ABSTRACT
OBJECTIVE: To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering. RESULTS: Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using ß-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R 2 = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for L-lysine overproduction. CONCLUSIONS: The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.
Subject(s)
Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Lysine/metabolism , Metabolic Engineering/methods , Promoter Regions, Genetic/genetics , DNA, Bacterial , Gene Expression Regulation, Bacterial/genetics , Genes, Reporter/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
A novel method based on pH value was proposed to simplify the substrate feeding method for glutamic acid fermentation. The linear relationship between the consumption amounts of ammonia (x) and that of glucose (y) was established (y = 7.4744x, R2 = 0.9989) which could be used as the ratio of the amount of ammonia and that of glucose in the feeding broth. Thus the concentration of glucose could be controlled through the adjustment of pH automatically. In the glutamic acid fermentation using the pH feedback-controlled glucose feeding method, the glucose concentration in fermentation broth was maintained between 12 and 21 g/L. Compare with the constant glucose concentration feeding method, the glucose conversion rate and glutamic acid productivity increased by 9.06% and 17.5% respectively, when the pH feedback-controlled glucose feeding method was employed, and fermentation period was shorten above 2 h.
