Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids

Layton, Donovan S.; Trinh, Cong T.

doi:10.1002/bit.25947

Cited by 58 publications

(84 citation statements)

References 42 publications

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“…The vaat gene from Fragaria vesca , the saat gene from F. ananassa and the atf1 gene from S. cerevisiae were amplified from plasmids pDL006, pDL001 and pDL004, respectively 3,22 . The atf1 ‘(the codon optimized atf1 gene), eht1 from S. cerevisiae 20 , and lipaseB from Candida antarctica 45 were all synthesized by GenScript (Piscataway, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, the production of high-value bioproducts which can be easily recovered from fermentation might be a solution to tackle the bottleneck in bioproduction. Fatty acid esters, or mono-alkyl esters, can be used as valuable fuels such as diesel components or specialty chemicals for food flavoring, cosmetic and pharmaceutical industries 3 . It is projected that the US market demand for fatty acid esters could reach $4.99 billion by 2025 4 .…”

Section: Mainmentioning

confidence: 99%

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Renewable Fatty Acid Ester Production in Clostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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44Endproduct toxicity is a key bottleneck for biofuel/biochemical production. Biosynthesis of 45 high-value derivatives that can easily separate would alleviate such toxicity and thus enhance 46 bioproduction efficiency. As a proof of principle, biosynthesis (along with in-line extractive 47 recovery) of fatty acid esters using clostridia was investigated in this study. We hypothesize 48 that solventogenic clostridia are excellent platforms for ester production, because they co-49 produce acyl-CoAs (acetyl-CoA and butyryl-CoA), acids (acetate and butyrate), and alcohols 50 (ethanol and butanol). Through rational screening for host strains and genes (encoding alcohol 51 acyl transferases and lipase), systematic metabolic engineering, and elimination of putative 52 prophages, we obtained strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl 53 butyrate respectively, which were both historically highest levels in microbial hosts. Our 54 principle for engineering microorganisms to produce high-value and easy-recoverable 55 endproducts is highly applicable to other bioprocesses, and could lead to breakthroughs in 56 biofuel/biochemical production and general bioeconomy. 57 58 59 60 61 Keywords: clostridia, biofuels and biochemicals, fatty acid ester, butyl acetate, butyl butyrate, 62 ethyl acetate, CRISPR-Cas9, hydrolysates, prophages 63 64 4 Main 65 Although tremendous efforts have been invested for biofuel and biochemical research, it 66is still challenging to generate robust strains that can produce target products at desirable 67 levels 1 . One key bottleneck is the intrinsic toxicity of endproducts to host cells 2 . Therefore, the 68 production of high-value bioproducts which can be easily recovered from fermentation might 69 be a solution to tackle the bottleneck in bioproduction. Fatty acid esters, or mono-alkyl esters, 70 can be used as valuable fuels such as diesel components or specialty chemicals for food 71 flavoring, cosmetic and pharmaceutical industries 3 . It is projected that the US market demand 72 for fatty acid esters could reach $4.99 billion by 2025 4 . In addition, esters, with fatty acid and 73 alcohol moieties, are generally hydrophobic and can easily separate from fermentation; thus 74 the production of ester can help mitigate endproduct toxicity to host cells and efficient 75 bioproduction can be achieved. 76 Conventionally, esters are produced through Fischer esterification which involves high 77 temperature and inorganic catalysts 5,6 . The reaction consumes a large amount of energy and 78 generates tremendous wastes, and thus is not environmentally friendly 5 . On the other hand, 79 ester production through biological routes is becoming more and more attractive because it is 80 renewable and environmentally benign. There are two primary biological pathways for ester 81 production: one is through esterification of fatty acid and alcohol catalyzed by lipases 7 , and the 82 other is based on condensation of acyl-CoA and alcohol catalyzed by alcohol acyl transfer...

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Section: Methodsmentioning

confidence: 99%

Section: Mainmentioning

confidence: 99%

Renewable Fatty Acid Ester Production in Clostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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“…[1,[8][9][10] This modular cell design approach, known as ModCell, uses multi-objective optimization to account for the competing cellular objectives when cellular metabolism is (re)designed in a modular fashion to produce a diverse class of target chemicals. ModCell has been experimentally demonstrated for biosynthesis of alcohols [8,11,12] and esters [13][14][15][16][17] in Escherichia coli.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Multi-objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

Garcia

Trinh

2019

Preprint

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A large space of chemicals with broad industrial and consumer applications could be synthesized by engineered microbial biocatalysts. However, the current strain optimization process is prohibitively laborious and costly to produce one target chemical and often requires new engineering efforts to produce new molecules. To tackle this challenge, modular cell design based on a chassis strain that can be combined with different product synthesis pathway modules has been recently proposed. This approach seeks to minimize unexpected failure and avoid task repetition, leading to a more robust and faster strain engineering process. The modular cell design problem was mathematically formulated using a multi-objective optimization framework.[1] In this study, we evaluated a library of the state-of-the-art multi-objective evolutionary algorithms (MOEAs) to identify the most effective method to solve the modular cell design problem. Using the best MOEA, we found better solutions for modular cells compatible with many product synthesis modules. Furthermore, the best performing algorithm could provide better and more diverse design options that might help increase the likelihood of successful experimental implementation. We identified key parameter configurations to overcome the difficulty associated with multi-objective optimization problems with many competing design objectives. Interestingly, we found that MOEA performance with a real application problem, e.g., the modular strain design problem, does not always correlate with artificial benchmarks. Overall, MOEAs provide powerful tools to solve the modular cell design problem for novel biocatalysis.

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“…For example, an Escherichia coli, engineered to use this biosynthetic pathway, could achieve high titer of isobutyl acetate [6, 7]. With appropriate expression of AATs and availability of alcohol and acyl-CoA moieties, various types of esters can be produced [2, 4]. Due to high volatility of esters, ester production at elevated temperature can benefit downstream product separation and hence reduce the process cost.…”

Section: Introductionmentioning

confidence: 99%

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

Seo

Lee

Garcia

et al. 2019

Preprint

Self Cite

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16Background. Esters are versatile chemicals and potential drop-in biofuels. To develop a 17 sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases 18 (AATs) has been studied for decades. Volatility of esters endows thermophilic production with 19 advantageous downstream product separation. However, due to the limited thermal stability of 20 AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, 21 developing thermostable AATs is important for thermophilic ester production directly from 22 lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., 23Clostridium thermocellum. 24Results. In this study, we engineered a thermostable chloramphenicol acetyltransferase from 25Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperature. 26We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly 27 conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that 28 F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, 29 we demonstrated the engineered C. thermocellum could produce isobutyl acetate directly from 30 cellulose. 31Conclusions. This study highlights that CAT is a potential thermostable AAT that can be 32 harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer 33 bioesters directly from lignocellulosic biomass. 34 35 Keywords: Alcohol acetyltransferase; thermostability; chloramphenicol acetyltransferase; 36 isobutyl acetate; esters; consolidated bioprocessing; Clostridium thermocellum. 37 38 39Esters are versatile chemicals which have been used as lubricants, solvents, food additives, 40 fragrances and potential drop-in fuels [1]. Currently, ester production largely relies on synthesis 41 from petroleum or extraction from plants, which makes it neither sustainable nor economically 42 feasible. Therefore, microbial production of esters has been studied for decades [2][3][4][5][6][7]. Most studies 43 have employed an alcohol acetyltransferase (E.C. 2.3.1.84, AAT), belonging to a broad 44 acetyltransferase class, that can synthesize a carboxylic ester by condensing an alcohol and an 45 acyl-CoA in a thermodynamically favorable aqueous environment [5]. For example, an 46Escherichia coli, engineered to use this biosynthetic pathway, could achieve high titer of isobutyl 47 acetate [6, 7]. With appropriate expression of AATs and availability of alcohol and acyl-CoA 48 moieties, various types of esters can be produced [2, 4]. Due to high volatility of esters, ester 49 production at elevated temperature can benefit downstream product separation and hence reduce 50 the process cost. Interestingly, it has recently been shown that for the same total carbon chain 51 length, short-chain esters are less toxic to microbial health than alcohols, which is potentially 52 beneficial for ester fermentation [8]. However, most of the AATs known to date a...

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Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids

Cited by 58 publications

References 42 publications

Renewable Fatty Acid Ester Production in Clostridium

Renewable Fatty Acid Ester Production in Clostridium

Comparison of Multi-objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

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