Production of poly(3-hydroxybutyrate) by Rhodococcus qingshengii N9T-4 using the oligotrophic gene expression system.

Rhodococcus qingshengii N9T-4 is a super oligotroph that grows on a completely inorganic basal medium without any additional carbon and nitrogen sources. This oligotrophy may facilitate low-carbon and low-cost bioprocessing for compounds of economic significance. In this study, an operon containing three Cupriavidus necator H16 genes that are involved in polyhydroxyalkanoate (PHA) biosynthesis, phaC1AB1, was expressed in N9T-4 cells under the promoter of aldA encoding an NAD-dependent aliphatic aldehyde dehydrogenase, which is highly expressed under the oligotrophic conditions. The three proteins were successfully expressed, and Nile Red-stained colonies of the transformants showed the remarkable fluorescence, suggesting PHA accumulation in the cells. Only 3-hydroxybutyrate was detected during fatty-acid methyl ester analysis, confirming that poly(3-hydroxybutyrate) (20.8 ± 1.4 wt% of the dry cell weight) was produced in N9T-4 cells expressing the pha genes. Furthermore, carbon and nitrogen sources were examined for the PHA production; ethanol was the most effective carbon source. Transmission electron microscopy of the transformants revealed PHA granules in the PHA-producing N9T-4 cells. Although the use of ethanol may not seem oligotrophic, it did not affect oligotrophic gene expression; that is, oligotrophic metabolism proceeded in cells grown on ethanol. Thus, this study demonstrates the oligotrophic production of PHA, providing low-carbon and low-cost production of PHA.