Please wait a minute...
 
 
食品与发酵工业  2022, Vol. 48 Issue (20): 22-28    DOI: 10.13995/j.cnki.11-1802/ts.030796
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
途径工程改造谷氨酸棒杆菌合成L-半胱氨酸
卞金玉1, 张晓梅1*, 徐国强2, 史劲松1, 许正宏2
1(江南大学 药学院,江苏 无锡,214122)
2(粮食与发酵国家工程实验室(江南大学),江苏 无锡,214122)
Pathway engineering modification of Corynebacterium glutamicum for L-cysteine synthesis
BIAN Jinyu1, ZHANG Xiaomei1*, XU Guoqiang2, SHI Jinsong1, XU Zhenghong2
1(School of Pharmaceutics Science, Jiangnan University, Wuxi 214122, China)
2(National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China)
下载:  HTML  PDF (2419KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 L-半胱氨酸是一种重要的含硫氨基酸,广泛应用在医药、食品和化妆品等行业。该文以实验室保藏的一株高产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)A36为出发菌株,通过加强表达丝氨酸O-乙酰基转移酶(serine O-acetyltransferase,SAT)、O-乙酰基-L-丝氨酸巯基化酶-A和转运蛋白Bcr编码基因强化L-半胱氨酸合成和转运,敲除L-半胱氨酸降解途径关键酶弱化其降解,构建系列重组菌株,其中重组菌S-C-7的L-半胱氨酸产量最高,为286.7 mg/L。进一步通过优化硫源提高菌株S-C-7的L-半胱氨酸产量,结果表明,最佳硫源为硫代硫酸钠,当发酵24 h时添加12 g/L硫代硫酸钠,L-半胱氨酸的产量最高,为581.6 mg/L,较优化前提高1.0倍。最后,在5 L发酵罐对S-C-7进行发酵评价,L-半胱氨酸产量达到1.2 g/L,是目前文献报道谷氨酸棒杆菌产L-半胱氨酸的最高产量;为实现谷氨酸棒杆菌发酵生产L-半胱氨酸奠定了基础。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
卞金玉
张晓梅
徐国强
史劲松
许正宏
关键词:  L-半胱氨酸  谷氨酸棒杆菌  合成途径  降解途径  转运蛋白    
Abstract: 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.
Key words:  L-cysteine    Corynebacterium glutamicum    synthetic pathway    degradation pathway    exporter
收稿日期:  2022-01-13      修回日期:  2022-02-27           出版日期:  2022-10-25      发布日期:  2022-11-18      期的出版日期:  2022-10-25
基金资助: 国家重点研发计划(2018YFA0901400);国家自然科学基金项目(32171470)
作者简介:  硕士研究生(张晓梅教授为通信作者,E-mail:zhangxiaomei@jiangnan.edu.cn)
引用本文:    
卞金玉,张晓梅,徐国强,等. 途径工程改造谷氨酸棒杆菌合成L-半胱氨酸[J]. 食品与发酵工业, 2022, 48(20): 22-28.
卞金玉,张晓梅,徐国强,et al. Pathway engineering modification of Corynebacterium glutamicum for L-cysteine synthesis[J]. Food and Fermentation Industries, 2022, 48(20): 22-28.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.030796  或          http://sf1970.cnif.cn/CN/Y2022/V48/I20/22
[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.
[1] 李洋, 张稳杰, 韩雨辰, 翟懿雪, 张成林. 生物法合成4-羟基异亮氨酸的代谢工程研究进展[J]. 食品与发酵工业, 2022, 48(5): 281-288.
[2] 宋卓琳, 黄明珠, 陈雪岚. 三磷酸腺苷供应优化促进谷氨酸棒杆菌产氨基酸的研究进展[J]. 食品与发酵工业, 2022, 48(3): 311-316.
[3] HABIMANA, 乔郅钠, 徐美娟, 杨套伟, 张显, 邵明龙, 饶志明. 重组谷氨酸棒杆菌全细胞催化一步法合成高果糖浆[J]. 食品与发酵工业, 2022, 48(2): 8-14.
[4] 来文梅, 谭书煜, 史锋. 动态调控谷氨酸棒状杆菌合成4-羟基异亮氨酸[J]. 食品与发酵工业, 2022, 48(17): 42-48.
[5] 上官玲玲, 卢慧芳, 夏会丽, 陈雄, 代俊. 谷氨酸棒杆菌细胞工厂构建与应用的研究进展[J]. 食品与发酵工业, 2022, 48(17): 313-320.
[6] 齐静静, 范炳森, 张萌, 许菲. 信号肽及发酵条件优化促进胶原蛋白在谷氨酸棒杆菌中分泌表达[J]. 食品与发酵工业, 2022, 48(15): 9-17.
[7] 张琦, 刘秀霞, 杨艳坤, 白仲虎. 谷氨酸棒杆菌中基因NCgl2632的敲除和过表达对三种外源蛋白表达的影响[J]. 食品与发酵工业, 2022, 48(14): 1-8.
[8] 魏敏华, 李宇虹, 张佳蓉, 孟静, 孟燕, 王浚哲, 张成林. 代谢工程构建谷氨酸棒杆菌合成5-氨基乙酰丙酸[J]. 食品与发酵工业, 2022, 48(14): 9-15.
[9] 魏舒宇, 史锋. 4-羟基异亮氨酸无抗发酵的菌株构建和上罐条件优化[J]. 食品与发酵工业, 2022, 48(12): 117-122.
[10] 芦楠, 李宇虹, 陈宁, 张成林. L-异亮氨酸及其衍生物代谢工程研究进展[J]. 食品与发酵工业, 2021, 47(9): 307-313.
[11] 耿雪营, 郭藏, 米生权, 张艳贞, 郭俊霞, 陈文. 单宁的血糖调节活性功能研究进展[J]. 食品与发酵工业, 2021, 47(7): 301-306.
[12] 熊海波, 陈志超, 曹华杰, 徐庆阳. 全营养流加对谷氨酸棒杆菌发酵产L-异亮氨酸的影响[J]. 食品与发酵工业, 2021, 47(6): 11-17.
[13] 熊海波, 刘云鹏, 徐庆阳. 超声对谷氨酸棒杆菌发酵L-异亮氨酸的影响[J]. 食品与发酵工业, 2021, 47(4): 40-46.
[14] 刘袆帆, 林诺怡, 成坚, 马路凯, 陈乃驿, 冯浩锴, 王琴. 微生物发酵法制备活性多糖的研究概述[J]. 食品与发酵工业, 2021, 47(3): 281-287.
[15] 孟丽虹, 刘秀霞, 杨艳坤, 白仲虎. 基于全基因组重测序策略对谷氨酸棒杆菌外源蛋白高产菌株相关基因的挖掘及初步验证[J]. 食品与发酵工业, 2021, 47(24): 1-6.
[1] YUAN Feng-jiao et al . Heterologous Expression of phenylpyruvate reductase from Lactobacillus plantarum and Its Application in the Preparation of Phenyllactic Acid[J]. Food and Fermentation Industries, 2017, 43(11): 16 -21 .
[2] ZHAO Xiang-ying et al. Effect of Glucose on xylitol fermentation byCandida tropicalis SFX - Y9[J]. Food and Fermentation Industries, 2017, 43(11): 107 .
[3] ZHANG Zhe-yuan.et al. Effects of different total solids of goat milk on quality of goat milk yogurt #br# [J]. Food and Fermentation Industries, 2017, 43(11): 112 .
[4] NI De-rang et al. Analysisofcharacteristiccompoundsofsteamedsorghum aroma[J]. Food and Fermentation Industries, 2017, 43(11): 202 .
[5] ZHANG Dong et al. Researchprogressonreducing sodium chlorideinmeatproducts[J]. Food and Fermentation Industries, 2017, 43(11): 238 .
[6] SONG Yuan-de. Establishmentandcomparisonofnew andoldeditionofqualification conditionsforfoodinspectionagency[J]. Food and Fermentation Industries, 2017, 43(11): 268 .
[7] . Isolation and identification of anaerobic bacteria in the process of Maotai-flavor liquor brewing[J]. Food and Fermentation Industries, 0, (): 1 .
[8] YU Qing-lin et al. Fermentation optimization of recombinant Yarrowia lipolytica for its efficient succinic acid production[J]. Food and Fermentation Industries, 0, (): 1 .
[9] Zheng Dan et al.. The inhibiting effect of flavonoid “astilbin” on pancreatic lipase[J]. Food and Fermentation Industries, 0, (): 1 .
[10] . Effect of Protein on Quality of Chinese Rice Wine #br# [J]. Food and Fermentation Industries, 0, (): 1 .
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn