Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene.
The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.
Valentin
H F
HF
Department of Biology, James Madison University, Harrisonburg, Virginia 22807, USA. Valenthe@vaxl.acs.jmu.cdu
Dennis
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D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001704:Biopolymers / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D003434:Crossing Over, Genetic; D045647:DNA Glycosylases; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D009615:Nocardia / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D001426:Bacterial Proteins; D001704:Biopolymers; D045647:DNA Glycosylases; D017931:DNA Primers; D004269:DNA, Bacterial; D008765:Methylmalonyl-CoA Mutase; D009699:N-Glycosyl Hydrolases; D011091:Polyesters; C052620:poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate); D000094122:Polyhydroxybutyrates; C079122:MutB protein, Salmonella typhimurium