Molecular Cloning Of The Gene Cluster ThuABCDEFG For Thuringiensin Biosynthesis And Analysis Of The Synthesis Pathway In Bacillus Thuringiensis Strain Ct-43

Thuringiensin,briefly named Thu,is a small molecular weight nucleotide insecticidal toxin produced by some strains of Bacillus thuringiensis.It has high toxic to dipteran,coleopteran, lepidopteran,orthoptera,hymenoptera and some species of nematode.Until now the separation and purification technologies for B.thuringiensis Thu have been improved a lot, however,its biosynthesis regulatory mechanisms and pathway remain unknown.In our study of Bacillus thuringiensis strain CT-43 which produces high production of Thu, we firstly located the Thu biosynthesis cluster on a large plasmid harboring crylB gene namely pBMB0558 by plasmids elimination assay and Southern blot experiment.Then,we cloned and sequenced pBMB0558 based on the construction of an E.coli-B.thuringiensis shuttle BAC library of strain CT-43.pBMB0558 is 109,464 bp plasmid in length.It has a NRPS\PKS cluster depended on an acyl carrier protein(ACP) protein,a typeâ…£secretion system-like gene domain with VirB4,VirB11 and VirD4 and some mobile genetic elements included a prophage and transposases.Next we carried out the heterogenous expression of those BAC clones in plasmid-cured B.thuringiensis BMB171 and obtained a clone BMB0542 which could produce high Thu.By reducing the insertion fragment of BMB0542,we located the Thu cluster on a 12-kb fragment and named it thuABCDEFG.The sequence determination showed this fragment is from pBMB0558.Based on the bioinformatics analysis of its sequence,we deduced Thu biosynthesis' pathway:biosynthesis of allose diacid and assembly of adenosine glucose allose diacid,the intermediate of Thu.The synthesis precursor of allose diacid was possibly 6-phosphate glucose.The assembly process is similar to the NRPS\PKS system.This pathway could be classified into three functional modules:Module 1,the recognition and hanging for allose diacid;Module 2,the transfer of glycoside;Module 3,the recognition and elongation with adenosine.In Module 1,there were three domains.One was ACP from PKS system while the adenylation domain A and condensation domain C were always found in NRPS system.We call this system RPS\PKS-like.Maybe the methyltransferase near the cluster relates to the post treatment of Thu.To confirm our deduced pathway,we carried out the interruption experiment on thuE gene in the cluster thuABCDEFG and compared both supernatants and intracellular metabolic products with CT-43 and thuE-interrupted mutant BMB0545 by LC-micrOTOF and LCMS-IT-TOF analysis.We found that adding phosphate group to adenosine glucose allose diacid is the last step during the Thu biosynthesis.