Metabolic engineering of microbes for branched-chain biodiesel production with low-temperature property

Biotechnology for Biofuels

Hui Tao⁴  Daoyi Guo³  Yuchen Zhang⁴  Zixin Deng¹  Tiangang Liu² 

[1] State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China

[2] Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan 430068, China

[3] Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China

[4] Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, China

关键词: Pichia pastoris;    E. coli;    WS/DGAT;    Branched-chain amino acid biosynthesis;    Metabolic engineering;    Branched-chain esters;    Biodiesel;   

Others  :  1219149
DOI  :  10.1186/s13068-015-0270-7

 received in 2015-03-17, accepted in 2015-06-05,  发布年份 2015

【 摘 要 】

Background

The steadily increasing demand for diesel fuels calls for renewable energy sources. This has attracted a growing amount of research to develop advanced, alternative biodiesel worldwide. Several major disadvantages of current biodiesels are the undesirable physical properties such as high viscosity and poor low-temperature operability. Therefore, there is an urgent need to develop novel and advanced biodiesels.

Results

Inspired by the proven capability of wax ester synthase/acyl-coenzyme A, diacylglycerol acyltransferase (WS/DGAT) to generate fatty acid esters, de novo biosynthesis of fatty acid branched-chain esters (FABCEs) and branched fatty acid branched-chain esters (BFABCEs) was performed in engineered Escherichia coli through combination of the (branched) fatty acid biosynthetic pathway and the branched-chain amino acid biosynthetic pathway. Furthermore, by modifying the fatty acid pathway, we improved FABCE production to 273 mg/L and achieved a high proportion of FABCEs at 99.3 % of total fatty acid esters. In order to investigate the universality of this strategy, Pichia pastoris yeast was engineered and produced desirable levels of FABCEs for the first time with a good starting point of 169 mg/L.

Conclusions

We propose new pathways of fatty acid ester biosynthesis and establish proof of concept through metabolic engineering of E. coli and P. pastoris yeast. We were able to produce advanced biodiesels with high proportions FABCEs and BFABCEs. Furthermore, this new strategy promises to achieve advanced biodiesels with beneficial low-temperature properties.

【 授权许可】

   
2015 Tao et al.

【 图 表 】

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【 参考文献 】

• [1]Hill J, Nelson E, Tilman D, Polasky S, Tiffany D: Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci U S A 2006, 103:11206-10.
• [2]Atsumi S, Liao JC: Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol 2008, 19:414-9.
• [3]Peralta-Yahya PP, Zhang F, Del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuels. Nature 2012, 488:320-8.
• [4]Dunn RO, Knothe G: Alternative diesel fuels from vegetable oils and animal fats. J Oleo Sci 2001, 50:415-26.
• [5]Knothe G, Matheaus AC, Ryan TW III: Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester. Fuel 2003, 82:971-5.
• [6]Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, et al.: Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010, 463:559-62.
• [7]Janßen HJ, Steinbüchel A: Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels 2014, 7:7. BioMed Central Full Text
• [8]Zhang F, Rodriguez S, Keasling JD: Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 2011, 22:775-83.
• [9]De Jong B, Siewers V, Nielsen J: Systems biology of yeast: enabling technology for development of cell factories for production of advanced biofuels. Curr Opin Biotechnol 2012, 23:624-30.
• [10]Nielsen J, Larsson C, van Maris A, Pronk J: Metabolic engineering of yeast for production of fuels and chemicals. Curr Opin Biotechnol 2013, 24:398-404.
• [11]Liu Q, Wu K, Cheng Y, Lu L, Xiao E, Zhang Y, et al.: Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction. Metab Eng 2015, 28:82-90.
• [12]Li X, Guo D, Cheng Y, Zhu F, Deng Z, Liu T: Overproduction of fatty acids in engineered Saccharomyces cerevisiae. Biotechnol Bioeng 2014, 111:1841-52.
• [13]Lu X, Vora H, Khosla C: Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 2008, 10:333-9.
• [14]Liu T, Vora H, Khosla C: Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 2010, 12:378-86.
• [15]Ruffing AM, Jones HD: Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942. Biotechnol Bioeng 2012, 109:2190-9.
• [16]Handke P, Lynch SA, Gill RT: Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals. Metab Eng 2011, 13:28-37.
• [17]Yu X, Liu T, Zhu F, Khosla C: In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli. Proc Natl Acad Sci 2011, 108:18643-8.
• [18]Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R: Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 2011, 476:355-9.
• [19]Xu P, Gu Q, Wang W, Wong L, Bower AG, Collins CH, et al.: Modular optimization of multi-gene pathways for fatty acids production in E coli. Nat Commun 2013, 4:1409.
• [20]Kalscheuer R, Uthoff S, Luftmann H, Steinbüchel A: In vitro and in vivo biosynthesis of wax diesters by an unspecific bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase from acinetobacter Calcoaceticus ADP1. Eur J Lipid Sci Technol 2003, 105:578-84.
• [21]Stöveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbüchel A: The wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 2005, 187:1369-76.
• [22]Kalscheuer R, Stolting T, Steinbüchel A: Microdiesel: Escherichia coli engineered for fuel production. Microbiology 2006, 152:2529-36.
• [23]Duan Y, Zhu Z, Cai K, Tan X, Lu X: De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation. PLoS ONE 2011., 6Article ID e20265
• [24]Zhang F, Carothers JM, Keasling JD: Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 2012, 30:354-9.
• [25]Thompson RA, Trinh CT: Enhancing fatty acid ethyl ester production in Saccharomyces cerevisiae through metabolic engineering and medium optimization. Biotechnol Bioeng 2014, 111:2200-8.
• [26]Shi S, Valle-Rodriguez JO, Khoomrung S, Siewers V, Nielsen J: Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production. Biotechnol Biofuels 2012, 5:7. BioMed Central Full Text
• [27]Shi S, Valle-Rodriguez JO, Siewers V, Nielsen J: Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters. Biotechnol Bioeng 2014, 111:1740-7.
• [28]Rodriguez GM, Tashiro Y, Atsumi S: Expanding ester biosynthesis in Escherichia coli. Nat Chem Biol 2014, 10:259-65.
• [29]Layton DS, Trinh CT: Engineering modular ester fermentative pathways in Escherichia coli. Metab Eng 2014, 26C:77-88.
• [30]Tai YS, Xiong M, Zhang K: Engineered biosynthesis of medium-chain esters in Escherichia coli. Metab Eng 2015, 27:20-8.
• [31]Guo D, Zhu J, Deng Z, Liu T: Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways. Metab Eng 2014, 22:69-75.
• [32]Lee I, Johnson LA, Hammond EG: Use of branched-chain esters to reduce the crystallization temperature of biodiesel. J Am Oil Chem Soc 1995, 72:1155-60.
• [33]Knothe G: Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol 2005, 86:1059-70.
• [34]Knothe G: Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ Sci 2009, 2:759.
• [35]Dunn R, Bagby M: Low-temperature properties of triglyceride-based diesel fuels: transesterified methyl esters and petroleum middle distillate/ester blends. J Am Oil Chem Soc 1995, 72:895-904.
• [36]Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008, 451:86-9.
• [37]Connor MR, Liao JC: Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 2008, 74:5769-75.
• [38]Cann AF, Liao JC: Production of 2-methyl-1-butanol in engineered Escherichia coli. Appl Microbiol Biotechnol 2008, 81:89-98.
• [39]Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC: Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 2010, 85:651-7.
• [40]Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, et al.: Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc Natl Acad Sci 2013, 110:7636-41.
• [41]Haushalter RW, Kim W, Chavkin TA, The L, Garber ME, Nhan M, et al.: Production of anteiso-branched fatty acids in Escherichia coli; next generation biofuels with improved cold-flow properties. Metab Eng 2014, 26C:111-8.
• [42]Jiang W, Jiang Y, Bentley GJ, Liu D, Xiao Y, Zhang F: Enhanced production of branched‐chain fatty acids by replacing β‐ketoacyl‐(acyl‐carrier‐protein) synthase III (FabH). Biotechnol Bioeng 2015.
• [43]Cho H, Cronan JE: Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 1995, 270:4216-9.
• [44]Zhang X, Li M, Agrawal A, San KY: Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases. Metab Eng 2011, 13:713-22.
• [45]Zheng Y, Li L, Liu Q, Qin W, Yang J, Cao Y, et al.: Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase. Biotechnol Biofuels 2012, 5:76-88. BioMed Central Full Text
• [46]Choi K-H, Heath RJ, Rock CO: β-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 2000, 182:365-70.
• [47]Kaneda T: Fatty acids of the genus Bacillus: an example of branched-chain preference. Bacteriol Rev 1977, 41:391.
• [48]Voelker TA, Davies HM: Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase. J Bacteriol 1994, 176:7320-7.
• [49]Runguphan W, Keasling JD: Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 2014, 21:103-13.
• [50]Valle-Rodríguez JO, Shi S, Siewers V, Nielsen J: Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid ethyl esters, an advanced biofuel, by eliminating non-essential fatty acid utilization pathways. Appl Energy 2014, 115:226-32.
• [51]Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM: Heterologous protein production using the Pichia pastoris expression system. Yeast 2005, 22:249-70.
• [52]Yan J, Zheng X, Du L, Li S: Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts. Biotechnol Biofuels 2014, 7:55. BioMed Central Full Text
• [53]Foglia TA, Nelson LA, Dunn RO, Marmer WN: Low-temperature properties of alkyl esters of tallow and grease. J Am Oil Chem Soc 1997, 74:951-5.
• [54]Zhang Y, Van Gerpen JH. Combustion analysis of esters of soybean oil in a diesel engine: SAE Technical Paper 1996. doi:10.4271/960765
• [55]Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD: Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 2009, 5:593-9.
• [56]Stöveken T, Steinbüchel A: Bacterial acyltransferases as an alternative for lipase-catalyzed acylation for the production of oleochemicals and fuels. Angew Chem Int Ed 2008, 47:3688-94.
• [57]Wältermann M, Stöveken T, Steinbüchel A: Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases. Biochimie 2007, 89:230-42.
• [58]Barney BM, Mann RL, Ohlert JM: Identification of a residue affecting fatty alcohol selectivity in wax ester synthase. Appl Environ Microbiol 2013, 79:396-9.
• [59]Li Y, Zhao Z, Bai F: High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb Technol 2007, 41:312-7.
• [60]Moser BR: Biodiesel production, properties, and feedstocks. Vitro Cell Dev Biol Plant 2009, 45:229-66.