidin 3-O-glucoside from glucose in controlled aerobic batch cultures. A similar study was performed and achieved mg/L level production of anthocyanins (delphinidin-3-O-glucoside, cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside) [37]. Although the production levels are very low, the de novo production demonstrates the possibility of microbial production of anthocyanins from cheap carbon sources, and opens up an avenue to efficient biosystems for anthocyanin production.
Production of unusual anthocyanins by bacterial strains Cyanidin 3-O-glucoside, one of the most commonly found anthocyanins in nature, has potential therapeutic roles in medical applications. To facilitate pharmacokinetic study of this compound, the synthesis of a deuterated version of this compound was recently demonstrated for the first time using recombinant E. coli. Specifically, an E. coli strain co-expressing ANS from Petunia hybrida and F3GT from Arabidopsis was grown in medium with D2O as the solvent and deuterated glycerol D8 as the carbon source. The strain produced deuterated cyanidin 3-O-glucoside when feeding normal catechin. This deuterated compound is more stable than the undeuterated one possibly due to the higher stability of the carbon-deuterium bonds than the carbon-hydrogen bonds [38]. Another unusual form of anthocyanins is pyranoanthocyanins, which are mostly found in red wines and are formed by the condensation of anthocyanins and compounds containing a polarizable double bond such as Current Opinion in Biotechnology 2020, 61:135–141
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acetaldehyde, pyruvic acid, hydroxycinnamic acids, and vinylphenols. To produce pyranoanthocyanins by microorganisms, an E. coli strain was engineered to produce 4-vinylphenol by expressing TAL and phenolic acid decarboxylase (PDC). Co-culturing this strain with a cyanidin 3-O-glucoside-producing E. coli strain and by optimizing co-culture parameters, including testing the presence of ethanol, generated 19.5 mg/L pyranocyanidin 3-O-glucoside-4-phenol. Additional expression of HpaBC in 4-vinylphenol-producing E. coli resulted in the production of another pyranoanthocyanin, pyranocyanidin 3-O-glucoside-4-catechol, and optimization of the co-culture conditions elevated its titer to 13 mg/L [39]. Pyranoanthocyanin production was also reported in L. lactis when using catechin to synthesize cyanidin 3O-glucoside. Interestingly, pyranoanthocyanins (including methylpyranocyanidin and methylpyranopeonidin) were generated concomitantly, which might be a spontaneous process given the potential reaction between cyanidin/peonidin (methylated cyanidin by unknown flavonoid methyltransferases in L. lactis) and the L. lactis metabolite acetoacetic acid. The same phenomenon was observed when using gallocatechin as the substrate, in which two new pyranoanthocyanins, that is, methylpyranodelphinidin and methylpyranopetunidin, were produced [21].
Conflict of interest statement
Conclusion Production of colorful anthocyanins by engineered microorganisms is a promising way of supplying these compounds for industrial use. With the continuously emerging techniques in metabolic engineering and synthetic biology, the production titers of some anthocyanins have been significantly increased through multidimensional engineering of pathway enzymes, global regulation of cosubstrate supply, transmembrane transportation of accumulated intracellular anthocyanins, and effective stabilization of newly synthesized anthocyanins. However, many problems still remain to be solved to further improve the production and to increase the commercial competitiveness of the microbial production. For example, the kinetics of the recombinant enzymes, their expression profiles, as well as the stability of the produced molecules have not been thoroughly studied and remain largely unknown. In addition, engineering of transporters for efficient secretion of intracellular anthocyanins into the media also lacks in-depth and mechanistic investigation. These points are all worth studying and can be interesting and useful foci of future exploration. Finally, the production of more complex anthocyanins with different colors (including the sought-after blue anthocyanins) is another important target for future metabolic engineering endeavors. Given the big strides on microbial anthocyanin production in the past years, it is highly expected that engineered microorganisms will become very competitive in supplying anthocyanins in the near future. Current Opinion in Biotechnology 2020, 61:135–141
Nothing declared.
Acknowledgements This work was supported by the startup funds from Shaanxi University of Science and Technology awarded to JZ and XW. The financial support from National Natural Science Foundation for Young Scientists of China (31900114) to XW is also acknowledged.
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