L-cysteine is an important sulfur-containing amino acid, which is widely used in pharmaceutical, food and cosmetic industries. In this study, C. glutamicum A36 was engineered to efficiently produce L-cysteine from sugar, and showed highest L-serine titer comparing with its' parent strain. In order to produce L-cysteine, two types of the serine O-acetyltransferase (gene encoded by cysE) were overexpressed in strain A36 respectively, strain S-C-1 and S-C-2 had been constructed, L-cysteine titer was 115.8 mg/L and 105.8 mg/L, respectively, and the parent strain A36 couldn't produce L-cysteine. Subsequently, in order to increase the titer of L-cysteine, several metabolic engineering strategies were performed, including overexpression of the OASS-A (gene encoded by cysK) and the L-cysteine exporter Bcr (gene encoded by bcr), and the deletion of the degradation pathway. A series recombinant strain had been constructed, and among all strains, S-C-7 showed the highest L-cysteine titer of 286.7 mg/L. The sulfur source was optimized to increase L-cysteine titer further, the addition of 12 g/L sodium thiosulfate at 24 h showed the highest L-cysteine titer of 581.6 mg/L, which was two times of that before optimization. Finally, in 5 L fermenter, the L-cysteine titer of strain S-C-7 could reach 1.2 g/L, which was the highest titer of L-cysteine produced by C. glutamicum up to now. It laid a foundation for the L-cysteine production by C. glutamicum.
卞金玉
,
张晓梅
,
徐国强
,
史劲松
,
许正宏
. Pathway engineering modification of Corynebacterium glutamicum for L-cysteine synthesis[J]. Food and Fermentation Industries, 2022
, 48(20)
: 22
-28
.
DOI: 10.13995/j.cnki.11-1802/ts.030796
[1] DUAN J, ZHANG Q, ZHAO H, et al.Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine[J].Biotechnology Letters, 2012, 34(6):1 101-1 106.
[2] TAKAGI H, OHTSU I.L-cysteine metabolism and fermentation in microorganisms[J].Advances in Biochemical Engineering/Biotechnology, 2017, 159:129-151.
[3] CALZETTA L, MATERA M G, ROGLIANI P, et al.Multifaceted activity of N-acetyl-L-cysteine in chronic obstructive pulmonary disease[J].Expert Review of Respiratory Medicine, 2018, 12(8):693-708.
[4] YIN J, REN, W K, YANG G, et al.L-cysteine metabolism and its nutritional implications[J].Molecular Nutrition and Food Research, 2016, 60(1):134-146.
[5] WENDISCH, VOIKER F.Metabolic engineering advances and prospects for amino acid production[J].Metabolic Engineering, 2020, 58:17-34.
[6] YOKOTA A, IKEDA M.Amino Acid Fermentation[M].Japan:Springer, 2017.
[7] POPA C N, POPESCU S, BEREHOIU R.Assessment of the E 920 additive (L-cysteine) in relation to some problems of modern food industry[J].Scientific Papers Series:Management, Economic Engineering in Agriculture and Rural Development, 2013, 13:413-418.
[8] WADA M, TAKAGI H.Metabolic pathways and biotechnological production of L-cysteine[J].Applied Microbiology and Biotechnology, 2006, 73(1):48-54.
[9] 朱加粉, 陈紫薇, 张晓梅, 等.调控NADH/NAD+对重组谷氨酸棒杆菌产L-丝氨酸的影响[J].食品与发酵工业, 2017, 43(6):54-59.
ZHU J F, CHEN Z W, ZHANG X M, et al.Effects of regulating NADH/NAD+ on L-serine production by recombinant Corynebacterium glutamicum[J].Food and Fermentation Industries, 2017, 43(6):54-59.
[10] LIU H, HOU Y H, WANG Y, et al.Fitness of chassis cells and metabolic pathways for L-cysteine overproduction in Escherichia coli[J].Journal of Agricultural and Food Chemistry, 2020, 68(50):14 928-14 937.
[11] 张伟. 谷氨酸棒杆菌外源蛋白分泌表达系统的开发及其应用研究[D].无锡:江南大学, 2019.
ZHANG W.Development and application of exogenous protein secretion and expression system of Corynebacterium glutamicum[D].Wuxi:Jiangnan University, 2019.
[12] 刘川. 谷氨酸棒状杆菌半胱氨酸转运蛋白的鉴定与特性研究以及半胱氨酸单细触物传感器的构建与应用[D].天津:天津科技大学, 2018.
LIU C.Identification and characterization of cysteine transporter in Corynebacterium glutamicum and construction and application of cysteine single-cell sensor[D].Tianjin:Tianjin University of Science and Technology, 2018.
[13] KISHINO M, KONDOH M, HIRASAWA T.Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum[J].Bioscience Biotechnology and Biochemistry, 2019, 83(12):2 390-2 393.
[14] JOO Y C, HYEON J E, HAN S O.Metabolic design of Corynebacterium glutamicum for production of L-cysteine with consideration of sulfur-supplemented animal feed[J].Journal of Agricultural and Food Chemistry, 2017, 65(23):4 698-4 707.
[15] LEE H S, HWANG B J.Methionine biosynthesis and its regulation in Corynebacterium glutamicum:Parallel pathways of transsulfuration and direct sulfhydrylation[J].Applied Microbiology and Biotechnology, 2003, 62(5-6):459-467.
[16] 贾慧慧, 李晓静, 陈涛,等.微生物发酵法生产L-丝氨酸及L-半胱氨酸研究进展[J].中国生物工程杂志, 2014, 34(5):100-106.
JIA H H, LI X J, CHEN T,et al.The research progress of microbial production of L-serine and L-cysteine[J].China Biotechnology, 2014, 34(5):100-106.
[17] TAKUMI K, ZIYATDINOV M K, SAMSONOV V, et al.Fermentative production of cysteine by Pantoea ananatis[J].Applied and Environmental Microbiology, 2017, 83(5):e02502-e02516.
[18] WEI L, WANG H, XU N, et al.Metabolic engineering of Corynebacterium glutamicum for L-cysteine production[J].Applied Microbiology and Biotechnology, 2019, 103(3):1 325-1 338.
[19] KAWA Y, OHTSU I, TAKUMI K, et al.Enhancement of L-cysteine production by disruption of yciW in Escherichia coli[J].Journal of Bioscience and Bioengineering, 2015, 119(2):176-179.
[20] MA M L, LIU T, WU H Y, et al.Enzymatic synthesis of L-cysteine by Escherichia coli whole-cell biocatalyst[C].International Conference on Applied Biotechnology, 2018.
[21] ZHANG X, ZHANG X M, XU G Q, et al.Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2018, 102(14):5 939-5 951.
[22] ZHU Q J, ZHANG X M, LUO Y C, et al.L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2015, 99(4):1 665-1 673.
