Codon Usage Optimization Aided α-Cyclodextrin Glycosyltransferase Expression In E. Coll And B. Megaterium

Cyclodextrin glycosyltransferase (EC2.4.1.19，CGTase) is extracellular enzyme, capableof converting starch or starch derivates to CD via a cyclization reaction. Because of its smallinternal cavity, high water solubility, and nonbioavailability,-CD has special applications inmolecular recognition, nanomaterials, and the pharmacy, agriculture, food, cosmetics industry.Nevertheless, the production level of CGTase is a key in-CD industry. In addition, manycarbohydrate derivatives and other compounds such as sugar alcohols, vitamins, glycosides,and polyols, can be used as acceptors in CGTase-catalyzed reactions, improving the propertiesand application of the products. For example,2-o--D-glucopyranosyl-L-ascorbic acid(AA-2G), which is a widely used, stable and non-reducible L-ascorbic acid derivative, can besynthesized enzymatically via transglycosylation with-CGTase from L-ascorbic acid and-cyclodextrin. So-CGTase has gained increasing industrial interest and extensive attentionin recent years.Current research mainly focuses on the selection and construction of-CGTase-producing strains. Escherichia coli has been the main host for the heterologousexpression of-CGTase. In this study, we focus on the aim of industrial application of-CGTase, we started our research from the aspects of systematic codon optimization andsafety concerns and the main contents are as follows:(1) The mature-cgt gene from Paenibacillus macerans JFB05-01was redesigned withsystematic codon optimization to preferentially match codon frequencies of Escherichia coli.Following synthesis, we obtained codon-optimized-cgt (E-co-cgt)(GenBank accession no.JX412224).The systematic codon optimization method was based on the “one amino acid–onecodon” method. Therefore, the most-preferred codons and second most-preferred codons wereused during codon optimization. E. coli-Codon-optimized-cgt (E-co-cgt) and wild type-cgt (wt-cgt) genes were cloned into pET-20b(+) and expressed in E. coli BL21(DE3). Thetotal protein yield of the synthetic gene was greater than wt-cgt expression (1710mg L-1) by2520mg L-1, with the extracellular enzyme activity being improved to55.3U mL-1in flaskfermentation. Gs at-3to+50of the pelB site of both genes were-19.10kcal mol-1. Thesefindings suggest that preferred codon usage is advantageous for translational efficiency toincrease protein expression.(2) Batch fermentation was applied, and the extracellular E-co-CGTase enzyme activitywas326%that of wt-CGTase. The results suggested that codon optimization is a reasonablestrategy to improve the yield of-CGTase for industrial application.(3) The Gram-positive bacterium Bacillus megaterium was chosen for production ofrecombinant-CGTase for safety concerns. Protein expression and secretion was ensured byusing the strong inducible promoter Pxyland the signal peptide SPLipA. However, no-CGTasewas detected in the culture supernatant, or in the intracellular soluble or insoluble proteinfractions by SDS-PAGE. Systematic codon usage optimization of the-cgt gene makesexpression of this gene in B. megaterium possible. Data from RT-PCR showed that the expression of-CGTase by codon optimization was mainly due to the enhanced efficiency oftranslation elongation. This is the first time this gene has been expressed in B. megaterium.(4) To enhance the production of-CGTase by B. megaterium, medium optimizationand induction strategies were performed with flask experiments. Modified TB was contained(in g L-1):12glycerol,24yeast extract, and12peptone, with17mM KH2PO4and72mMK2HPO4. Modified induction strategies was as follows: xylose (5g/L) was added into thegrowth medium when the OD578reached0.5, to induce the expression of the recombinantprotein, and the mixture was incubated from37to32C. Batch and simple fed-batchfermentation were performed to enhance-CGTase yield to48.9U/ml, the highest activityreported in a Bacillus species.