Development of classical and recombinant DNA techniques for genetic studies on the pleuromutilin producing basidiomycete Clitopilus passeckerianus (Pilat) Singer Strain NRRL3100

• Main Authors: Raymundo, A.K., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Biological Sciences, Tavanlar, M.A.T., Philippines Univ. Los Baños, College, Laguna (Philippines). National Inst. of Molecular Biology and Biotechnology, Papa, I.A, Zulaybar, T.O., Philippines Univ. Los Baños, College, Laguna (Philippines). National Inst. of Molecular Biology and Biotechnology, Barnes, A.A., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Biological Sciences, Aurin, C.LM, Teves, F., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Biological Sciences, Zarate, J.T., Philippines Univ. Los Baños, College, Laguna (Philippines). Inst. of Biological Sciences
• Format: Publication
• Language: English
• Published: College, Laguna (Philippines)
• Subjects: ANIMAL HEALTH; BASIDIOMYCOTINA; http://www.fao.org/aos/agrovoc#c_13629; http://www.fao.org/aos/agrovoc#c_23897; http://www.fao.org/aos/agrovoc#c_33270; http://www.fao.org/aos/agrovoc#c_34079; http://www.fao.org/aos/agrovoc#c_431; http://www.fao.org/aos/agrovoc#c_5014; MUTACION; MUTATION; PCR; PROTOPLASTE; PROTOPLASTOS; PROTOPLASTS; SANIDAD ANIMAL; SANTE ANIMALE; TIAMULIN; TIAMULINA; TIAMULINE

Pleuromutilin, the precursor for the semi-synthetic Tiamulin used solely for animal health, is produced by Clitopilus passeckerianus and some other organisms. Its activity is mainly against penicillin and streptomycin-resistant staphylococci; Streptococcus, Enterococcus, Bacilus subtilis and some strains of Escherichia coli, Ebertthella typhii and Vibrio cholera. This research was initiated to develop classical and molecular approaches in the study of the genetic system of C. passeckerianus. Mutagenesis of C. passeckerianus NRRL 3100 was done using N-Methyl-N'-Nitro-N-Nitrosoguanidine (NTG) and acridine orange (AO). Four NTG mutants with the highest levels of antibiotic production and four non-producing AO mutants shown by cup cylinder assay were further studied. After several months in storage, the supposed non-producers, LP5 and LP6, reverted back to antibiotic production but at levels 8-17% lower than the wild type. All four non- or low producers were induced to produce conidia as opposed to the wild type and the high producers which did not conidiate. By comparing mycelial dry weight, utilization patterns of vitamins, carbon and nitrogen compounds, differences were observed among the mutants. These indicated that certain metabolic pathways may have been affected by mutation. Subtractive hybridization of Sau3A-digested DNA of wild type strain and Pst1-digested DNA of LP5, a low producer, did not result in the cloning of any subtracted fragment using BamH1-digested pBS as vector. Cloning of certain genes in the biosynthetic pathway was done through polymerase chain reaction (PCR). The initial gene studied was geranyl transferace (GGT) gene, an enzyme involved in the biosynthetic pathway to pleuromutilin. The sequence of the gene was obtained from GENBANK database and several sets of primers were designed. Using degenerate primers, the non-producing mutant Cp M5 yielded 8 bands while the wild type yielded only 3 bands as PCR products. The possibility of insertion is considered in the light of such results. With the use of primers based on the inward sequence of GGT, a 200-bp PCR product was obtained. This 200-bp product was cloned into E. coli competent cell using the TOPO cloning kit. Positive clones were viewed as white colonies on Luria-Bertani agar medium containing 5-bromo-4-chloro-3-indolyl-Beta-D-galactoside (X-gal) and 50 ug/ml ampicillin. The 200-bp product was also submitted for sequencing using an ABI PRISM 3100 Sequencer. To be able to introduce any gene, a transformation system had to be developed. Protoplast preparation has then to be optimized to be used for transformation. Protoplasts at the rate of 11.5 × 106/ml after 2.5 h incubation with Novozym 234 at 37 deg C were obtained. Protoplasts regenerants were observed on Mycological Agar supplemented with the osmotic stabilizer, sorbitol at 0.4 and 0.6 M concentrations. Co-transformation with a marked plasmid known to integrate into the chromosome of many fungi will be done to determine success of integration. Southern blotting will also be done to confirm integration.