Engineered Production Of Albomycins In Heterologous Streptomyces Hosts

Albomycins,a peptidyl nucleoside antibiotic,have potent antimicrobial activities against many Gram-negative bacteria and Gram-positive bacteria.Albomycins consist of a iron carrier,a unique thiofuranose and a highly modified cytosine.This allows albomycins to be actively transported via the bacterial iron uptake system into bacterial cells,where they are hydrolyzed the“Trojan horse”antibiotic albomycin to release SB-217452 by host peptidases.SB-217452 has been established as an inhibitor of bacterial seryl-t RNA synthetase,providing albomycins with potent and intriguing biological activity.They serve as promising candidates for the development of new antibacterial drugs or drug leads.However,application of albomycins are limited by the fact that yield of albomycins in the native producer Streptomyces griseus ATCC 700974 in quite low.Therefore,to increase titers of albomycins,this study aims to construct a high-yield strain by means of molecular biology.The main results of this article are as follows:1.The albomycins biosynthetic gene cluster were cloned by the Transformation-Associated Recombination in Yeast(TAR)system to p CAP01.The gene cluster is about34.3 kb in size and contains 18 structural genes with no regulatory genes.To facilitate genetic manipulation in the host,the gene cluster was transferred to p SET152 by PCR targeting to generate pGN001.2.The recombinant plasmid pGN001 was introduced into Streptomyces coelicolor CH999,S.coelicolor M1146,S.coelicolor M1152,S.coelicolor M1154,S.lividans TK23,S.lividans TK24,and S.albus J1074 by E.coli-Streptomyces interspecies conjugation.The albomycin gene cluster was successfully expressed in all heterologous hosts,and S.coelicolor M1146 was selected as the best suitable heterologous host.3.To increase the production of albomycins in a heterologous host,the promoters of the key structural genes within the gene cluster were replaced with constitutive promoters.The result showed that abmJ and abmK in the gene cluster driven by P_{hrd B} and P_{Kas O*}respectively,albomycins production increased only on the second day,and there was no significant difference in the highest output.Abm J and abmK in the gene cluster driven by P_SF14 and P_{hrd B} respectively,the production of albomycins increased.4.To further increase the production of albomycins,a series of the genetically modified gene clusters were introduced into S.coelicolor M1246,M1346,and M1446 to obtain recombinant strains containing 2,3 and 4 copies of the gene clusters,respectively.The results shows that the production of albomycins were increased in these strains compared with that with a single copy of the gene cluster.In summary,the biosynthetic gene cluster for albomycins biosynthesis were successfully expressed in several Streptomyces hosts.A high-yielding strain producting albomycins were constructed through genetic engineering,which laid a good foundation for subquent heterogeneous production.