A0461, A1526, and B0724 are genes for putative β-oxidation multifunctional enzymes. A high number of genes in R. eutropha H16 are annotated as enzymes that potentially functions in fatty acid β-oxidation, which indicates the possible versatility of this strain for degradation of various hydrophobic compounds. Based on a detailed domain search, we identified 51 genes for acyl-CoA synthetase (ACS), 54 genes for acyl-CoA dehydrogenase (ACDH),
53 genes for enoyl-CoA hydratase (ECH), 3 genes for 3-hydroxyacyl-CoA dehydrogenase (3HCDH), Selleck GDC0449 and 21 genes for β-ketothiolase (KT). In fact, our RNA-seq examination revealed that many genes for putative β-oxidation enzymes were even expressed on fructose, as shown in Figure 4. The previous microarray study revealed that the two gene clusters of H16_A0459-A0464 and H16_A1526-A1531 were induced and in deed played important roles during β-oxidation in the cells grown
on trioleate [18]. It was observed that the cluster H16_A0459-A0464 (which contains ACDH, 3HCDH-ECH fusion, KT, and ECH) was expressed weakly throughout cultivation on fructose, while the cluster H16_A1526-A1531 (which contains TGF-beta family ECH-3HCDH fusion, KT, and ACDH) exhibited approximately 8.5 to 11.4-fold increased expression in the PHA production phase compared with that in the growth phase. fadD3 (H16_A3288), which has been reported very to be induced on trioleate [18], was moderately and constitutively expressed on fructose. H16_B1148, which encodes another ACS, was extremely induced in the PHA production phase. The cluster H16_A1067-A1070 was also induced in the PHA production phase. In particular, the induction ratio and expression levels of H16_A1067 and A1068, both encoding ACDH, were very high in F26. Both of H16_A1069 and A1070 were identified as genes that encode homologs of (R)-specific enoly-CoA hydratase (R-ECH), and the product of H16_A1069 (PhaJ4a) has been
demonstrated to be an R-ECH that is specific to mcl-enoyl-CoAs [11]. These results strongly suggested that fatty acid β-oxidation was functional even in the presence of fructose in R. eutropha H16, and it may have a role in the active turnover of acyl moieties derived from lipids. Tsuge et al. reported that when R. eutropha PHB-4 expressed laboratory-evolved phaC1 from Pseudomonas sp. 61-3, it accumulated PHA co-polyester which contained a small fraction of mcl-3-hydroxyalkanoate units from fructose [15]. It was assumed that the mcl-(R)-3-hydroxyacyl-CoA monomers were provided through the activated β-oxidation linked with lipid turnover when the cells were grown on fructose. The detection of the mcl-CoA-thioesters in R. eutropha H16 cells grown on fructose according to the metabolomic analysis [23] was consistent with this expectation.