Cloning And Enzymological Research On CefG Gene Of Cephalosporium Acremonium

Cephalosporin C(CPC), one of the industrially important β-lactam antibiotics, has a high demand for producing anti-infectious and semi-synthetic antibiotics. Cephalosporium acremonium is the major filamentous fungi to manufacture CPC by fermentation in industry. To date,the improvement of CPC- producing strain is usually involved manipulation of the key enzyme-coding gene in CPC biosynthesis pathway. For example, cefEF was cloned and reintroduced into the host to improve the productivity and activity of enzyme. The final step, transformation of deacetylcephalosporin C (DAC) into CPC in the biosynthesis pathway of CPC is catalyzed by an acetyltransferase encoded by cefG.Since cefG is a gene with two introns in Cephalosporium acremonium genome, RT-PCR was performed to amplify ～1.1Kb cefG after good quality of total RNA was obtained by an adapted method from CPC-producing strain. Alignment with published sequences in GenBank showed only one base difference at DNA level and identical amino acid sequence.The cefG gene was cloned into an E.coli vector pLY-5 for high expression of acetyltransferase. However, it was found that the cloned gene could not express in E.coli ,which may result from the differences of codon bias beween two strains. An obvious band was observed in SDS-PAGE electrophoresis when cefG was cloned into plasmid pET28a for fusion expression. The optimal conditions for the soluble expression of this fusion protein were determined. The soluble protein is bioactive, and DAC was partly transformed to CPC in vitro.Furthermore, NTA resin was used to purify the recombinant protein. Purified DAC acetyltransferase was acquired after washing and elution with buffer NTA-60 and NTA-200. Meanwhile, the optimal enzyme activity was obtained in 50mmol/L CaCl₂, potassium phosphate buffer, pH 7.0 at 44℃. Then maximum transformation can be seen at 10 minutes when substrate concentration was lower than 5mmol/L, while Michaelis constant (Km) of recombinant DAC-acetyltransferase was 7.649×10^-4mol/L by double reciprocal plotting. This work gives a promising perspective for in vivo expression of cefG to increase the transformation efficiency of final CPC in biosynthesis pathway as well as CPC production.