Genetically modified strains of Ralstonia eutropha H16 with β-ketothiolase gene deletions for production of copolyesters with defined 3-hydroxyvaleric acid contents.
β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate, Ralstonia eutropha H16 produces the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]. In R. eutropha, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373-5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (phaA, bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, and pcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-type R. eutropha and the mutants. In contrast, during the growth on oleate of a multiple mutant lacking phaA, bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16ΔphaA and all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Nicole
N
Lindenkamp
Elena
E
Volodina
Alexander
A
Steinbüchel
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000102:Acetyl-CoA C-Acyltransferase / Q000172:deficiency* / Q000235:genetics; D001483:Base Sequence; D002210:Caprylates; D020118:Cupriavidus necator / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D019301:Oleic Acid; D009841:Oligonucleotides / Q000235:genetics; D010421:Pentanoic Acids / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011422:Propionates; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014631:Valerates; D000094122:Polyhydroxybutyrates
D000105:Acetyl Coenzyme A; D000102:Acetyl-CoA C-Acyltransferase; D002210:Caprylates; D005227:Fatty Acids; D006885:Hydroxybutyrates; D019301:Oleic Acid; D009841:Oligonucleotides; D010421:Pentanoic Acids; D011091:Polyesters; D011422:Propionates; D014631:Valerates; C013056:beta-hydroxyvaleric acid; C016173:undecanoic acid; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C029658:propionic acid; C031492:octanoic acid