Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E. coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710 mg L-1) by 2,520 mg L-1, with the extracellular enzyme activity being improved to 55.3 U mL-1 ... More
Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E. coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710 mg L-1) by 2,520 mg L-1, with the extracellular enzyme activity being improved to 55.3 U mL-1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were -19.10 kcal mol-1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326 % that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.
