Chemoenzymatic Synthesis Technics Of Oxazole-containing Polyketone Derivatives

Conglobatin is an oxazole-containing polyketide with 16-membered macrodiolide originally isolated from Streptomyces conglobatus.The biosynthesis of oxazole-containing conglobatin is directed by a multi-enzyme assembly line of non-ribosomal peptide synthetase（NRPS）and polyketide synthase（PKS）,in which an uncanonical iterative-acting C-terminal thioesterase domain,Cong-TE,ligates two fully elongated chains/conglobatin-monomer（5）on the terminal acyl carrier protein and forms the linear conglobatin-dimer（6）,and subsequently cyclized the resulting dimer to a C₂-symmetric macrodiolide.Screening of the conglobatin producer for secondary metabolites led to the discovery of two new compounds conglactones A（1）and B（2）featuring the ester-bond linked hybrid structure consisting of an aromatic polyketide benwamycin I（3）and one（for 1）/two（for 2）molecules of 5.To investigate the biosynthetic origin of 1 and 2,we constructed two mutants of S.conglobatus containing respective in-frame deletions of cong E,a phosphohydrolase homologous gene in conglobatin biosynthesis gene cluster（cong BGC）,and nft M1,a PKS gene in the BGC of 3（nft BGC）.The mutation results combined with the gene complementation of mutantΔcong E confirmed that the production of compounds 1 and 2 was associated with cong BGC and nft BGC.Based on the above results,we hypothesized that Cong-TE could employ free alcohols,e.g.,compound 3,for nucleophilic attack to theα-carbonyl of cong-monomer or cong-dimer bound on Cong-TE,and release the hybrid polyketides of 1 and 2.Biochemical analysis revealed that 1 and 2 were produced by Cong-TE from 3 and an N-acetylcysteamine thioester form of 5（7）.Furthermore,the substrate compatibility of Cong-TE was demonstrated by enzymatically generating bunch of ester products with 7 and exotic alcohols.We tested sixty-nine alcohol substrates and forty-three of them were successfully to be converted into hybrid polyketide molecules.This property of Cong-TE was further validated by producing thirty-six hybrid esters in the fermentation of conglobatin producer fed with non-indigenous alcohols.The compatibility of Cong-TE showed a prospect for scaling up the production of hybrid polyketides via fermentation strategy.In conclusion,we discovered two new oxazole-containing hybrid polyketides,conglactones A（1）and B（2）and revealed the biosynthetic mechanism of them.We also characterized the potential to develop the Cong-TE as a biocatalytic tool to subsequently green synthesis active oxazole-containing hybrid polyketides.It will creat the conditions for the technology development of green synthesis of new structured oxazole polyketide medicinal molecules.