产丙谷二肽重组大肠杆菌的构建及发酵优化

Construction of a L-Alanyl-L-Glutamine Producing Recombinant Strain and Optimization of Its Fermentation Conditions

SAET基因编码的α-氨基酸酯酰基转移酶能够以丙氨酸甲酯盐酸盐和谷氨酰胺为底物缩合生成L-丙氨酰-L-谷氨酰胺.为了使α-氨基酸酯酰基转移酶能够在大肠杆菌中过量表达,在保证氨基酸序列不变的前提下,优化密码子和m RNA二级结构,共改变了396个碱基,GC含量由原来的42.15%升高到48.22%;将优化后的基因分别与phoC启动子和色氨酸串联启动子相连并插入到不同质粒中,将重组质粒转入大肠杆菌,DH5α/p ET21a-phoC-SAET产量最高。通过对重组大肠杆菌生产L-丙氨酰-L-谷氨酰胺的条件研究发现,最佳培养基是:葡萄糖15 g/L,酵母提取物10 g/L,胰蛋白胨10 g/L,NH_4AC 5 g/L,KH_2PO_46.75 g/L,K_2HPO_42.25 g/L,MgSO_4·7H_2O0.5 g/L。最适转化条件是:将发酵液加入到含Ala-OMe·HCl 100 mmol/L,Gln 50 mmol/L的pH 9的溶液中,25℃反应1.5 h。优化后产量达到383 mg/L,是优化前的3.9倍。

α-Amino acid ester acyltransferase,which is encoded by SAET,can synthesize L-alanyl-L-glutamine from L-alanine methyl ester hydrochloride and L-glutamine.To improve the expression level of α-amino acid ester acyltransferase in Escherichia coli,we optimized the codons and the mRNA secondary structure of SAET in the translation initiation region.Total of 396 nucleotides were changed,and the G＋C ratio was simultaneously increased from 42.15% to 48.22%after optimization.Codon-optimized SAET gene was cloned into expression vectors with a tryptophan tandem promoter or phosphate promoter.DH5α/pET21a-phoC-SAET is the strain with the highest yield.The composition of the optimized culture medium for the genetic engineered Escherichia coli to produce L-alanyl-L-glutamine is as follows：glucose 15 g/L,yeast extract 10 g/L,tryptone 10 g/L,NH4AC 5 g/L,KH2PO4 6.75 g/L,K2HPO4 2.25 g/L and MgSO4·7H2O 0.5 g/L.The optimal reaction conditions are：AlaOMe ·HC1 100mmol/L,Gln 50 mmol/L,pH 9,at 25 ℃ for 90 min.The yield is 383 mg/L,which showed 3.9 fold improvement over that of the initial condition.