Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization.
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.
National Engineering Laboratory for Cereal Fermentation Technology-NELCF, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China.
Hua
H
Liu
Jianghua
J
Li
Guocheng
G
Du
Jingwen
J
Zhou
Jian
J
Chen
D000595:Amino Acid Sequence; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics; D059370:RNA Folding; D047391:alpha-Cyclodextrins / Q000378:metabolism
D003062:Codon; D047391:alpha-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C032613:alpha-cyclodextrin