Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse ß-oxidation from glucose in Ralstonia eutropha H16.

BACKGROUND: Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. RESULTS: This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse ß-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), ß-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. CONCLUSION: These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.

Surface Modification of Sponge-like Porous Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/Gelatine Blend Scaffolds for Potential Biomedical Applications.

In this study, we described the preparation of sponge-like porous scaffolds that are feasible for medical applications. A porous structure provides a good microenvironment for cell attachment and proliferation. In this study, a biocompatible PHA, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was blended with gelatine to improve the copolymer's hydrophilicity, while structural porosity was introduced into the scaffold via a combination of solvent casting and freeze-drying techniques. Scanning electron microscopy results revealed that the blended scaffolds exhibited higher porosity when the 4HB compositions of P(3HB-co-4HB) ranged from 27 mol% to 50 mol%, but porosity decreased with a high 4HB monomer composition of 82 mol%. The pore size, water absorption capacity, and cell proliferation assay results showed significant improvement after the final weight of blend scaffolds was reduced by half from the initial 0.79 g to 0.4 g. The pore size of 0.79g-(P27mol%G10) increased three-fold while the water absorption capacity of 0.4g-(P50mol%G10) increased to 325%. Meanwhile, the cell proliferation and attachment of 0.4g-(P50mol%G10) and 0.4g-(P82mol%G7.5) increased as compared to the initial seeding number. Based on the overall data obtained, we can conclude that the introduction of a small amount of gelatine into P(3HB-co-4HB) improved the physical and biological properties of blend scaffolds, and the 0.4g-(P50mol%G10) shows great potential for medical applications considering its unique structure and properties.

Genetic incorporation of oil-utilizing ability in Cupriavidus malaysiensis USMAA2-4 for sustainable polyhydroxyalkanoates production from palm olein and 1-pentanol.

The sustainability in polyhydroxyalkanoates (PHA) production is drawing increasing attention as the effort to increase the economic feasibility for commercialization pursues. Oleic acid is widely preferred by bacteria but its employment for PHA production makes sustainability rather dubious. This study showed promising poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] content of 68 wt % by lipase genes-harbouring Cupriavidus malaysiensis USMAA2-4 transformant from palm olein and 1-pentanol. High oleic acid content and low oil saturation caused palm olein to outperform crude palm oil, crude palm kernel oil and soybean oil due to its preference for oleic acid shown by previous screening. The transformant showed 8-fold and 40-fold higher lipase activity compared to C. necator H16 and its wild-type respectively. The transformant was unaffected by Co2+ but the growth of C. necator H16 was inversely proportional to Co2+ concentration and the employment of 1-pentanol also ceased its growth and PHA accumulation. Although the inhibitory effect of Fe2+, Cu2+ and Zn2+ at high molarity on LipA decreased PHA content of C. malaysiensis USMAA2-4 transformant by 23-24 wt %, the lipase activity was restorable with high molarity of Ca2+, thus resulted in higher PHA content. The transformant enabled the employment of low-cost 1-pentanol as the precursor for cost-effective PHA production and its preference for palm olein contributed to higher sustainability.

P(3HB-co-4HB) as high value polyhydroxyalkanoate: its development over recent decades and current advances.

Polyhydroxyalkanoate (PHA) is a biogenic polymer that has the potential to substitute synthetic plastic in numerous applications. This is due to its unique attribute of being a biodegradable and biocompatible thermoplastic, achievable through microbial fermentation from a broad utilizable range of renewable resources. Among all the PHAs discovered, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] stands out as a next generation healthcare biomaterial for having high biopharmaceutical and medical value since it is highly compatible to mammalian tissue. This review provides a critical assessment and complete overview of the development and trend of P(3HB-co-4HB) research over the last few decades, highlighting aspects from the microbial strain discovery to metabolic engineering and bioprocess cultivation strategies. The article also outlines the relevance of P(3HB-co-4HB) as a material for high value-added products in numerous healthcare-related applications.

High PHA density fed-batch cultivation strategies for 4HB-rich P(3HB-co-4HB) copolymer production by transformant Cupriavidus malaysiensis USMAA1020.

P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).

Production of high molecular weight poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer by Cupriavidus malaysiensis USMAA1020 utilising substrate with longer carbon chain.

Long carbon chain alkanediols are used in the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], however these substrates possess high toxicity towards bacterial cells. This study demonstrated the effective utilisation of a long carbon chain alkanediol, namely 1,8-octanediol, to enhance the yield and production of a copolymer with a high molecular weight of over 1000 kDa, which is desirable for novel applications in medical and biopharmaceuticals. The increased PHA content (47-61 wt%) and concentration (1.7-4.5 g/L) was achieved by additional feeding of a combination of C4 substrates at C/N 10, with 1,8-octanediol + γ-butyrolactone producing P(3HB-co-22 mol% 4HB) with a high molecular weight (1060 kDa) and elongation at break of 970%. The DO-stat feeding strategy of C/N 10 has shown an increment of PHA concentration for both carbon combination, 0.45-4.27 g/L and 0.32-3.36 g/L for 1,8-octanediol + sodium 4-hydroxybutyrate (4HB-Na) and 1,8-octanediol + γ-butyrolactone, but with a slight reduction on molecular weight and mechanical strength. Nonetheless, further study revealed that a nitrogen-absence feeding strategy could retain the high molecular weight and elongation at break of the copolymer, and simultaneously improving the overall P(3HB-co-4HB) production.

A fed-batch strategy to produce high poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) terpolymer yield with enhanced mechanical properties in bioreactor.

This study reports an efficient fed-batch strategy to improve poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer production by Cupriavidus sp. USMAA2-4 with enhanced mechanical properties in bioreactor. The cultivations have been performed by combining oleic acid with γ-butyrolactone at different concentration ratios with 1-pentanol at a fixed concentration. The batch and fed-batch fermentations have resulted in P(3HB-co-3HV-co-4HB) with compositions of 9-35 mol% 3HV and 4-24 mol% 4HB monomers. The DO-stat fed-batch fermentation strategies have significantly improved the production with a maximum 4.4-fold increment of cell dry weight (CDW). Besides, appropriate feeding of the substrates has resulted in an increment of terpolymer productivity from 0.086-0.347 g/L/h, with a significantly shortened cultivation time. The bacterial growth and terpolymer formation have been found to be affected by the concentration of carbon sources supplied. Characterization of P(3HB-co-3HV-co-4HB) has demonstrated that incorporation of 3HV and 4HB monomer has significantly improved the physical and thermodynamic properties of the polymers, by reducing the polymer's crystallinity. The tensile strength, Young's modulus of the terpolymer has been discovered to increase with the increase of M w. The fed-batch fermentation strategies employed in this study have resulted in terpolymers with a range of flexible materials having improved tensile strength and Young's modulus as compared to the terpolymer produced from batch fermentation. Possession of lower melting temperature indicates an enhanced thermal stability which broadens the polymer processing window.

Pilot scale production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) biopolymers with high molecular weight and elastomeric properties.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [(P(3HB-co-4HB)] copolymer receives attention as next generation biomaterial in medical application. However, the exploitation of the copolymer is still constrained since such copolymer has not yet successfully been performed in industrial scale production. In this work, we intended to establish pilot production system of the copolymer retaining the copolymer quality which has recently discovered to have novel characteristic from lab scale fermentation. An increase of agitation speed has significantly improved the copolymer accumulation efficiency by minimizing the utilization of substrates towards cell growth components. This is evidenced by a drastic increase of PHA content from 28 wt% to 63 wt% and PHA concentration from 3.1 g/L to 6.5 g/L but accompanied by the reduction of residual biomass from 8.0 g/L to 3.8 g/L. Besides, fermentations at lower agitation and aeration have resulted in reduced molecular weight and mechanical strength of the copolymer, suggesting the role of sufficient oxygen supply efficiency in improving the properties of the resulting copolymers. The KLa-based scale-up fermentation was performed successfully in maintaining the yield and the quality of the copolymers produced without a drastic fluctuation. This suggests that the scale-up based on the KLa values supported the fermentation system of P(3HB-co-4HB) copolymer production in single-stage using mixed-substrate cultivation strategy.

Microbial-based synthesis of highly elastomeric biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thermoplastic.

This study reports the production of P(3HB-co-4HB) [Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)] in possession of high molecular weight and elastomeric properties by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate cultivation system. 1,4-butanediol and 1,6-hexanediol are found to be efficient substrate mixture that has resulted in high copolymer yield, occupying a maximum of 70wt% of the total biomass and producing higher 4HB monomer composition ranging from 31mol% to 41mol%. In substrate mixtures involving 1,6-hexanediol, cleavage of the 6-hydroxyhexanoyl-CoA produces Acetyl-CoA and 4-hydroxybutyryl-CoA. Acetyl-CoA is instrumental in initiating the cell growth in the single-stage fermentation system, preventing 4-hydroxybutyryl-CoA from being utilized via ß-oxidation and retained the 4HB monomer at higher ratios. Macroscopic kinetic models of the bioprocesses have revealed that the P(3HB-co-4HB) formation appears to be in the nature of mixed-growth associated with higher formation rate during exponential growth phase; evidenced by higher growth associated constants, α, from 0.0690g/g to 0.4615g/g compared to non-growth associated constants, ß, from 0.0092g/g/h to 0.0459g/g/h. The P(3HB-co-31mol% 4HB) produced from the substrate mixture exhibited high weight-average molecular weight, Mw of 927kDa approaching a million Dalton, and possessed elongation at break of 1637% upon cultivation at 0.56wt% C. This is the first report on such properties for the P(3HB-co-4HB) copolymer. The copolymer is highly resistant to polymer deformation after being stretched.

Synthesis of high 4-hydroxybutyrate copolymer by Cupriavidus sp. transformants using one-stage cultivation and mixed precursor substrates strategy.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.

En route to economical eco-friendly solvent system in enhancing sustainable recovery of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer.

Separation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco-balance as P(3HB-co-4HB) is widely used for biomedical applications. Therefore, an efficient and eco-based extraction of P(3HB-co-4HB) using a combination of NaOH and Lysol in digesting the non-polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB-co-4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.

Biosynthetic enhancement of single-stage Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production by manipulating the substrate mixtures.

Two-stage fermentation was normally employed to achieve a high poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] productivity with higher 4HB molar fraction. Here, we demonstrated single-stage fermentation method which is more industrial feasible by implementing mixed-substrate cultivation strategy. Studies on bioreactor scale show a remarkably high PHA accumulation of 73 wt%, contributing to a high PHA concentration and product yield of 8.6 g/L and 2.7 g/g, respectively. This fermentation strategy has resulted in copolymers with wider range of 4HB monomer composition, which ranges from 12 to 55 mol%. These copolymers show a broad range of weight average molecular weight (M w ) from 119.5 to 407.0 kDa. The copolymer characteristics were found to be predominantly affected by the nature of the substrates and the mixture strategies, regardless of the 4HB monomer compositions. This was supported by the determination of copolymer randomness using (13)C-NMR analysis. The study warrants significantly in the copolymer scale-up and modeling at industrial level.