Cloning Of The Antibiotic HYGROCINS Biosynthesis Gene Cluster

Streptomyces hygroscopicus ATCC 29253, the main producer of rapamycin isolated from Easter Island soil sample by Vezina, was found to produce a new antibiotic hygrocins in 2005. Hygrocins including hygrocin A and hygrocin B were first discovered by cocrystallization with rapamycin during purification process of rapamycin. According to chemistry structure, hygrocins belongs to ansamycins family and should have the antibacterial activity.In order to study the biosynthetic pathway of hygrocins, a genomic cosmid library of S. hygroscopicus ATCC 29253 was constructed and screened by PCR using the primer pairs that were designed for amplification of 3-amino-5-hydroxybenzoic acid (AHBA) synthase genes according to amino acid sequence of their conserved motifs. AHBA synthase is an essential enzyme catalyzing production of AHBA precursors for anamycins biosynthesis. Overlapping analysis and subcloning sequencing of the selected seven positive cosmid clones, cosmid 13F11 containing AHBA catalytic enzyme gene and polyketide synthase (PKS) gene was chosen for shotgun sequencing. Base on the flanking sequence of cosmid 13F11, more putative hygrocins biosynthetic genes were fished out by chromosome walking. So far, two cosmids,55.444kb in total were fully sequenced, including five necessary genes for AHBA synthesis, five polyketide synthase genes for the catalytic aliphatic bridge formation, three oxidoreductase and others genes involved in postmodification, regulation and transportation. To investigate function of the cloned gene cluster, the AHBA synthase gene hgnI and a polyketide synthase gene hgnE were disrupted by single homologous recombination in S. hygroscopicus chromosome. HPLC analysis found that two disruption mutations lost the ability of hygrocins production. It confirmed that the cloned gene cluster is response for catalyzing biosynthesis of hygrocins.