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Fatty Acid Production by Enhanced Malonyl-CoA Supply in Escherichia coli

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Abstract

The expression of exogenous genes encoding acetyl-CoA carboxylase (Acc) and pantothenate kinase (CoaA) in Escherichia coli enable highly effective fatty acid production. Acc-only strains grown at 37 °C or 23 °C produced an approximately twofold increase in fatty acid content, and additional expression of CoaA achieved a further twofold accumulation. In the presence of pantothenate, which is the starting material for the CoA biosynthetic pathway, the size of the intracellular CoA pool at 23 °C was comparable to that at 30 °C during cultivation, and more than 500 mg/L of culture containing cellular fatty acids was produced, even at 23 °C. However, the highest yield of cellular fatty acids (1100 mg/L of culture) was produced in cells possessing the gene encoding type I bacterial fatty acid synthase (FasA) along with the acc and coaA, when the transformant was cultivated at 30 °C in M9 minimal salt medium without pantothenate or IPTG. This E. coli transformant contained 141 mg/L of oleic acid attributed to FasA under noninducible conditions. The increased fatty acid content was brought about by a greatly improved specific productivity of 289 mg/g of dry cell weight. Thus, the effectiveness of the foreign acc and coaA in fatty acid production was unambiguously confirmed at culture temperatures of 23 °C to 37 °C. Cofactor engineering in E. coli using the exogenous coaA and acc genes resulted in fatty acid production over 1 g/L of culture and could effectively function at 23 °C.

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References

  1. Milke L, Marienhagen J (2020) Engineering intracellular malonyl-CoA availability in microbial hosts and its impact on polyketide and fatty acid synthesis. Appl Microbiol Biotechnol 104:6057–6065. https://doi.org/10.1007/s00253-020-10643-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Cronan JE Jr, Waldrop GL (2002) Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 41:407–435. https://doi.org/10.1016/s0163-7827(02)00007-3

    Article  CAS  PubMed  Google Scholar 

  3. Rathnasingh C, Raj SM, Lee Y, Catherine C, Ashok S, Park S (2012) Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains. J Biotechnol 157:633–640. https://doi.org/10.1016/j.jbiotec.2011.06.008

    Article  CAS  PubMed  Google Scholar 

  4. Cheng Z, Jiang J, Wu H, Li Z, Ye Q (2016) Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli. BioresourTechnol 200:897–904. https://doi.org/10.1016/j.biortech.2015.10.107

    Article  CAS  Google Scholar 

  5. Maharjan S, Park JW, Yoon YJ, Lee HC, Jae Kyung Sohng JK (2010) Metabolic engineering of Streptomyces venezuelae for malonyl-CoA biosynthesis to enhance heterologous production of polyketides. Biotechnol Lett 32:277–282. https://doi.org/10.1007/s10529-009-0152-9

    Article  CAS  PubMed  Google Scholar 

  6. Zha W, Rubin-Pitel SB, Shao Z, Zhao H (2009) Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering. Metab Eng 11:192–198. https://doi.org/10.1016/j.ymben.2009.01.005

    Article  CAS  PubMed  Google Scholar 

  7. Wattanachaisaereekul S, Lantz AE, Nielsen ML, Nielsen J (2008) Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply. Metab Eng 10:246–254. https://doi.org/10.1016/j.ymben.2008.04.005

    Article  CAS  PubMed  Google Scholar 

  8. Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S (2005) Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl Microbiol Biotechnol 68:498–504. https://doi.org/10.1007/s00253-005-1916-3

    Article  CAS  PubMed  Google Scholar 

  9. Miyahisa I, Funa N, Ohnishi Y, Martens S, Moriguchi T, Horinouchi S (2006) Combinatorial biosynthesis of flavones and flavonols in Escherichia coli. Appl Microbiol Biotechnol 71:53–58. https://doi.org/10.1007/s00253-005-0116-5

    Article  CAS  PubMed  Google Scholar 

  10. Leonard E, Lim K-H, Saw P-N, Koffas MA (2007) Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 73:3877–3886. https://doi.org/10.1128/AEM.00200-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Leonard E, Yan Y, Fowler ZL, Li Z, Lim C-G, Lim K-H, Koffas MA (2008) Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol Pharm 5:257–265. https://doi.org/10.1021/mp7001472

    Article  CAS  PubMed  Google Scholar 

  12. Kim BG, Lee H, Ahn J-H (2014) Biosynthesis of pinocembrin from glucose using engineered Escherichia coli. J Microbiol Biotechnol 24:1536–1541. https://doi.org/10.4014/jmb.1406.06011

    Article  CAS  PubMed  Google Scholar 

  13. Davis MS, Solbiati J, Cronan JE Jr (2000) Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem 275:28593–28598. https://doi.org/10.1074/jbc.M004756200

    Article  CAS  PubMed  Google Scholar 

  14. Lu X, Vora H, Khosla C (2008) Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 10:333–339. https://doi.org/10.1016/j.ymben.2008.08.006

    Article  CAS  PubMed  Google Scholar 

  15. Ruenwai R, Cheevadhanarak S, Laoteng K (2009) Overexpression of acetyl-CoA carboxylase gene of Mucor rouxii enhanced fatty acid content in Hansenula polymorpha. Mol Biotechnol 42:327–332. https://doi.org/10.1007/s12033-009-9155-y

    Article  CAS  PubMed  Google Scholar 

  16. Jeon E, Lee S, Won JI, Han SO, Kim J, Lee J (2011) Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism. Enzyme Microb Technol 49:44–51. https://doi.org/10.1016/j.enzmictec.2011.04.001

    Article  CAS  PubMed  Google Scholar 

  17. Meng X, Yang J, Cao Y, Li L, Jiang X, Xu X, Liu W, Xian M, Zhang Y (2011) Increasing fatty acid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms. J Ind Microbiol Biotechnol 38:919–925. https://doi.org/10.1007/s10295-010-0861-z

    Article  CAS  PubMed  Google Scholar 

  18. Xu P, Gu Q, Wang W, Wong L, Bower AG, Collins CH, Koffas MA (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 4:1409. https://doi.org/10.1038/ncomms2425

    Article  CAS  PubMed  Google Scholar 

  19. Cao Y, Liu W, Xu X, Zhang H, Wang J, Xian M (2014) Production of free monounsaturated fatty acids by metabolically engineered Escherichia coli. Biotechnol Biofuels 7:59. https://doi.org/10.1186/1754-6834-7-59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Shin KS, Lee SK (2017) Introduction of an acetyl-CoA carboxylation bypass into Escherichia coli for enhanced free fatty acid production. Bioresour Technol 245:1627–1633. https://doi.org/10.1016/j.biortech.2017.05.169

    Article  CAS  PubMed  Google Scholar 

  21. Chen D, Yuan X, Liang L, Liu K, Ye H, Liu Z, Liu Y, Huang L, He W, Chen Y, Zhang Y, Xue T (2019) Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii. Biotechnol Lett 41:1133–1145. https://doi.org/10.1007/s10529-019-02715-0

    Article  CAS  PubMed  Google Scholar 

  22. Xu P, Ranganathan S, Fowler ZL, Maranas CD, Koffas MAG (2011) Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng 13:578–587. https://doi.org/10.1016/j.ymben.2011.06.008

    Article  CAS  PubMed  Google Scholar 

  23. Janßen HJ, Steinbüchel A (2014) Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels 7:7. https://doi.org/10.1186/1754-6834-7-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kurosawa K, Boccazzi P, de Almeida NM, Sinskey AJ (2010) High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J Biotechnol 147:212–218. https://doi.org/10.1016/j.jbiotec.2010.04.003

    Article  CAS  PubMed  Google Scholar 

  25. Gande R, Dover LG, Krumbach K, Besra GS, Sahm H, Oikawa T, Eggeling L (2007) The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis. J Bacteriol 189:5257–5264. https://doi.org/10.1128/JB.00254-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Satoh S, Ozaki M, Matsumoto S, Nabatame T, Kaku M, Shudo T, Asayama M, Chohnan S (2020) Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli. Biotechnol Lett 42:2595–2605. https://doi.org/10.1007/s10529-020-02996-w

    Article  CAS  PubMed  Google Scholar 

  27. Vallari DS, Jackowski S, Rock CO (1987) Regulation of pantothenate kinase by coenzyme A and its thioesters. J Biol Chem 262:2468–2471. https://doi.org/10.1016/S0021-9258(18)61527-3

    Article  CAS  PubMed  Google Scholar 

  28. Brand LA, Strauss E (2005) Characterization of a new pantothenate kinase isoform from Hricobacter pylori. J Biol Chem 280:20185–20188. https://doi.org/10.1074/jbc.C500044200

    Article  CAS  PubMed  Google Scholar 

  29. Hong BS, Yun MK, Zhang Y-M, Chohnan S, Rock CO, White SW, Jackowski S, Park H-W, Leonardi R (2006) Prokaryotic type II and type III pantothenate kinases: the same monomer fold creates dimers with distinct catalytic properties. Structure 14:1251–1261. https://doi.org/10.1016/j.str.2006.06.008

    Article  CAS  PubMed  Google Scholar 

  30. Ogata Y, Chohnan S (2015) Prokaryotic typeIII pantothenate kinase enhances coenzyme A biosynthesis in Escherichi coli. J Gen Appl Microbiol 61:266–269. https://doi.org/10.2323/jgam.61.266

    Article  CAS  PubMed  Google Scholar 

  31. Schweizer E, Hofmann J (2004) Microbial type I fatty acid synthase (FAS): major players in a network of cellular FAS systems. Microbiol Mol Biol Rev 68:501–517. https://doi.org/10.1128/MMBR.68.3.501-517.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ariga N, Maruyama K, Kawaguchi A (1984) Comparative studies of fatty acid synthases of corynebacteria. J Gen Appl Microbiol 30:87–95. https://doi.org/10.2323/jgam.30.87

    Article  CAS  Google Scholar 

  33. Stuible H-P, Wagner C, Andreou I, Hunter G, Haselmann J, Schweizer E (1996) Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes. J Bacteriol 178:4787–4793. https://doi.org/10.1128/jb.178.16.4787-4793.1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Radmacher E, Alderwick LJ, Besra GS, Brown AK, Gibson KJC, Sahm H, Eggeling L (2005) Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum. Microbiology 151:2421–2427. https://doi.org/10.1099/mic.0.28012-0

    Article  CAS  PubMed  Google Scholar 

  35. Haushalter RW, Groff D, Deutsch S, The L, Chavkin TA, Brunner SF, Katz L, Keasling JD (2015) Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production. Metab Eng 30:1–6. https://doi.org/10.1016/j.ymben.2015.04.003

    Article  CAS  PubMed  Google Scholar 

  36. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. https://doi.org/10.1139/o59-099

    Article  CAS  PubMed  Google Scholar 

  37. Takamura Y, Nomura G (1988) Changes in the intracellular concenrration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12. J Gen Microbiol 134:2249–2253. https://doi.org/10.1099/00221287-134-8-2249

    Article  CAS  PubMed  Google Scholar 

  38. Takamura Y, Kitayama Y, Arakawa A, Yamanaka S, Tosaki M, Ogawa Y (1985) Malonyl-CoA: acetyl-CoA cycling. A new micromethod for determination of acyl-CoAs with malonate decarboxylase. Biochim Biophys Acta 834:1–7. https://doi.org/10.1016/0005-2760(85)90170-5

    Article  CAS  PubMed  Google Scholar 

  39. Chohnan S, Takamura Y (1991) A simple micromethod for measurement of CoASH and its use in measuring intracellular levels of CoASH and short chain acyl-CoAs in Escherichia coli K12 cells. Agric Biol Chem 55:87–94. https://doi.org/10.1271/bbb1961.55.87

    Article  CAS  Google Scholar 

  40. R Core Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/.

  41. Amiri-Jami M, Griffiths MW (2010) Recombinant production of omega-3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1. J Appl Microbiol 109:1897–1905. https://doi.org/10.1111/j.1365-2672.2010.04817.x

    Article  CAS  PubMed  Google Scholar 

  42. Amiri-Jami M, Abdelhamid AG, Hazaa M, Kakuda Y, Griffiths MW (2015) Recombinant production of omega-3 fatty acids by probiotic Escherichia coli Nissle 1917. FEMS Microbiol Lett 362:fnv166. https://doi.org/10.1093/femsle/fnv166

    Article  CAS  PubMed  Google Scholar 

  43. Peng YF, Chen WC, Xiao K, Xu L, Wang L, Wan X (2016) DHA production in Escherichia coli by expressing reconstituted key genes of polyketide synthase pathway from marine bacteria. PLoS ONE 11:e0162861. https://doi.org/10.1371/journal.pone.0162861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Marr AG, Ingraham JL (1962) Effect of temperature on the composition of fatty acids in Escherichia coli. J Bacteriol 84:1260–1267. https://doi.org/10.1128/jb.84.6.1260-1267.1962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sullivan KH, Hegeman GD, Cordes EH (1979) Alteration of the fatty acid composition of Escherichia coli by growth in the presence of normal alcohols. J Bacteriol 138:133–138. https://doi.org/10.1128/jb.138.1.133-138.1979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. de Mendoza D, Garwin JL, Croban JE Jr (1982) Overproduction of cis-vaccenic acid and altered temperature control of fatty acid synthesis in a mutant of Escherichia coli. J Bacterial 151:1608–1611. https://doi.org/10.1128/jb.151.3.1608-1611.1982

    Article  Google Scholar 

  47. Jackowski S, Rock CO (1981) Regulation of coenzyme A biosynthesis. J Bacteriol 148:926–932. https://doi.org/10.1128/jb.148.3.926-932.1981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jackowski S, Rock CO (1984) Metabolism of 4’-phosphopantetheine in Escherichia coli. J Bacteriol 158:115–120. https://doi.org/10.1128/jb.158.1.115-120.1984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Liu T, Vora H, Khosla C (2010) Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 12:378–386. https://doi.org/10.1016/j.ymben.2010.02.003

    Article  CAS  PubMed  Google Scholar 

  50. Xu L, Wang L, Zhou XR, Chen WC, Singh S, Hu Z, Huang FH, Wan X (2018) Stepwise metabolic engineering of Escherichia coli to produce triacylglycerol rich in medium-chain fatty acids. Biotech Biofuels 11:177. https://doi.org/10.1186/s13068-018-1177-x

    Article  CAS  Google Scholar 

  51. Zhang F, Ouellet M, Batth TS, Adams PD, Petzold CJ, Mukhopadhyay A, Keasling JD (2012) Enhancing fatty acid production by the expression of the regulatory transcription factor FadR. Metab Eng 14:653–660. https://doi.org/10.1016/j.ymben.2012.08.009

    Article  CAS  PubMed  Google Scholar 

  52. Okuyama H, Orikasa Y, Nishida T, Watanabe K, Morita N (2007) Bacterial genes responsible for the biosynthesis of eicosapentaenoic acid and docosahexaenoic acids and their heterologous expression. Appl Environ Microbiol 73:665–670. https://doi.org/10.1128/AEM.02270-06

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Enago (www.enago.jp) for the English language review.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (C) JP19K05761 from The Japan Society for the Promotion of Science.

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  1. Moena Kaku and Mei Ishidaira contributed equally to this study.

Authors and Affiliations

  1. Department of Food and Life Sciences, Ibaraki University College of Agriculture, 3-21-1 Chuo, Ami, Ibaraki, 300-0393, Japan

    Moena Kaku, Mei Ishidaira, Shusaku Satoh, Miho Ozaki, Daisuke Kohari & Shigeru Chohnan

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  1. Moena Kaku
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MK performed the experimental work, data collection, and analysis. MI was a major contributor to the experimental work. SS and MO performed the experimental work. DK performed the statistical analysis. SC designed the study and wrote the manuscript.

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Correspondence to Shigeru Chohnan.

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Kaku, M., Ishidaira, M., Satoh, S. et al. Fatty Acid Production by Enhanced Malonyl-CoA Supply in Escherichia coli. Curr Microbiol 79, 269 (2022). https://doi.org/10.1007/s00284-022-02969-4

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