Strategic Use of Vegetable Oil for Mass Production of 5-Hydroxyvalerate-Containing Polyhydroxyalkanoate from δ-Valerolactone by Engineered Cupriavidus necator

Abstract

Although efforts have been undertaken to produce polyhydroxyalkanoates (PHA) with various monomers, the low yield of PHAs because of complex metabolic pathways and inhibitory substrates remains a major hurdle in their analyses and applications. Therefore, we investigated the feasibility of mass production of PHAs containing 5-hydroxyvalerate (5HV) using δ-valerolactone (DVL) without any pretreatment along with the addition of plant oil to achieve enough biomass. We identified that PhaC_BP-M-CPF4, a PHA synthase, was capable of incorporating 5HV monomers and that C. necator PHB^-4 harboring phaC_BP-M-CPF4 synthesized poly(3HB-co-3HHx-co-5HV) in the presence of bean oil and DVL. In fed-batch fermentation, the supply of bean oil resulted in the synthesis of 49 g/L of poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol%5HV) from 66 g/L of biomass. Thermophysical studies showed that 3HHx was effective in increasing the elongation, whereas 5HV was effective in decreasing the melting point. The contact angles of poly(3HB-co-3HHx-co-5HV) and poly(3HB-co-3HHx) were 109 and 98°, respectively. In addition, the analysis of microbial degradation confirmed that poly(3HB-co-3HHx-co-5HV) degraded more slowly (82% over 7 days) compared to poly(3HB-co-3HHx) (100% over 5 days). Overall, the oil-based fermentation strategy helped produce more PHA, and the mass production of novel PHAs could provide more opportunities to study polymer properties.

Keywords: 5-hydroxyvalerate; plant oil; polyhydroxyalkanoates; δ-valerolactone.

Figure 1

The overall methodological framework of the experiment. The experiment proceeded in the following order: establishing a strain capable of producing poly(3HB-co-5HV), confirming the increase in PHA production containing 5HV through vegetable oil feeding, verifying the feasibility of mass-producing PHA containing 5HV through vegetable oil-based fermentation, and finally, analyzing the physical properties of the produced PHA.

Figure 2

Validation of the 5HV polymerization ability of wild-type Cupriavidus necator H16 and C. necator PHB⁻⁴ harboring various PHA synthases. (a) Wild-type C. necator H16 (b) C. necator PHB⁻⁴ harboring phaC_Ac (c) C. necator PHB⁻⁴ harboring phaC_Ra (d) C. necator PHB⁻⁴ harboring phaC_BP-M-CPF4. Statistical analysis was performed by applying ANOVA with the level of significance at 5%.

Figure 3

Confirmation of increased production of poly(3HB-co-3HHx-co-5HV) after supplying bean oil. (a) 5-hydroxyvaleric acid (5HVA) or (b) δ-valerolactone (DVL) was used as the 5HV precursor. (c) N-source optimization for poly(3HB-co-3HHx-co-5HV) production. (d) DVL feeding time optimization. Statistical analysis was performed by applying ANOVA with a level of significance at 5%.

Figure 4

Fed-batch Poly(3HB-co-3HHx-co-5HV) production by C. necator PHB⁻⁴ harboring phaC_BP-M-CPF4 in a 5-L jar fermenter. The initial culture conditions included 1% fructose, 0.5% bean oil, and 0.1% NH₄NO₃. From 10 h to 20 h of culture, 100 g/L of bean oil was supplied, and 5 g/L of DVL was added after 48 h. The changes in (a) DCW (Dry Cell Weight) and PHA, as well as (b) the molar fractions of 3HHx and 5HV, were monitored over the cultivation period. Statistical analysis was performed by applying ANOVA with the level of significance at 5%.

Figure 5

Time-dependent degradation rate of (a) poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol% 5HV) and (b) poly(3HB-co-7.1 mol% 3HHx) films by Microbulbifer sp. Sol66. Statistical analysis was performed by applying ANOVA with the level of significance at 5%.

Figure 6

Analysis of changes in contact angle over time for extracted (a) poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol% 5HV) and (b) poly(3HB-co-7.1 mol% 3HHx) films. Statistical analysis was performed by applying ANOVA with the level of significance at 5%.