Journal of Microbiology and Biotechnology

  • 제31권1호
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  • Pages.154-162
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  • 2021
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Engineering of Biosynthesis Pathway and NADPH Supply for Improved L-5-Methyltetrahydrofolate Production by Lactococcus lactis

  • Lu, Chuanchuan (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Liu, Yanfeng (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Li, Jianghua (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Liu, Long (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University) ;
  • Du, Guocheng (Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University)
  • 투고 : 2019.11.04
  • 심사 : 2019.12.17
  • 발행 : 2021.01.28
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초록

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 ㎍/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 ㎍/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 ㎍/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 ㎍/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 ㎍/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

키워드

참고문헌

  1. Arcot J, Shrestha A. 2005. Folate: methods of analysis. Trends Food Sci. Technol. 16: 253-266. https://doi.org/10.1016/j.tifs.2005.03.013
  2. Asrar FM, O'Connor DL. 2005. Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets. J. Nutr. Biochem. 16: 587-593. https://doi.org/10.1016/j.jnutbio.2005.02.006
  3. Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. 1997. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am. Clin. Nutr. 65: 1790-1795. https://doi.org/10.1093/ajcn/65.6.1790
  4. Bailey LB, Gregory III JF. 1999. Folate metabolism and requirements. J. Nutr. 129: 779-782. https://doi.org/10.1093/jn/129.4.779
  5. Kim Y-I. 2000. Methylenetetrahydrofolate reductase polymorphisms, folate, and cancer risk: a paradigm of gene-nutrient interactions in carcinogenesis. Nutr. Rev. 58: 205-209. https://doi.org/10.1111/j.1753-4887.2000.tb01863.x
  6. Blom HJ, Smulders Y. 2011. Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects. J. Inherit. Metab. Dis. 34: 75-81. https://doi.org/10.1007/s10545-010-9177-4
  7. Hubner R, Houlston R. 2009. Folate and colorectal cancer prevention. Br. J. Cancer 100: 233-239. https://doi.org/10.1038/sj.bjc.6604823
  8. Poe M, Hensens O, Hoogsteen K. 1979. 5-Methyl-5, 6, 7, 8-tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring. J. Biol. Chem. 254: 10881-10884. https://doi.org/10.1016/S0021-9258(19)86604-8
  9. Nduati E, Diriye A, Ommeh S, Mwai L, Kiara S, Masseno V, et al. 2008. Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer. Parasitol. Res. 102: 1227-1234. https://doi.org/10.1007/s00436-008-0897-4
  10. Das JS, Duttagupta C, Ali E, Dhar TK. 1995. Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis. J. AOAC Int. 78: 1173-1177. https://doi.org/10.1093/jaoac/78.5.1173
  11. Lin M, Young C. 2000. Folate levels in cultures of lactic acid bacteria. Int. Dairy J. 10: 409-413. https://doi.org/10.1016/S0958-6946(00)00056-X
  12. Delchier N, Reich M, Renard CM. 2012. Impact of cooking methods on folates, ascorbic acid and lutein in green beans (Phaseolus vulgaris) and spinach (Spinacea oleracea). LWT-Food Sci. Technol. 49: 197-201. https://doi.org/10.1016/j.lwt.2012.06.017
  13. Berry RJ, Bailey L, Mulinare J, Bower C, Dary O. 2010. Fortification of flour with folic acid. Food Nutr. Bull. 31: S22-S35. https://doi.org/10.1177/15648265100311s103
  14. Blancquaert D, Storozhenko S, Loizeau K, De Steur H, De Brouwer V, Viaene J, et al. 2010. Folates and folic acid: from fundamental research toward sustainable health. Crit. Rev. Plant Sci. 29: 14-35. https://doi.org/10.1080/07352680903436283
  15. Wright AJ, Dainty JR, Finglas PM. 2007. Folic acid metabolism in human subjects revisited: potential implications for proposed mandatory folic acid fortification in the UK. Br. J. Nutr. 98: 667-675. https://doi.org/10.1017/S0007114507777140
  16. Ohrvik VE, Witthoft CM. 2011. Human folate bioavailability. Nutrients 3: 475-490. https://doi.org/10.3390/nu3040475
  17. Christensen KE, Mikael LG, Leung K-Y, Levesque N, Deng L, Wu Q, et al. 2015. High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice. Am. J. Clin. Nutr. 101: 646-658. https://doi.org/10.3945/ajcn.114.086603
  18. Hu J, Wang B, Sahyoun NR. 2016. Application of the key events dose-response framework to folate metabolism. Crit. Rev. Food Sci. Nutr. 56: 1325-1333. https://doi.org/10.1080/10408398.2013.807221
  19. Choi KE, Schilsky RL. 1988. Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography. Anal. Biochem. 168: 398-404. https://doi.org/10.1016/0003-2697(88)90335-1
  20. Rossi M, Raimondi S, Costantino L, Amaretti A. 2016. Folate: Relevance of chemical and microbial production. pp.103-128. Industrial Biotechnology of Vitamins, Biopigments, and Antioxidants.
  21. Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, Hugenholtz J. 2003. Increased production of folate by metabolic engineering of Lactococcus lactis. Appl. Environ. Microbiol. 69: 3069-3076. https://doi.org/10.1128/AEM.69.6.3069-3076.2003
  22. Zhu T, Pan Z, Domagalski N, Koepsel R, Ataai M, Domach M. 2005. Engineering of Bacillus subtilis for enhanced total synthesis of folic acid. Appl. Environ. Microbiol. 71: 7122-7129. https://doi.org/10.1128/AEM.71.11.7122-7129.2005
  23. Serrano-Amatriain C, Ledesma-Amaro R, Lopez-Nicolas R, Ros G, Jimenez A, Revuelta JL. 2016. Folic acid production by engineered Ashbya gossypii. Metab. Eng. 38: 473-482. https://doi.org/10.1016/j.ymben.2016.10.011
  24. Song AA-L, In LLA, Lim SHE, Rahim RA. 2017. A review on Lactococcus lactis: from food to factory. Microb. Cell Fact. 16: 55. https://doi.org/10.1186/s12934-017-0669-x
  25. LeBlanc JG, Milani C, de Giori GS, Sesma F, van Sinderen D, Ventura M. 2013. Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr. Opin. Biotechnol. 24: 160-168. https://doi.org/10.1016/j.copbio.2012.08.005
  26. Bermingham A, Derrick JP. 2002. The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. Bioessays 24: 637-648. https://doi.org/10.1002/bies.10114
  27. Hanson AD, Gregory Iii JF. 2002. Synthesis and turnover of folates in plants. Curr. Opin. Plant Biol. 5: 244-249. https://doi.org/10.1016/S1369-5266(02)00249-2
  28. Hugenschmidt S, Schwenninger SM, Lacroix C. 2011. Concurrent high production of natural folate and vitamin B12 using a co-culture process with Lactobacillus plantarum SM39 and Propionibacterium freudenreichii DF13. Process Biochem. 46: 1063-1070. https://doi.org/10.1016/j.procbio.2011.01.021
  29. Maniatis T, Fritsch EF, Sambrook J. 1983. Molecular cloning : a laboratory manual. Anal. Biochem. 186: 182-183.
  30. Holo H, Nes IF. 1989. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55: 3119-3123. https://doi.org/10.1128/AEM.55.12.3119-3123.1989
  31. Mancini R, Saracino F, Buscemi G, Fischer M, Schramek N, Bracher A, et al. 1999. Complementation of the fol2 Deletion in Saccharomyces cerevisiae by human and Escherichia coli genes encoding GTP cyclohydrolase I. Biochem. Biophys. Res. Commun. 255: 521-527. https://doi.org/10.1006/bbrc.1998.9951
  32. Li S, Zhang J, Xu H, Feng X. 2016. Improved xylitol production from D-Arabitol by enhancing the coenzyme regeneration efficiency of the pentose phosphate pathway in Gluconobacter oxydans. J. Agric. Food Chem. 64: 1144-1150. https://doi.org/10.1021/acs.jafc.5b05509
  33. Leeming R, Pollock A, Melville L, Hamon C. 1990. Measurement of 5-methyltetrahydrofolic acid in man by high-performance liquid chromatography. Metabolism 39: 902-904. https://doi.org/10.1016/0026-0495(90)90298-Q
  34. Nandania J, Kokkonen M, Euro L, Velagapudi V. 2018. Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid chromatography-tandem mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 1092: 168-178. https://doi.org/10.1016/j.jchromb.2018.06.008
  35. Sybesma W, Burgess C, Starrenburg M, van Sinderen D, Hugenholtz J. 2004. Multivitamin production in Lactococcus lactis using metabolic engineering. Metab. Eng. 6: 109-115. https://doi.org/10.1016/j.ymben.2003.11.002
  36. Jabrin S, Ravanel S, Gambonnet B, Douce R, Rebeille F. 2003. One-carbon metabolism in plants. regulation of tetrahydrofolate synthesis during germination and seedling development. Plant Physiol. 131: 1431-1439. https://doi.org/10.1104/pp.016915
  37. Yang J, Ogawa Y, Camakaris H, Shimada T, Ishihama A, Pittard A. 2007. folA, a new member of the TyrR regulon in Escherichia coli K-12. J. Bacteriol. 189: 6080-6084. https://doi.org/10.1128/JB.00482-07
  38. Plamann M, Stauffer G. 1989. Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine. J. Bacteriol. 171: 4958-4962. https://doi.org/10.1128/jb.171.9.4958-4962.1989
  39. Basset GJ, Quinlivan EP, Ravanel S, Rebeille F, Nichols BP, Shinozaki K, et al. 2004. Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids. Proc. Natl. Acad. Sci. USA 101: 1496-1501. https://doi.org/10.1073/pnas.0308331100
  40. Jin X-M, Chang Y-K, Lee JH, Hong S-K. 2017. Effects of increased NADPH concentration by metabolic engineering of the pentose phosphate pathway on antibiotic production and sporulation in Streptomyces lividans TK24. J. Microbiol. Biotechnol. 27: 1867-1876. https://doi.org/10.4014/jmb.1707.07046
  41. Christodoulou D, Link H, Fuhrer T, Kochanowski K, Gerosa L, Sauer U. 2018. Reserve flux capacity in the pentose phosphate pathway enables Escherichia coli's rapid response to oxidative stress. Cell Syst. 6: 569-578. e567. https://doi.org/10.1016/j.cels.2018.04.009
  42. Rui B, Shen T, Zhou H, Liu J, Chen J, Pan X, et al. 2010. A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress. BMC Syst. Biol. 4: 122. https://doi.org/10.1186/1752-0509-4-122
  43. Jägerstad M, Jastrebova J. 2013. Occurrence, stability, and determination of formyl folates in foods. J. Agric. Food Chem. 61: 9758-9768. https://doi.org/10.1021/jf4028427
  44. Kim J-H, Sung M-W, Lee E-H, Nam K-H, Hwang K-Y. 2008. Crystallization and preliminary X-ray diffraction analysis of 5, 10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum DSM 1728. J. Microbiol. Biotechnol. 18: 283-286.
  45. Solem C, Dehli T, Jensen PR. 2013. Rewiring Lactococcus lactis for ethanol production. Appl. Environ. Microbiol. 79: 2512-2518. https://doi.org/10.1128/AEM.03623-12
  46. Li Y, Hugenholtz J, Sybesma W, Abee T, Molenaar D. 2005. Using Lactococcus lactis for glutathione overproduction. Appl. Microbiol. Biotechnol. 67: 83-90. https://doi.org/10.1007/s00253-004-1762-8
  47. van de Guchte M, Van der Vossen J, Kok J, Venema G. 1989. Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 55: 224-228. https://doi.org/10.1128/AEM.55.1.224-228.1989