BTR Home --> Services --> Metabolic Engineering --> NAG Example 1 --> Page 4 of 11

glmS Gene Over-Expression in a Modified E. coli Host

 

E. coli K-12 strain W3110 was modified for glmS expression and GlcN production. Briefly, the manXYZ operon and nag regulon were inactivated in order to minimize the transport and metabolism of GlcN and GlcNAc. The defective λDE3 prophage containing a T7 RNA polymerase gene under lacUV5 promoter control was also introduced through λDE3 lysogenization, generating strain 7101-17 (DE3). Glucosamine synthase genes from E. coli, Bacillus subtilis, Candida albicans and Saccharomyces cerrevisiae were inserted into plasmid pET24d(+). The expression cassette of the E. coli glmS gene was integrated into the chromosome at the lacZ locus in 2123-12.

Strains containing a free-replicating plasmid or an integrated expression cassette were grown in shake flasks and induced with IPTG (isopropylthio-β-D-galactoside) in a defined mineral salt medium (M9A) supplemented with glucose. With the strain hosting an empty vector, glucosamine in the growth medium was about 5 mg/L. Over-expression of bacterial glmS and yeast GFA1 genes led to higher levels of glucosamine production (see Table 1). The highest levels of enzyme activity and GlcN production were seen with the Bacillus glmS gene, which encodes an enzyme more resistant to inhibition by GlcN-6-P than the E. coli homologue.

Table 1. Over-expression of different glucosamine synthase genes (glmS and GFA1) in E. coli, and accumulation of glucosamine in the growth medium

Over-Expression of glmS gene and Accumulation of GlcN in Medium

Top