Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production

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• 作者：Santiago Comba ; Martín Sabatini ; Simón Menendez-Bravo…
• 关键词：TAG biosynthesis ; Phosphatidate phosphatase ; Escherichia coli ; Oil production ; Triacylglycerol
• 刊名：Biotechnology for Biofuels
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：7
• 期：1
• 全文大小：1,176 KB
• 参考文献：1. Alvarez, HM, Steinbuchel, A (2002) Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 60: pp. 367-376 CrossRef
  2. Comba, S, Arabolaza, A, Gramajo, H (2012) Emerging engineering principles for yield improvement in microbial cell design. Comput Struct Biotechnol J 3: pp. e201210016 CrossRef
  3. Kalscheuer, R, Stoveken, T, Luftmann, H, Malkus, U, Reichelt, R, Steinbuchel, A (2006) Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters. Appl Environ Microbiol 72: pp. 1373-1379 CrossRef
  4. Choi, YJ, Lee, SY (2013) Microbial production of short-chain alkanes. Nature 502: pp. 571-574 CrossRef
  5. Peralta-Yahya, PP, Zhang, F, Cardayre, SB, Keasling, JD (2012) Microbial engineering for the production of advanced biofuels. Nature 488: pp. 320-328 CrossRef
  6. Liang, MH, Jiang, JG (2013) Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology. Prog Lipid Res 52: pp. 395-408 CrossRef
  7. Kurosawa, K, Boccazzi, P, Almeida, NM, Sinskey, AJ (2010) High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J Biotechnol 147: pp. 212-218 CrossRef
  8. Rucker, J, Paul, J, Pfeifer, BA, Lee, K (2013) Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions. Appl Microbiol Biotechnol 97: pp. 2753-2759 CrossRef
  9. Janssen, HJ, Steinbuchel, A (2014) Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels 7: pp. 7 CrossRef
  10. Lin, F, Chen, Y, Levine, R, Lee, K, Yuan, Y, Lin, XN (2013) Improving fatty acid availability for bio-hydrocarbon production in Escherichia coli by metabolic engineering. PLoS One 8: pp. e78595 CrossRef
  11. Janssen, HJ, Steinbuchel, A (2014) Production of triacylglycerols in Escherichia coli by deletion of the diacylglycerol kinase gene and heterologous overexpression of atfA from Acinetobacter baylyi ADP1. Appl Microbiol Biotechnol 98: pp. 1913-1924 CrossRef
  12. Comba, S, Menendez-Bravo, S, Arabolaza, A, Gramajo, H (2013) Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor. Microb Cell Fact 12: pp. 9 CrossRef
  13. Raetz, CR, Newman, KF (1978) Neutral lipid accumulation in the membranes of Escherichia coli mutants lacking diglyceride kinase. J Biol Chem 253: pp. 3882-3887
  14. Raetz, CR, Newman, KF (1979) Diglyceride kinase mutants of Escherichia coli: inner membrane association of 1,2-diglyceride and its relation to synthesis of membrane-derived oligosaccharides. J Bacteriol 137: pp. 860-868
  15. Arabolaza, A, Rodriguez, E, Altabe, S, Alvarez, H, Gramajo, H (2008) Multiple pathways for triacylglycerol biosynthesis in Streptomyces coelicolor. Appl Environ Microbiol 74: pp. 2573-2582 CrossRef
  16. Rotering, H, Raetz, CR (1983) Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase. J Biol Chem 258: pp. 8068-8073
  17. Dillon, DA, Wu, WI, Riedel, B, Wissing, JB, Dowhan, W, Carman, GM (1996) The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity. J Biol Chem 271: pp. 30548-305
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Biotechnology
  Plant Breeding/Biotechnology
  Renewable and Green Energy
  Environmental Engineering/Biotechnology
  
• 出版者：BioMed Central
• ISSN：1754-6834

文摘

Background Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations. Results The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lppβ genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L-1 at the end of the process. Conclusions This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.