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

Abstract:

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·HCl 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.