Effect of single- gene knockout of L- serine transport system on L- serine production in Escherichia coli
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摘要: 氨基酸转运系统改造是一种重要的氨基酸菌种选育方式。sda C,cyc A,sst T和tdc C是目前报道的大肠杆菌中与L-丝氨酸转运吸收相关的四个基因,本研究以实验室前期构建的L-丝氨酸工程菌SWCH-05为基础,采用Red重组系统,分别构建了sda C,cyc A,sst T和tdc C单基因敲除菌,并通过补料分批发酵实验考察了转运吸收基因缺失对菌株产L-丝氨酸的影响。发酵结果表明,sda C敲除菌L-丝氨酸产量达到了16.3 g/L,与出发菌株相比提高了43%,cyc A敲除菌L-丝氨酸产量为14.1 g/L,与出发菌株相比提高了25%,而sst T和tdc C基因敲除菌的L-丝氨酸产量均与对照菌相近。Abstract: Transport system modification is an important method in strain improvement.It has been reported that the uptake of L- serine was regulated by four genes of sda C,cyc A,sst T and tdc C in Escherichia coli.In this study,the four single- gene knockout mutant strains of sda C,cyc A,sst T and tdc C were constructed,respectively. The fermentation results showed that the sda C and cyc A knockout mutants produced 16.3 g / L and 14.1 g / L L- serine,respectively,which were 43% and 25% higher than that of the control strain. However,the production of sst T and tdc C mutants were both similar to the control strain.
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Keywords:
- transport system /
- L-serine /
- Escherichia coli /
- gene knockout
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[1] 刘岩,王慧,史吉平,等.微生物法生产L-丝氨酸代谢工程研究进展[J].生物技术通报,2015,31(8):44-49. [2] 周芳,李洪军,杨复习,等.磷脂酰丝氨酸研究进展[J].食品工业科技,2008(5):297-300. [3] Gu P,Yang F,Su T,et al.Construction of an L-serine producing Escherichia coli via metabolic engineering[J].Journal of Industrial Microbiology&Biotechnology,2014,41(9):1443-1450.
[4] Xu G,Jin X,Guo W,et al.Characterization,modification,and overexpression of 3-phosphog-lycerate dehydrogenase in Corynebacterium glutamicum for enhancing L-serine production[J].Annals of Microbiology,2014,65(2):929-935.
[5] 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. [6] Burkovski A,Krmer R.Bacterial amino acid transport proteins:occurrence,functions,and significance for biotechnological applications[J].Applied Microbiology and Biotechnology,2002,58(3):265-274.
[7] Lee K H,Park J H,Kim T Y,et al.Systems metabolic engineering of Escherichia coli for L-t hreonine production[J].Molecular Systems Biology,2007,3(1):149.
[8] Vrljic M,Sahm H,Eggeling L.A new type of transporter with a new type of cellular function:l-lysine export from Corynebacterium glutamicum[J].Molecular Microbiology,1996,22(5):815-826.
[9] Hori H,Yoneyama H,Tobe R,et al.Inducible L-alanine exporter encoded by the novel gene yga W(ala E)in Escherichia coli[J].Applied and Environmental Microbiology,2011,77(12):4027-4034.
[10] 赵志军,陈晟,吴丹,等.大肠杆菌色氨酸转运系统单基因敲除对色氨酸生产的影响[J].生物工程学报,2011,27(12):1765-1772. [11] Shao Z Q,Lin R T,Newman E B.Sequencing and characterizeation of the sda C gene and identification of the sda CB operon in Escherichia coli K12[J].European Journal of Biochemistry,1994,222(3):901-907.
[12] Hsieh J M,Besserer G M,Madej M G,et al.Bridging the gap:A GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini[J].Protein Science,2010,19(4):868-880.
[13] Schneider F,Krmer R,Burkovski A.Identification and characterization of the mainβ-alanine uptake system in Escherichia coli[J].Applied Microbiology and Biotechnology,2004,65(5):576-582.
[14] Kim Y M,Ogawa W,Tamai E,et al.Purification,reconstitution,and characterization of Na+/serine symporter,Sst T,of Escherichia coli[J].Journal of Biochemistry,2002,132(1):71-76.
[15] Ogawa W,Kayahara T,Tsuda M,et al.Isolation and characterization of an Escherichia coli mutant lacking the major serine transporter,and cloning of a serine transporter gene[J].Journal of Biochemistry,1997,122(6):1241-1245.
[16] Na D,Park J H,Jang Y S,et al.Systems metabolic engineering of Escherichia coli for chemicals,materials,biofuels,and pharmaceuticals[M].Systems Metabolic Engineering.Springer Netherlands,2012:117-149.
[17] Simic P,Sahm H,Eggeling L.L-Threonine export:use of peptides to identify a new translocator from Corynebacterium glutamicum[J].Journal of Bacteriology,2001,183(18):5317-5324.
[18] Baba T,Ara T,Hasegawa M,et al.Construction of Escherichia coli K-12 in-frame,single-gene knockout mutants:the Keio collection[J].Molecular Systems Biology,2006,2(1):1-11.
[19] Zhao Z J,Zou C,Zhu Y X,et al.Development of L-tryptophan production strains by defined genetic modification in Escherichia coli[J].Journal of Industrial Microbiology&Biotechnology,2011,38(12):1921-1929.
[20] Wang D,Zhou J,Chen C,et al.R-acetoin accumulation and dissimilation in Klebsiella pneumoniae[J].Journal of Industrial Microbiology and Biotechnology,2015,42(8):1105-1115.
[21] 杨智,阳利龙,祝文兵,等.异硫氰酸苯酯柱前衍生化RP-HPLC法测定人血浆中10种氨基酸的浓度[J].中国临床药理学与治疗学,2011,16(5):549-552.
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