L-serine is widely used in medicine, food and cosmetic industry, however, the industrial production of L-serine by microbial fermentation has not yet been realized. In previous studies, we found that there had a significant difference in L-serine titer by Corynebacterium glutamicum A using sucrose and glucose. In this paper, comparative metabolomics analysis method was used to study the differences of intracellular metabolites of C. glutamicum A in different carbon sources, revealed the key metabolites of L-serine synthesis, and then investigated the influence of the addition of key metabolites on L-serine production. Through GC-MS, principal component analysis, and partial least squares discriminant analysis, 15 kinds of intracellular metabolites were found to be closely related to L-serine synthesis, including glucose, glucose 6-phosphate, 3-phosphoglycerate, pyruvate, fructose 6-phosphate, alanine, succinic acid, fumaric acid, citric acid, malic acid, phenylalanine, tryptophan, proline, glycine and methionine, among which fumaric acid, succinic acid, α-ketoglutaric acid, malic acid, and citric acid in the tricarboxylic acid cycle had great influence on the synthesis of L-serine. The fermentation results showed that the addition of 2.5 g/L fumaric acid had the significant inhibition on the cell growth, and the biomass (OD562) decreased by 33.9%. Although fumaric acid was added to inhibit the cell growth, it was significantly beneficial to L-serine production. When 2 g/L fumaric acid was added, L-serine titer increased by 29.8%. These results showed that the optimal medium based on comparative metabolomics analysis could improve the L-serine production.
[1] ZHANG X M, XU G Q, SHI J S, et al.Microbial production of L-serine from renewable feedstocks[J].Trends in Biotechnology, 2018, 36(7):700-712.
[2] 朱林江, 李崎.L-丝氨酸的微生物法制备研究进展[J].食品与发酵工业, 2015,41(1):181-185.
ZHU L J, LI Q.Microbial production of L-serine[J].Food and Fermentation Industries, 2015, 41(1):181-185.
[3] WENDISCH V F.Metabolic engineering advances and prospects for amino acid production[J].Metabolic Engineering, 2020, 58:17-34.
[4] RENNIG M, MUNDHADA H, WORDOFA G G, et al.Industrializing a bacterial strain for L-serine production through translation initiation optimization[J].ACS Synthetic Biology, 2019, 8(10):2347-2358.
[5] PETERS-WENDISCH P, NETZER R, EGGELING L, et al.3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum:the C-terminal domain is not essential for activity but is required for inhibition by L-serine[J].Applied Microbiology and Biotechnology, 2002, 60(4):437-441.
[6] PETERS-WENDISCH P, STOLZ M, ETTERICH H, et al.Metabolic engineering of Corynebacterium glutamicum for L-serine production[J].Applied and Environmental Microbiology, 2005, 71(11):7139-7144.
[7] STOLZ M, PETERS-WENDISCH P, ETTERICH H, et al.Reduced folate supply as a key to enhanced L-serine production by Corynebacterium glutamicum[J].Applied and Environmental Microbiology, 2007, 73(3):750-755.
[8] 徐国强, 窦文芳, 许泓瑜, 等.直接发酵法生产L-丝氨酸高产菌株的选育[J].食品与生物技术学报, 2014, 33(11):1191-1195.
XU G Q, DOU W F, XU H Y, et al.Breeding of high-yield L-serine producing strains which can ferment sugar directly[J].Journal of Food Science and Biotechnology, 2014, 33(11):1191-1195.
[9] 赖联贺. 改造谷氨酸棒杆菌非磷酸转移酶葡萄糖转运途径高产L-丝氨酸[D].无锡:江南大学, 2017.
LAI L H.Engineering of the non-PTS glucose transport system for enhancing L-serine production by Corynebacterium glutamicum[D].Wuxi:Jiangnan University, 2017.
[10] 曹鹏, 胡栋, 张君, 等.基于比较代谢组学的理性优化方法提高阿维菌素产量[J].微生物学报, 2017, 57(2):281-292.
CAO P, HU D, ZHANG J, et al.Enhanced avermectin production by rational feeding strategies based on comparative metabolomics[J].Acta Microbiologica Sinica, 2017, 57(2):281-292.
[11] 冯鑫, 程睿, 王珮玥, 等.电喷雾检测器、蒸发光散射检测器与示差折光检测器测定食品中5种糖成分的方法比较[J].食品安全质量检测学报, 2021, 12(4):1513-1518.
FENG X, CHENG R, WANG P Y, et al.Comparison of determination of 5 carbohydrates in food by charged aerosol detector, evaporative light scattering detector and refractive index detector[J].Journal of Food Safety and Quality, 2021, 12(4):1513-1518.
[12] CHEN Q, WANG Q, WEI G Q, et al.Production in Escherichia coli of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources[J].Applied and Environmental Microbiology, 2011, 77(14):4886-4893.
[13] PINU F, VILLAS-BOAS S, AGGIO R, et al.Analysis of intracellular metabolites from microorganisms:Quenching and extraction protocols[J].Metabolites, 2017, 7(4):53.
[14] WANG J B, PU S B, SUN Y, et al.Metabolomic profiling of autoimmune hepatitis:The diagnostic utility of nuclear magnetic resonance spectroscopy[J].Journal of Proteome Research, 2014, 13(8):3792-3801.
[15] KIEFFER D A, PICCOLO B D, VAZIRI N D, et al.Resistant starch alters gut microbiome and metabolomic profiles concurrent with amelioration of chronic kidney disease in rats[J].American Journal of Physiology-Renal Physiology, 2016, 310(9):F857-F871.
[16] HASPEL J A, CHETTIMADA S, SHAIK R S, et al.Circadian rhythm reprogramming during lung inflammation[J].Nature Communications, 2014, 5:4753.
[17] RAO G D, SUI J K, ZHANG J G.Metabolomics reveals significant variations in metabolites and correlations regarding the maturation of walnuts (Juglans regia L.)[J].Biology Open, 2016, 5(6):829-836.
[18] KIEFER P, HEINZLE E, ZELDER O, et al.Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose[J].Applied and Environmental Microbiology, 2004, 70(1):229-239.
[19] NIE L B, XU K X, ZHONG B, et al.Enhanced L-ornithine production from glucose and sucrose via manipulation of the fructose metabolic pathway in Corynebacterium glutamicum[J].Bioresources and Bioprocessing, 2022, 9(1):11.
[20] 许娟, 许琳, 郝宁.代谢组学分析提高谷氨酸棒杆菌L-瓜氨酸产量[J].生物加工过程, 2018, 16(4):8-15.
XU J, XU L, HAO N.Improve L-citrulline production in Corynebacterium glutamicum by metabolomics analysis[J].Chinese Journal of Bioprocess Engineering, 2018, 16(4):8-15.
[21] JIA S S, WANG Y, HU J H.et al.Mineral and metabolic profiles in tea leaves and flowers during flower development[J].Plant Physiology and Biochemistry, 2016, 106:316-326.
[22] RAO, J, CHENG F, HU C Y, et al.Metabolic map of mature maize kernels[J].Metabolomics, 2014, 10(5):775-787.
[23] ZHANG X M, YAO L P, XU G Q, et al.Enhancement of fructose utilization from sucrose in the cell for improved L-serine production in engineered Corynebacterium glutamicum[J].Biochemical Engineering Journal, 2017, 118:113-122.
[24] SAWADA K, ZEN-IN S, WADA M, et al.Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032[J].Metabolic Engineering, 2010, 12(4):401-407.
[25] 乔郅钠, 徐美娟, 龙梦飞, 等.TCA循环关键节点对L-谷氨酸合成的影响[J].生物工程学报, 2020, 36(10):2113-2125.
QIAO Z N, XU M J, LONG M F, et al.Effect of key notes of TCA cycle on L-glutamate production[J].Chinese Journal of Biotechnology, 2020, 36(10):2113-2125.
[26] MAN Z W, XU M J, RAO Z M, et al.Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production[J].Scientific Reports, 2016, 6:28629.
[27] 吴洁琴. 代谢工程改造大肠杆菌强化乙酰辅酶A合成L-精氨酸的研究[D].无锡:江南大学, 2022.
WU J Q.Metabolic engineering of Escherichia coil to enhance the synthesis of L-arginine by acetyl-CoA[D].Wuxi:Jiangnan University, 2022.
