Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Enhancement of polyhydroxybutyrate production by introduction of heterologous phasin combination in Escherichia coli.

Phasin is a surface-binding protein of polyhydroxyalkanoate (PHA) granules that is encoded by the phaP gene. As its expression increases, PHA granules become smaller, to increase their surface area, and are densely packed inside the cell, thereby increasing the PHA content. A wide range of PHA-producing bacteria have phaP genes; however, their PHA productivity differs, although they are derived from the cognate bacterial host cell. Modulating phasin expression could be a new strategy to enhance PHA production. This study aimed to characterize the effect of heterologous phasins on the reconstitution of E. coli BL21(DE3) and determine the best synergistic phaP gene combination to produce polyhydroxybutyrate (PHB). We identified novel phasins from a PHB high-producer strain, Halomonas sp. YLGW01, and introduced a combination of phaP genes into Escherichia coli. The resulting E. coli phaP1,3 strain had enhanced PHB production by 2.9-fold, leading to increased cell mass and increased PHB content from 48 % to 65 %. This strain also showed increased tolerance to inhibitors, such as furfural and vanillin, enabling the utilization of lignocellulose biosugar as a carbon source. These results suggested that the combination of phaP1 and phaP3 genes from H. sp. YLGW01 could increase PHB production and robustness.

Finding of novel polyhydroxybutyrate producer Loktanella sp. SM43 capable of balanced utilization of glucose and xylose from lignocellulosic biomass.

Polyhydroxybutyrate (PHB) is a potential substitute for plastics derived from fossil fuels, owing to its biodegradable and biocompatible properties. Lignocellulosic biomass could be used to reduce PHB production costs; however, the co-utilization of sugars, such as glucose and xylose, without catabolite repression is a difficult problem to be solved. Here, we selected a novel Loktanella sp. SM43 from a marine environment and optimized the conditions for PHB production. Loktanella sp. SM43 showed high PHB production (66.5% content) from glucose. When glucose and xylose were used together, this strain showed high utilization of both substrates compared to other high PHB-producers such as Halomonas sp. and Cupriavidus necator, which showed glucose preference. Loktanella sp. SM43 showed high growth and PHB production with lignocellulosic hydrolysates. When pine tree hydrolysates were used, PHB production was the highest at 3.66 ± 0.01 g/L, followed by Miscanthus (3.46 ± 0.09 g/L) and barley straw hydrolysate (3.36 ± 0.36 g/L). Overall, these results reveal the potential of Loktanella sp. SM43 to produce PHB using various lignocellulosic hydrolysates as feedstock and the first systematic study for PHB production with Loktanella sp. The approach of screening novel strains is a strategy to overcome co-utilization of sugars without genetic engineering.

Novel Poly(butylene adipate-co-terephthalate)-degrading Bacillus sp. JY35 from wastewater sludge and its broad degradation of various bioplastics.

Poly(butylene adipate-co-terephthalate) (PBAT), a bioplastic consisting of aliphatic hydrocarbons and aromatic hydrocarbons, was developed to overcome the shortcomings of aliphatic and aromatic polyesters. Many studies report the use of PBAT as a blending material for improving properties of other bioplastics. However, there are few studies on microorganisms that degrade PBAT. We found six kinds of PBAT-degrading microorganisms from various soils. Among these, Bacillus sp. JY35 showed superior PBAT degradability and robustness to temperature. We monitored the degradation of PBAT films by Bacillus sp. JY35 using scanning electron microscopy, field emission scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. GC-MS was used to measure the PBAT film degradation rate at different temperatures and with additional NaCl and carbon sources. Certain additional carbon sources improve the growth of Bacillus sp. JY35. However, this did not increase PBAT film degradation. Time-dependent PBAT film degradation rates were measured during three weeks of cultivation, after which the strain achieved almost 50% degradation. Additionally, various bioplastics were applied to solid cultures to confirm the biodegradation range of Bacillus sp. JY35, which can degrade not only PBAT but also PBS, PCL, PLA, PHB, P(3HB-co-4HB), P(3HB-co-3HV), P(3HB-co-3HHx), and P(3HB-co-3HV-co-3HHx), suggesting its usability as a superior bioplastic degrader.