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<p>Polyhydroxyalkanoate (PHA) is a bioplastic attracting interest as an alternative to petroleum-based plastics. Particularly, poly (3-hydroxybutyrate-<i>co</i>-3-hydroxyhexanoate) (P(3HB-<i>co</i>-3HHx)), which shows notable polymeric properties, is usually produced using the engineered <i>Cupriavidus necator</i>. Currently, production of P(3HB-<i>co</i>-3HHx) is primarily possible by engineering <i>phaC</i>, however, relatively rare study of controlling the expression of enoyl-CoA hydratase (<i>phaJ</i><sub><i>Pa</i></sub>), which is directly involved in 3-hydroxyhexanoate (3HHx) monomers synthesis, was shown to control 3HHx mole fraction. As a result, we aimed to verify this by constructing vectors housing <i>phaC</i><sub>BP-M-CPF4</sub> and <i>phaJ</i><sub><i>Pa</i></sub> with different ribosome-binding site (RBS) to control PhaJ translation. When different constructions were applied, the fluctuation in the 3HHx molar fraction was directly related to the <i>phaJ</i><sub><i>Pa</i></sub> RBS sequence and it was shown that varying the RBS sequence to AAAGGAGATATAG produces increased 3HHx mole fraction (3.6–6.2%). When fermentation was performed for 168&#xa0;h to verify the capacity of the engineered strain (H16/pSJ-3) for mass production, it produced 194.9&#xa0;g/L dry cell weight and 155.4&#xa0;g/L of P(3HB-<i>co</i>-9.5&#xa0;mol% 3HHx). Overall, this study presents a different approach of altering polymer properties for manipulating the 3HHx mole fraction of P(3HB-<i>co</i>-3HHx) by controlling PhaJ translation.</p>

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Controlling 3-hydroxyhexanoate mole fraction in poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) by altering enoyl-CoA hydratase (phaJ) ribosome-binding site in Cupriavidus necator H16

  • 期刊论文

  • 发表日期：

    2025年10月11日

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  • Yuni Shin,
  • Gaeun Lim,
  • Yebin Han,
  • Jeong Chan Joo,
  • Hee-Taek Kim,
  • Jong-Min Jeon,
  • Jeong-Jun Yoon,
  • Shashi Kant Bhatia,
  • Yung-Hun Yang

摘要

Polyhydroxyalkanoate (PHA) is a bioplastic attracting interest as an alternative to petroleum-based plastics. Particularly, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), which shows notable polymeric properties, is usually produced using the engineered Cupriavidus necator. Currently, production of P(3HB-co-3HHx) is primarily possible by engineering phaC, however, relatively rare study of controlling the expression of enoyl-CoA hydratase (phaJPa), which is directly involved in 3-hydroxyhexanoate (3HHx) monomers synthesis, was shown to control 3HHx mole fraction. As a result, we aimed to verify this by constructing vectors housing phaCBP-M-CPF4 and phaJPa with different ribosome-binding site (RBS) to control PhaJ translation. When different constructions were applied, the fluctuation in the 3HHx molar fraction was directly related to the phaJPa RBS sequence and it was shown that varying the RBS sequence to AAAGGAGATATAG produces increased 3HHx mole fraction (3.6–6.2%). When fermentation was performed for 168 h to verify the capacity of the engineered strain (H16/pSJ-3) for mass production, it produced 194.9 g/L dry cell weight and 155.4 g/L of P(3HB-co-9.5 mol% 3HHx). Overall, this study presents a different approach of altering polymer properties for manipulating the 3HHx mole fraction of P(3HB-co-3HHx) by controlling PhaJ translation.