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<p><i>Rhodococcus qingshengii</i> 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 <i>Cupriavidus necator</i> H16 genes that are involved in polyhydroxyalkanoate (PHA) biosynthesis, <i>phaC1AB1</i>, was expressed in N9T-4 cells under the promoter of <i>aldA</i> encoding an NAD<sup>+</sup>-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 <i>pha</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月22日

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  • Akihiro Otsuka,
  • Izumi Orita,
  • Hiroya Yurimoto,
  • Nobuyuki Yoshida

摘要

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.