Site-Directed Mutagenesis And Functional Analysis Of A Glycosyltransferase Med-ORF8 During Medermycin Biosynthesis

Medermycin isolated from Streptomyces sp. AM-7161 is an aromatic polyketide antibiotic possessing antitumor and antibacterial activity. The structure of medermycin features in a rare C-C glycosidic bond which joins a polyketide skeleton with a novel angolasamine (deoxyhexose). med-ORF8 located in the medermycin biosynthetic gene cluster was previously proved as an indispensable gene for medermycin biosynthesis. It was deduced to be responsible for the C-glycosylation. By far the structure and the catalytic mechanism of Med-ORF8 still remain unknown. In the present study, bioinformatics analysis, site-directed mutagenesis and prokaryotic expression of Med-ORF8 were carried out and main results are summarized as follows:(1) Bioinformatics analysis of Med-ORF8To determine the catalytic site of Med-ORF8 for mutagenesis, homology analysis showed that Med-ORF8 belongs to the C-glycosyltransferase family; Analysis of the basic parameters of Med-ORF8, such as composition of amino acid, molecular weight, theoretical isoelectric point and hydrophobicity indicated that Med-ORF8 should be an unstable, acidic and hydrophobic protein. Secondary structure analysis showed that Med-ORF8 consists of 11β-strands and 14α-helices and a glycosyltransferase domain. Three-dimensional structure modeling of Med-ORF8 showed that it resembles the structure of UrdGT2 (a C-glycosyltransferase for urdermycin biosynthesis).(2) Improvement of the megaprimer site-directed PCR mutagenesis method and incorporation of a site mutation into Med-ORF8Amino acid sequences of three types of glycosyltransferases were aligned using online clustalW (http://align.genome.jp/) to search for the difference and similarity. Based on the bioinformatics analysis, we decided to change the amino acid I to D (at the position 307) of Med-ORF8.To incorporate the site mutation into Med-ORF8, a megaprimer PCR method was improved, including:selection of a plasmid containing the target gene as template instead of a target gene. The resultant mutant PCR product with different size could be distinguished from the template plasmid on electrophoresis; Avoiding using a normal primer pair in one reaction for amplification of a full length gene; Choosing different vectors with different selection markers for cloning of wild type and mutant gene respectively. Therefore, contamination of wild type genes was completely eliminated for subsequent selection of recombinant strains on antibiotics-containing medium. We mutated I307 of Med-ORF8 as D307 successfully by the improved megaprimer PCR method and the resultant gene with the site mutation was named as med-ORF8*(3) Establishment and optimization of the prokaryotic expression system of mutated med-ORF8*The resultant glycosyltransferase gene (med-ORF8*) was inserted into the prokaryotic expression vector pET-28a to generate the prokaryotic expression plasmid pHSL74-6 which then transformed into E.coli BL21 (DE3) strain. The protein of a molecular weight of 43 KD with His-tag was successfully expressed with a high efficiency, but mainly in the form of inclusion body. To solve this problem, systemic optimization of expression conditions for med-ORF8* was subsequently conducted, including induction temperature (8℃), inducer concentration (0.05 mM for IPTG or 0.05 %for lactose) and induction duration (20-24 h).(4) Functional analysis of Med-ORF8*The med-ORF8* gene with a point mutation was cloned into the Streptomyces expression vector pWHM4* to obtain the Streptomyces expression plasmid pHSL73-2 which then transformed into Streptomyces coelicolor CH999/pHSL3, which carries the medermycin biosynthetic gene cluster with replacement of med-ORF8 gene by the apramycin-resistant gene. The resultant transformants can grow on selective medium and produce indicative pigments, indicating that Med-ORF8* is functional. Further analysis of the metabolitic products of resultant transformants is under the way to determine the influence of the mutation (I307 to D307) of Med-ORF8.(5) Characterization of a new metabolite related to medermycin biosynthesis in Streptomyces sp. AM-7161HPLC/MS analysis revealed the obvious accumulation of a new compound X as well as medermycin in the fermentation broth of both Streptomyces sp. AM-7161 and heterologous system of medermycin biosynthetic gene cluster, indicating that X is related to the biosynthesis of medermycin. Based on the full-wavelength absorption pattern and molecular weight analysis, X is supposed to be an intermediate during medermycin biosynthesis. The purification conditions and structure determination of X is in progress.