Construction Of Genetic Engineering Strain For Production Of Dalbavancin Precursor A40926 B

A40926 is secondary metabolisms of Nonomuraea sp.ATCC 39727 used as precursor of a semi-synthetic glycopeptide antibiotic Dalbavancin,which is structurally similar to Teicoplanin and has stronger activity and longer half-life period in clinical trials.The aim of this study is to construct a strain producing A40926 B by genetic engineering based on A416-B4,a mutant of Nonomuraea sp.ATCC 39727 obtained by UV,NTG and protoplast mutation.The results of the paper could simplify the process for production and improve the yield of A40926.It also supplies a basic foundation for industrial-scale production of A40926 B.First,the genetic transformation system(gene knockout)of A416-B4 was optimized according to the reported method.Then,a knockout plasmid pKdbv23 was introduced into A416-B4 to knockout dbv23 gene coding acyltransferase through homologous recombination double-cross.A strain A416-? dbv23 was obtained which mainly produced A40926 B components and no longer produced A40926 PB components.The titer of A40926 B was 1577mg/L in shake flasks culture for A416-?dbv23,an increase by 94% compared with the A416-B4.Two regulator genes dbv3,dbv4 were inserted into integrated expression vector pEva-152 containing erythromycin resistance gene promoter,and transformed into A416-?dbv23 to obtain strains A416-?dbv23-dbv3 and A416-?dbv23-dbv4.The result showed that the effect of dbv4 gene on A40926 B production was slightly better than the dbv3.A416-? dbv23-dbv4 produced 1707mg/L A40926 B in shake flasks culture,an increase by 8.2% compared with the A416-?dbv23 and an increase by110% compared with the A416-B4.