The Research On Cuevaene Biosynthesis And The Function Of Asm43and Asm44in Ansamitocin Biosynthesis

The paper contains two parts, with one related to the research on the cuevaene biosynthesis in Streptomyces sp. LZ35strain, and the other, concentrating on the function of two enzymes involved in the biosynthesis of3-amino-5-hydroxyl benzoic acid(3-HBA), which is the precursor of ansamitocin in Actionosynnema pretiosum ATCC31565.Streptomyces sp. LZ35is a strain of marine actinomycete. Our laboratory identified the compound cuevaene in2010, whose structure has been reported. However, the biosynthesis of it remains to be uncovered. The purpose of the study is to reveal the mechanisms of its biosynthesis and regulation. The work can be devided into three parts. Firstly, knock out eight possible biosynthetic genes and four negative regulator genes of cuv cluster in LZ35Agdm strain and then determine the function of genes according to the differences in the metabolites. Our results showed that chorismate hydrolase(Cuv10) and FAD-binding monooxygenase(Cuv18) are the essencial enzymes in the cuevaene biosynthesis. Secondly, examine the in vitro reaction of Cuv10to confirm its role in the3-hydroxybenzoic acid formation. Thirdly, conduct the constitutive overexpression of two positive regulator genes cuv15(LAL) and cuv20(SARP) in LZ35△gdm strain and then check the differences in cuevaene production.Ansamitocin, produced by A. pretiosum ATCC31565, has strong anticancer activity. It has been reported that the precursor of mC7N starter unit shared by ansamycins, AHBA, is synthesized via a novel aminoShikimate Pathway paralleling to the Shikimate Pathway. Kanosamine has been confirmed to be the precursor of AHBA, while the transformation of UDP-glucose to kanosamine hasnot been totally proved. The second part of the article tried to testify the roles of Asm43and Asm44in the transformation mentioned above. Asm43and asm44were expressed in E.coli and then catalyzed the in vitro reaction. However, our endeavor to reconstruct the transformation of UDP-glucose to UDP-kanosamine or kanosamine was in vain. Finally, the possible combination of Asm43and Asm44is testified by pull-down experiments and unfortunately, the result was far from our expectation.