Boosting D-carbamoylase activity of recombinant Bacillus subtilis by adjusting gene dosage and central carbon metabolism

Abstract

D-p-hydroxyphenylglycine (D-HPG) as an intermediate of semisynthetic antibiotics has an important value in the pharmaceutical industry. The high pollution and high costs of chemical synthesis make D-HPG production by biocatalysis more promising. The hydantoinase method requires D-hydantoinase and D-carbamoylase (DCase) to convert D,L-p-hydroxyphenylhydantoin (D,L-HPH) into D-HPG. The recombinant Bacillus subtilis used for the whole-cell catalysis in this process needs to improve the activity and stability of DCase. The gene dosage and cell metabolism of DCase affect its activity, and this study intends to reduce the acidification effect caused by carbon catabolite repression at the genetic level. Among strains with different gene dosages, the double-copy integrated strain DN02 had the highest DCase average activity of 132 U/g dry cell weight (gDCW). When glucose was used as the carbon source, weakening glucose absorption can significantly alleviate the acidification of fermentation broth. The glcT mutant reduced the average glucose absorption rate by about 57%, whereas the DCase activity increased to about 518 U/gDCW. In addition, modifying the CcpA-binding site in citZ and the CodY-binding site in citB to increase their expression levels can also relieve the acidification of fermentation broth, which reduced the accumulation of acetate by 24% and 17%, respectively. The DCase activity of derivative strains DN16 and DN17 can reach about 615 and 641 U/gDCW. Comparing the catalytic activity of strains to dual-enzyme activities to produce D-HPG, the average whole-cell activity of strain DN17/pUBS was about fivefold higher than that of DN02/pUBS. These strategies might also be useful for other recombinant strains to express heterologous enzymes. Isolation of nucleic acids from various cells is a step of PCR. In this study, magnetic nanoparticles can be used to extract genomic DNA and total RNA due to their paramagnetism and biocompatibility. The amount and accuracy of DNA and total RNA extracted were verified by Polymerase Chain Reaction (PCR). The method has the advantages of removing dangerous reagents such as phenol and chloroform, replacing inorganic coating such as silica with organic oil, and shortening reaction time.