[Expression of glycerol dehydrogenase gene in Escherichia coli by codon optimization]

To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization.

For immediately downstream region of initiation codon in gldA, we designed optimized sequence by choosing higher AT-content synonymous, in order that this region's AT-content was increased without changing the corresponding amino acids. Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR, so that physically optimized gene gldA-4 was acquired. We cloned gldA-4 into pET-32a(+) to construct expression plasmid pET-gldA4, which was transformed into Escherichia coli BL21 (DE3) for gaining engineering bacteria E. coli-4, by contrast engineering bacteria involved gldA-WT named E. coli-WT. After E. coli-4 and E. coli-WT were fermented in shake flasks,we measured enzyme activities of expression products with glycerol as substrate.

Four gldA-4's bases in the second, fifth and sixth codon were different with gldA-WT, so AT-content of the optimized gene was up to 80.0% higher than the wild gene's 53.3%. Furthermore, enzyme activity of E. coli-4's crude extract was 191.3 U/mL more three times than E. coli-WT's 48.3 U/mL.

This optimization scheme was quick and easy, but indeed increased dehydrogenase's activity. It possible becomes a universal method to improve heterogenous expression level of target genes.