Cloning And Functional Analysis Of The PKS Gene In Maituolaimycin Biosynthesis From Streptomyces Luteogriseus

Maituolaimycin is a novel antibiotic produced by Streptomyces luteogriseus, itsstructural information and anti-bacterial performance raises commercial and academicvalues. The cloning of maituolaimycin biosynthetic genes is required in metabolicengineering and productivity improvement. The early results in our lab suggested thatmaituolaimycin synthesis is associated with the PKSs pathway. Based on theinformation of PKSs genes available, here, we try to clone maituolaimycinbiosynthetic genes with PCR, and further to study the function with a gene's disruptionmutant.With optimized procedure of PCR, a fragment of three genes with total length of3659 bp was cloned from Streptomyces luteogriseus genome, designated mai-PKS. Itshows that are four genes in mai-PKS by the sequence analysis with Blast search andCluster alignment with genes available in genebank from other microbes. Four genesare orderly designated mai-MT, mai-KS1, mai-ACP, and mai-KS2.The length of mai-MT is 906 bp, encodes a putative polypeptide of 301 aminoacids. The length of mai-KS1 is 1047 bp, and encodes a putative polypeptide of 348amino acids. Mai-ACP, the smallest gene of four, possesses 249 bp in length andencodes a putative polypeptide of 82 amino acids. The longest gene, mai-KS2 has1341 bp and encodes a putative polypeptide of 447 amino acids. The amino acidsequence of mai-MT shows homologue of 60% to 80% with ACP malonyreansferases.The amino acid sequence of mai-KS1 shows homologue of 65% to 92% with3-oxoacyl-ACP synthases. The amino acid sequence of mai-ACP shows homologue68% to 92% with acyl carrier proteins. The amino acid sequence of mai-KS2 showshomologue of 62% to 90% with β-ketoacyl synthases. All of four genes are similar toPKSs, and highest homologous with corresponding genes from Streptomycesavermitilis. A mutant strain of Streptomyces luteogriseus by disrupting mai-KS2 wasconstructed with the homogenous exchange methods. Metabolites of this mutant wasdetected with LC-MS, the results shows that the mutant does not producemaituolaimycin, suggesting that KS, even mai-PKS, is related with maituolaimycinbiosynthesis in Streptomyces luteogriseus. This study was first time of cloning genesof maituolaimycin biosynthesis from Streptomyces luteogriseus with PCRamplification, proving maituolaimycin biosynthesis is PKSs pathway. It is helpful andpromising for further study on the maituolaimycin biosynthetic gene clusters,molecular engineering of Streptomyces luteogriseus and combinatorial biosynthesisfor hybrid-antibiotics.