Multi-step metabolic engineering Corynebacterium glutamicum ATCC13032 to produce L-methionine

Abstract

L-Methionine is widely used in food, agricultural and pharmaceutical industries. In this study, the L-methionine production in Corynebacterium glutamicum ATCC13032 was promoted by eliminating the feedback inhibition of key rate-limiting enzymes, blocking L-threonine biosynthesis, and strengthening the downstream pathway of L-homoserine. ATCC13032 does not accumulate L-threonine, we found that overexpressing the genes lysC and hom^G378S could accumulate 0.6 g/L L-threonine. Deleting the genes thrB, McbR, and metD in ATCC13032 could accumulate 0.49 g/L L-methionine. Next, enhancing oxaloacetate supply, overexpressing brnFE, and deleting Ncgl2640 that involved in the repression of sulphuric metabolism could accumulate 0.92 g/L L-methionine. Further overexpressing the genes related to L-homoserine downstream pathway, the resulting strain ZBW011/pEC-metYX could produce 1.82 g/L L-methionine. Finally, the gene pyk2 was deleted and the final strain ZBW014/pEC-metYX produced 7.06 g/L L-methionine in a 2.4-L fermenter. The strategies presented in this study would be useful to engineer C. glutamicum for industrial L-methionine production.

Keywords

Corynebacterium glutamicum / Fermentation / L-methionine production / Promoter substitution

References

[1] WangH, LiY, CheY, et al. . Production of L-methionine from 3-methylthiopropionaldehyde and O-acetylhomoserine by catalysis of the yeast O-acetylhomoserine sulfhydrylase[J]. J Agric Food Chem, 2021, 69287932-7.

[2] MitsuhashiS. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides. Curr Opin Biotechnol, 2014, 26: 38-44.

[3] TangXL, DuXY, ChenLJ, et al. . Enhanced production of L-methionine in engineered Escherichia coli with efficient supply of one carbon unit. Biotechnol Lett, 2020, 42: 429-36.

[4] NeubauerC, LandeckerH. A planetary health perspective on synthetic methionine[J]. Lancet Planet Health, 2021, 58e560-9.

[5] Kim H, et al. Comparative life cycle assessment for the sustainable production of fermentation-based L-methionine. J Clean Prod. 2024;142700. https://doi.org/10.1016/j.jclepro.2024.142700.

[6] ParkJH, LeeSY. Metabolic pathways and fermentative productionof L-aspartate family amino acids[J]. Biotechnol J, 2010, 56560-77.

[7] XuJ, HanM, ZhangJ, et al. . Improvement of L-lysine production combines with minimization of by-products synthesis in Corynebacterium glutamicum. J Chem Technol Biotechnol, 2014, 89121924-33.

[8] ZhangY, LiuY, ZhangS, et al. . Metabolic engineering of Corynebacterium glutamicum WM001 to improve L-isoleucine production[J]. Biotechnol Appl Chem, 2021, 683568-84.

[9] FangY, WangJ, MaW, et al. . Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system[J]. Metab Eng, 2020, 61: 33-46.

[10] LiN, LiL, YuS, et al. . Dual-channel glycolysis balances cofactor supply for L-homoserine biosynthesis in Corynebacterium glutamicum. Bioresour Technol, 2023, 369: 128473.

[11] Cai M, Liu Z, Zhao Z, et al. Microbial production of L-methionine and its precursors using systems metabolic engineering[J]. Biotechnol Adv. 2023;108260. https://doi.org/10.1016/j.biotechadv.2023.108260.

[12] QinT, HuX, HuJ, et al. . Metabolic engineering of Corynebacterium glutamicum strain ATCC13032 to produce L-methionine[J]. Biotechnol Appl Chem, 2015, 624563-73.

[13] LiY, CongH, LiuB, et al. . Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level[J]. Antonie Van Leeuwenhoek, 2016, 109: 1185-97.

[14] HuangJF, LiuZQ, et al. . Metabolic engineering of Escherichia coli for microbial production of L-methionine. Biotechnol Bioeng, 2017, 1144843-51.

[15] HuangJF, et al. . Systematic analysis of bottlenecks in a multibranched and multilevel regulated pathway: the molecular fundamentals of L-methionine biosynthesis in Escherichia coli. ACS Synth Biol, 2018, 7112577-89.

[16] NiuK, FuQ, MeiZL, et al. . High-level production of L-methionine by dynamic deregulation of metabolism with engineered nonauxotroph Escherichia coli[J]. ACS Synth Biol, 2023, 122492-501.

[17] LiZ, LiuQ, SunJ, et al. . Multivariate modular metabolic engineering for enhanced L-methionine biosynthesis in Escherichia coli[J]. Biotechnol Biofuels Bioprod, 2023, 161101.

[18] OhnishiJ, et al. . A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl Microbiol Biotechnol, 2002, 58: 217-23.

[19] ReinscheidDJ, EikmannsBJ, SahmHE. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase. J Bacteriol, 1991, 173103228-30.

[20] North JA, Narrowe AB, Xiong WL, A nitrogenase-like enzyme system catalyzes methionine, ethylene, and methane biogenesis[J]. Science, 2020;369(6507):1094-1098. https://doi.org/10.1126/science.abb6310.

[21] Han G et al. H X, Q T, Metabolic engineering of Corynebacterium glutamicum ATCC13032 to produce S-adenosyl- L-methionine. Enzyme and microbial technology 83 (2016): 14–21. https://doi.org/10.1016/j.enzmictec.2015.11.001\n

[22] KrömerJO, WittmannC, et al. . Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum. Metab Eng, 2006, 84353-69.

[23] WangT, LiY, LiJ, et al. . An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum. Microb Biotechnol, 2019, 125907-19.

[24] LiN, XuS, DuG, et al. . Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux. Biochem Eng J, 2020, 161: 107665.

[25] RückertC, MilseJ, AlbersmeierA, et al. . The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules. BMC Genomics, 2008, 9: 1-8.

[26] LiY, WeiH, WangT, et al. . Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives. Bioresour Technol, 2017, 245: 1588-602.

[27] MuQ, ZhangS, MaoX, et al. . Highly efficient production of L-homoserine in Escherichia coli by engineering a redox balance route. Metab Eng, 2021, 67: 321-9.

[28] TrötschelC, DeutenbergD, BatheB, et al. . Characterization of methionine export in Corynebacterium glutamicum. J Bacteriol, 2005, 187113786-94.

[29] SchwentnerA, FeithA, MünchE, et al. . Metabolic engineering to guide evolution–creating a novel mode for L-valine production with Corynebacterium glutamicum[J]. Metab Eng, 2018, 47: 31-41.

[30] MampelJ, SchröderH, HaefnerS, et al. . Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum. Appl Microbiol Biotechnol, 2005, 68: 228-36.

[31] Koutmos M, Datta S, Pattridge KA et al. Insights into the reactivation of cobalamin-dependent methionine synthase. Proceedings of the National Academy of Sciences. 2009;106(44):18527-32. https://doi.org/10.1073/pnas.0906132106

[32] FerlaMP, et al. . Bacterial methionine biosynthesis. Microbiology, 2014, 16081571-84.

[33] MatthewsRG, KoutmosM, DattaS. Cobalamin-dependent and cobamide-dependent methyltransferases. Curr Opin Struct Biol, 2008, 186658-66.

[34] ShenZY, WangYF, WangLJ, et al. . Thorough research and modification of one-carbon units cycle for improving L-methionine production in Escherichia coli[J]. 3 Biotech, 2023, 136203.

[35] RuanL, LiL, ZouD, et al. . Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle. Biotechnol Biofuels, 2019, 12: 1-2.

Funding

National Key Research and Development Program of China(2021YFC2100900)

RIGHTS & PERMISSIONS

Jiangnan University