Functional characterization and enzymatic properties of glycosyltransferase gene ssp803 in carrimycin producing strain
Objective To characterize the function and enzymatic properties of glycosyltransferase gene ssp803 in carrimycin producing bacteria and its effect on carrimycin biosynthesis through in vivo gene inactivation and in vitro enzymatic reaction.Methods The ssp803 gene was inactivated by the CRISPR-Cas9 gene editing system in the carrimycin producing strain 54-IA.Both ssp803 and its homologous gene oleD under the control of the constitutive promoter kasOp* were transformed into the 54-IA and ∆803 mutant,respectively.The changes of isovalerylspiramycins(the major components of carrimycin)production in the ∆803,the complementary strains and ssp803/oleD overexpression strains were detected by HPLC.The ssp803 gene was cloned into expression vector pColdI,expressed in Transetta(DE3).The purified Ssp803 glycosylation assays were performed in the enzymatic reaction with macrolide antibiotics(as aglycones)and uridine diphosphate-glucose(as a glycosyl donor).The glycosylation products were detected by HPLC-MS,and the enzymatic kinetic parameters of Ssp803 were determined using the UDP-Glo assay kit.Results The ssp803 in-frame deletion mutant ∆803 was screened by PCR.The production of isovalerylspiramycins(the major components of carrimycin)in ∆803 was similar to that of starting strain 54-IA.However,isovalerylspiramycins yield was significantly decreased in both ∆803 and 54-IA strains containing highly expressed ssp803 gene,and the production was reduced even more in oleD overexpression in the two strains.Through in vitro enzymatic reaction,the optimal temperature and pH for Ssp803 catalysis were 37℃and 9.06,respectively.The Ssp803 could inactivated all tested macrolides by glycosylation.Its catalytic activity for 16-membered macrolide antibiotics,such as isovalerylspiramycin I,was lower than that of 14-membered macrolides,and the highest catalytic efficiency was achieved for solithromycin.Conclusions The Ssp803 is a glycosyltransferase for macrolides inactivation,being more specific for 14-membered macrolide antibiotics,and optimal aglycone is solithromycin.However,overexpression of ssp803 and oleD genes caused the inhibition of isovalerylspiramycins biosynthesis in carrimycin producing strain.
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