Optimizing Biosynthetic Pathway Of Daptomycin In Streptomyces Roseosporus

The drug-resistance of pathogenic bacteria is becoming more and more serious,due to the widely used of antibiotics in clinic.Now,highly resistant gram-positive bacterial pathogens appear constantly,such as methicillin-resistant Staphylococcus aureus,vancomycin-resistant enterococcus and penicillin-resistant Streptococcus pneumonia.New antibiotics against resistant-pathogens are required urgently.Daptomyicn is a kind of cyclic lipopeptides,extracted from fermentation broth of Streptomyces roseosporus.It is considered as the best alternative to vancomycin,because of its high antibacterial activity to gram-positive pathogens.At present,there are profound studies of daptomyicn on the structure,property and function mechanism as well as the synthesis process.However,up till the present moment,the production of daptomycin was only improved slightly,by metabolic engineering and random mutation.We analyzed the common cyclic lipopeptides and their biosynthesis genes involved in activation of fatty acid side chains,and speculated that LptEF was the fusion protein of acyl CoA ligase and acyl carrier protein.We expressed and purified the DptE(acyl CoA ligase)and DptF(ACP)as a fusion protein.We found the catalytic efficiency of fusion protein DptEF is obviously increasing in vitro.Then we fused dptE and dptF in vivo,the flask fermentation results showed that the daptomycin production of FdptEF was obviously improved(26.7%),as the fermentation period is shorten by 24 hours.After adjusting the feeding medium from 0.1%to 0.15%,we improved daptomycin production by 59.4%.The 13th amino acid of daptomycin nucleus,kynurenine,is a nonprotein amino acid,which produced by the metabolism of tryptophan.In Streptomyces,tryptophan is oxidized to N-formyl-L-kynurenine by tryptophan-2,3-dioxgenase,subsequently hydrolysed to kynurenine by formamidase.Eventually kynurenine is hydrolysed by kynureninase.By deleting the gene sro3365,we confirmed there was only one kynurenine formamidase in S.roseosporus L30.We found that sro3367 was transcribed constantly in bacteria growth,while the transcript of dptJ increased along with daptpmycin production.We overexpressed dptJ,sro3367 and sro3365 respectively,and deleted sro3366.In these mutant strains,daptomyicn production increased in different degrees.And we combined all the manipulations including knockout sro3366 and overexpressed dptJ,sro3367 and sro3365,then improved daptomyicn production by 92%.Finally,we improved daptomyicn production by 140%,through combined the two optimizing approaches.