This study was conducted to increase the titer of L-methionine. The effects of sulfur module on L-methionine production were investigated, and the fermentation condition was optimized. The native promoter of cysK was replaced with trc promoter on the genome, and glpE and nrdH were co-expressed on plasmid pA*H. In order to further study the effects of strong promoter on L-methionine synthesis, the p32 promoter was obtained from the PG3 promoter library. The fermentation media was optimized to improve the supply of thiosulfate to increase the yield of L-methionine. After 48 h fermentation of the strain Escherichia coli W3110 JAHFEBL trc-cysK/pA*H-p32-nrdH-glpE, the titer of L-methionine was 1.3 g/L, which increased by 41.5% in comparison to that of the control. Using optimized medium, the titer of L-methionine reached 2.3 g/L, which was 77.0% higher than that before optimization. Therefore, this paper provides a basis for biosynthesising other sulfur-containing amino acids.
JIN Liqun
,
JIN Weirong
,
LIU Zhiqiang
. Optimized promoter in sulfur assimilation module and fermentation condition enhanced L-methionine production[J]. Food and Fermentation Industries, 2019
, 45(19)
: 8
-16
.
DOI: 10.13995/j.cnki.11-1802/ts.021127
[1] WILLKE T.Methionine production - a critical review[J]. Applied Microbiology and Biotechnology, 2014, 98(24): 9 893-9 914.
[2] 党万利, 金利群,郑裕国,等. 蛋氨酸生产工艺研究进展[J]. 食品与发酵工业, 2012, 38(4): 152-158.
[3] BECKER J, WITTMANN C.Systems and synthetic metabolic engineering for amino acid production-the heartbeat of industrial strain development[J]. Current Opinion in Biotechnology, 2012, 23(5): 718-726.
[4] GOMES J, KUMAR D.Production of L-methionine by submerged fermentation: A review[J]. Enzyme & Microbial Technology, 2005, 37(1): 3-18.
[5] 李华. 系统代谢工程改造大肠杆菌生产L-蛋氨酸[D]. 无锡:江南大学, 2017.
[6] HUANG J F, SHEN Z Y, MAO Q L, et al.Systematic analysis of bottlenecks in a multibranched and multilevel regulated pathway: The molecular fundamentals of L-methionine biosynthesis in Escherichia coli[J]. ACS Synthetic Biology, 2018, 7(11):2 577-2 589.
[7] NAKATANI T, OHTSU I, NONAKA G, et al.Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli[J]. Microbial Cell Factories, 2012, 11(1): 62.
[8] KAWANO Y, ONISHI F, SHIROYAMA M, et al.Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli[J]. Applied Microbiology & Biotechnology, 2017, 101(4): 1-11.
[9] MAIER T H P. Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway[J]. Nature Biotechnology, 2003, 21(4): 422-427.
[10] ARISTI P, ARNE H, ALEXIOS V G.Protein levels of Escherichia coli thioredoxins and glutaredoxins and their relation to null mutants, growth phase, and function[J]. Journal of Biological Chemistry, 2002, 277(21): 18 561-18 567.
[11] KAWANO Y, ONISHI F, SHIROYAMA M, et al.Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli[J]. Appl Microbiol Biotechnol, 2017, 101(18): 6 879-6 889.
[12] BOGICEVIC B.CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity[J]. Appl Microbiol Biotechnol, 2012, 94(5): 1 209-1 220.
[13] HRYNIEWICZ M M, KREDICH N M.Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium[J]. Journal of Bacteriology, 1994, 176(12): 3 673.
[14] ZHANG C, SEOW V Y, CHEN X, et al.Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli[J]. Nat Commun, 2018, 9(1): 1 858.
[15] ZHANG C, CHEN X, LINDLEY N D, et al.A "plug-n-play" modular metabolic system for the production of apocarotenoids[J]. Biotechnol Bioeng, 2018, 115(1): 174-183.
[16] CLOMBURG J M, GONZALEZ R.Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology[J]. Appl Microbiol Biotechnol, 2010, 86(2): 419-434.
[17] REDDEN H, MORSE N, ALPER H S.The synthetic biology toolbox for tuning gene expression in yeast[J]. Fems Yeast Research, 2014, 15(1): 1.
[18] ZHOU S, DU G, KANG Z, et al.The application of powerful promoters to enhance gene expression in industrial microorganisms[J]. World Journal of Microbiology and Biotechnology, 2017, 33(2): 23.
[19] HUANG J F, LIU Z Q, JIN L Q, et al.Metabolic engineering of Escherichia coli for microbial production of L-methionine[J]. Biotechnology & Bioengineering, 2017, 114(4): 843.
[20] JIANG Y, CHEN B, DUAN C, et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J]. Appl Environ Microbiol, 2015, 81(7): 2 506-2 514.
[21] JIAO S, LI X, YU H, et al.In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters[J]. Biotechnology & Bioengineering, 2017, 114(4): 832.
[22] 陈可泉, 马江锋,姜岷,等. 氮源对重组大肠杆菌发酵产L-精氨酸的影响[J]. 生物加工过程, 2011, 9(6): 11-14.
