Engineering of β-oxidation pathway to tailor polyhydroxyalkanoate (PHA) production in Bacillus thermoamylovorans

Abstract

Polyhydroxyalkanoate (PHA) is a type of microbial polyester that is stored within cells as a carbon and energy source. This study evaluated the effects of knockout gene mutation on the production and modification of PHA in Bacillus thermoamylovorans. The genes fadB and fadA, which encode 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase, respectively, in B. thermoamylovorans were deleted to suppress the β-oxidation pathway. The potential of the wild type and mutant to produce PHA under various fatty acids was subsequently studied. The wild type produced the highest biomass and PHA content, measuring 4.5 ± 0.3 g/l and 41.4 ± 1.0%, respectively, when grown on sodium octanoate. The similar pattern was observed in mutant strain, which yielded 3.8 ± 0.2 g/l of biomass and 41.0 ± 1.0% PHA under the same condition. In addition, heterogeneous PHA was detected in both the wild-type and mutant strains. These findings indicate that fadB and fadA are crucial for fatty acid degradation. However, deleting fadB and fadA did not negatively affect biomass or PHA production. Furthermore, the production of PHA was scaled up in a 3-l fermenter. High values of the specific growth rate, biomass productivity, and maximum productivity were detected in the wild-type. Interestingly, the mutant strain produced medium-co-long-chain-length (mcl-co-lcl) PHA, whereas the wild-type strain primarily synthesized short-chain-length (scl) PHA. Compared with scl-PHA, mcl-PHA is more advantageous because of its superior elasticity, reduced crystallinity and tensile strength, and elevated melting point. This study represents the first report of enhanced PHA production in B. thermoamylovorans through knockout of the fadB and fadA genes.