Improvement Of Ansamitocins By Adaptive Evolution And Culture Conditions Optimization

Ansamitocins,a kind of antitumor macrolide antibiotic,are produced by Actinosynnema pretiosum spp.auranticum ATCC 31565.Ansamitocin P-3(AP-3)manifests the highest anticancer activity in ansamitocins derivatives.Currently,a novel anticancer drug named Kadcyla(T-DM1)has been approved by FDA,where ansamitocins could be used as its payload.However,the production of ansamitocins is limited in microorganisms.It is therefore necessary to improve the biosynthesis of ansamitocins.The aim of this study was to explore the strategies of improving AP-3 production by adaptive evolution and bioprocess optimization.Firstly,A.pretiosum spp.auranticum was used as starting strain in adaptive evolution.Improvement of AP-3 by adaptive evolution was conducted with the appropriate addition of purified ansamitocin P-3,rifampin,streptomycin and paromomycin,respectively.Two strains with enhanced production,namely Str 4 and Par 1,have been obtained.The AP-3 contents increased by 33.4%and 31.7%,and ansamitocin yield increased by 52.2%and 46.4%,respectively,compared to those of the starting strain.Next,the transcriptional differences between evoved mutants and starting strain were analyzed.A few genes on ansamitocin biosynthetic gene clusters were up-regulated to different extent.The results exhibited that secondary metabolism of ansamitocins was enhanced obviously.Meanwhile,rel A which is related to biosynthesis of ppGpp synthetase was also up-regulated,indicating that ppGpp biosynthesis was enhanced and along with the overproduction of ansamitocins.Finally,some culture conditions of the evolved mutants were adjusted for better production of target metabolite.The results of phosphate supplementation suggested that in comparison with the starting strain,the evolved mutants showed less responses to phosphate level in the medium.As the methylation donor and osmotic pressure regulator,betaine was found to promote the metabolism of this strain.The production of AP-3 was further enhanced by the addition of 0.1%betaine to the evolved mutants.The AP-3 productions of two evolved strains were increased by 54.6%and 47.4%,respectively and the yields were increased by 79.1%and 66.7%respectively,indicating the favorable effects of betaine on the production of ansamitocins.