PHBV Accumulation Research Articles

Characterization of the conversion system of natural rubber to poly(3-Hydroxyalkanoate) in Piscinibacter gummiphilus strain NS21T

Poly(3-hydroxyalkanoate) (PHA), a bacteria-synthesized biodegradable polyester, is a useful alternative to fossil resources, and current systems for its production rely predominantly on edible resources, raising concerns about microbial competition for nutrients. Therefore, we investigated mechanisms underlying PHA production from non-edible resources by Piscinibacter gummiphilus strain NS21T. Strain NS21T can utilize natural rubber as a carbon source on solid media and potentially produces PHA. Gas chromatography and nuclear magnetic resonance analyses of NS21T cell extracts revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate) from natural rubber and glucose, respectively. Transcriptional analysis suggested that phaC is involved in PHA production. An increased PHBV accumulation rate under nitrogen-limiting conditions indicates the potential of this strain to be used as a PHBV production enhancement strategy. Furthermore, the disruption of PHA depolymerase genes resulted in enhanced PHA production, indicating the involvement of these genes in PHA degradation. These findings highlight the potential of NS21T for PHBV production from natural rubber, a non-edible resource.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Exploring New Strategies for Optimizing the Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from Methane and VFAs in Synthetic Cocultures and Mixed Methanotrophic Consortia.

In this work, the potential of a synthetic coculture and a mixed methanotrophic consortium to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from renewable and waste-based feedstocks was assessed batchwise. Methylocystis parvuscocultivated with Rhodococcus opacus and a Methylocystis-enriched culture previously grown on methane were subjected to nutrient starvation in a medium enriched with valeric acid (30% w w-1 of Ctot) or with a VFAs mixture containing acetic, propionic, butyric, and valeric acids (15% w w-1 of Ctot) under a CH4:O2 or air atmosphere. For all test series, pH was adjusted to 7 after adding the cosubstrates, and a negligible substrate consumption or polymer production was considered the end point of the trial. Results showed that valeric acid promoted PHBV accumulation in both cultures regardless of the atmosphere. Interestingly, the mixture of VFAs supported PHBV accumulation only in the presence of methane. The highest PHBV contents for the coculture and the mixed consortium, equal to 73.7 ± 2.5% w w-1 and 49.6 ± 13% w w-1, respectively, were obtained with methane and the VFAs mixture. This study demonstrates the suitability of cocultures and biobased cosubstrates for the sustainable production of the biodegradable polymer PHBV.

Biorefinery of volatile fatty acids for the synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Paracoccus sp. TOH: Fermentation performance and metabolic pathway

The residual microbes and uncertain volatile fatty acids (VFAs) components in anaerobic fermentation hydrolysate cause microbial contamination and multi-type polyhydroxyalkanoates (PHA) generation in pure microbial synthesis systems. Meanwhile, the regulation of biosynthesis of poly(3-hydroxybuturate-co-3-hydroxyvalerate) (PHBV), one type of excellent PHA, by re-utilization of waste-organics as carbon substrates is still elusive. In this study, four pretreatment methods for sludge hydrolysate containing 4746 mg-COD·L−1 of VFAs were established to assess the accumulation of PHBV by using Paracoccus sp. TOH as the chassis. Results showed that the autoclaving system exhibited an optimal PHBV concentration of 202.9 mg L−1 with the highest relative abundance of Paracoccus sp. TOH (92.4%). Valeric acid was identified as the primary substrate for 3-hydroxyvalerate accumulation, and the highest PHBV production of 3.76 g L−1 was achieved using 3 g L−1 valeric acid and 5 g L−1 glycerol as co-substrates. The increased carbon metabolism flows through the sequential action of acyl-coenzyme A dehydrogenase and enoyl-coenzyme A hydratase in the fatty acid β-oxidation pathway facilitated PHBV accumulation. This study provides a blueprint for the microbial synthesis of PHBV with oriented acid production from organic wastes.

Enhanced poly(3-hydroxybutyrateco-3-hydroxyvalerate) production from high-concentration propionate by a novel halophile Halomonas sp. YJ01: Detoxification of the 2-methylcitrate cycle

As a carbon substrate, propionate can be used to synthesize poly(3-hydroxybutyrateco-3-hydroxyvalerate) [PHBV] biopolymer, but high concentrations can inhibit PHBV production. Therefore, novel PHBV producers that can utilize high propionate concentrations are needed. Here, a novel halophile, Halomonas sp. YJ01 was applied to PHBV production via a propionate-dependent pathway, and optimal culture growth conditions were determined. The maximum poly(3-hydroxybutyrate) [PHB] content and yield in the presence of glucose were 89.5 wt% and 5.7 g/L, respectively. This strain utilizes propionate and volatile fatty acids (VFAs) for PHBV accumulation. Multiple genes related to polyhydroxyalkanoate (PHA) synthesis were identified using whole-genome annotation. The PHBV yield and 3HV fraction obtained by strain YJ01 utilizing 15 g/L propionate were 0.86 g/L and 29 mol%, respectively, but in cultures with glucose-propionate, it decreased its copolymer dry weight. This indicates that propionyl-CoA was converted to pyruvate through the 2-methylcitrate cycle (2MCC), which reduced propionate detoxification for the strain.

The co-conversion of methane and mixtures of volatile fatty acids into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) expands the potential of an integrated biorefinery

In this work, the potential of Methylocystis hirsuta to simultaneously use methane and volatile fatty acids mixtures for triggering PHBV accumulation was assessed for the first time batchwise. Biotic controls carried out with CH4 alone confirmed the inability of Methylocystis hirsuta to produce PHBV and achieved 71.2 ± 7 g m−3d−1 of PHB. Pure valeric acid and two synthetic mixtures simulating VFAs effluents from the anaerobic digestion of food waste at 35 °C (M1) and 55 °C (M2) were supplied to promote 3-HV inclusion. Results showed that pure valeric acid supported the highest polymer yields of 105.8 ± 9 g m−3d−1 (3-HB:3-HV=70:30). M1 mixtures led to a maximum of 103 ± 4 g m−3d−1 of PHBV (3-HB:3-HV=85:15), while M2 mixtures, which did not include valeric acid, showed no PHV synthesis. This suggested that the synthesis of PHBV from VFAs effluents depends on the composition of the mixtures, which can be tuned during the anaerobic digestion process.

Replication Origin Deletion Enhances Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloarchaea.

Although the use of multiple replication origins for chromosome replication has been widely characterized in haloarchaea, whether it is possible to manipulate the chromosome copy number by their genetic engineering is not known, and how it would affect the cell functioning is poorly understood. Here, we demonstrate that deletion of the three active chromosomal origins in Haloferax mediterranei remarkably reduces its DNA amounts and ploidy numbers. Consequently, the mutant strain H. mediterranei Δ123 is more sensitive to UV and mitomycin C. Surprisingly, the cell size increases by 21.2%, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in shake flask culture enhances from 7.23 to 8.11 g/L in ΔEPSΔ123, although there is also a decrease in cell growth. In this mutant, the chromosomal copy number decreases, whereas the pha-encoding pHM300 megaplasmid copy number increases. Moreover, our transcriptome analysis reveals that the genes involved in primary metabolisms are significantly downregulated in ΔEPSΔ123, whereas those responsible for starch utilization and precursor supplying for PHBV monomers are upregulated. This indicates that more energy and carbon flux is redirected from primary metabolism to PHBV synthesis, thereby enhancing its PHBV accumulation. These findings may therefore provide a rational design to enhance PHBV synthesis by simply tuning the replication origins to modulate the chromosome/megaplasmid copy number ratio and subsequently influence cellular metabolism and physiological functions. IMPORTANCE The haloarchaeon Haloferax mediterranei is a potential producer of PHBV (100% biodegradable plastic) from inexpensive carbon sources. We previously reported that H. mediterranei possessed three active chromosomal origins and, when these origins were deleted, a dormant origin was activated to initiate the replication of chromosome. In this context, in the present study, we first found a close connection between replication initiation and PHBV accumulation. We describe the potential industrial advantages of the strain H. mediterranei ΔEPSΔ123, which includes the enlargement of cell volume by 21.2% and enhancement of PHBV production by 11.2%. We further reveal the possible mechanism that contributes to the greater PHBV production in the ΔEPSΔ123 strain. Overall, we provide here a conceptual advance in the field of synthetic biology by modulating chromosome replication to improve the production of bio-based chemicals.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxvalerate) from volatile fatty acids by Cupriavidus necator.

A promising strategy to alleviate the plastic pollution from traditional petroleum-based plastics is the application of biodegradable plastics, in which polyhydroxyalkanoates (PHAs) have received increasing interest owing to their considerable biodegradability. In the PHAs family, poly(3-hydroxybutyrate-co-3-hydroxvalerate) (PHBV) has better mechanical properties, which possesses broader application prospects. With this purpose, the present study adopted Cupriavidus necator to synthesize PHBV utilizing volatile fatty acids (VFAs) as sole carbon sources. Results showed that the concentration and composition of VFAs significantly influenced the production of PHAs. Especially, even carbon VFAs (acetate and butyrate) synthesized only poly(3-hydroxybutyrate) (PHB), while the addition of odd carbon VFAs (propionate and valerate) resulted in PHBV production. The 3-hydroxyvalerate (3HV) contents in PHBV were directly determined by the specific VFAs compositions, in which valerate was the preferred substrate for 3HV accumulation. After optimization by response surface methodology, the highest PHBV accumulation achieved 79.47% in dry cells, and the conversion efficiency of VFAs to PHBV reached 40%, with the PHBV production of 1.20 ± 0.05 g/L. This study revealed the metabolic rule of VFAs converting into PHAs by C. necator and figured out the optimal VFAs condition for PHBV accumulation, which provides a valuable reference for developing downstream strategies of PHBV production in industrial applications in future.

Bioconversion of waste activated sludge hydrolysate into polyhydroxyalkanoates using Paracoccus sp. TOH: Volatile fatty acids generation and fermentation strategy

The expensive carbon matrix is a bottleneck restricting the industrialization of polyhydroxyalkanoates (PHAs). Volatile fatty acids (VFAs) derived from waste activated sludge via anaerobic fermentation might be alternative carbon matters for PHAs synthesis. In this study, the effect of enzymes on VFAs yields and the feasibility of the produced VFAs for PHAs fermentation by Paracoccus sp. TOH were investigated. The optimum cumulative VFAs concentration reached 4076.6 mg-COD·L−1 in the lysozyme treatment system. Correspondingly, the highest poly(3-hydroxybuturate-co-3-hydroxyvalerate) (PHBV) concentration (119.1 mg·L−1) containing 20.3 mol% 3-hydroxyvalerate was obtained. It proved that Paracoccus sp. TOH possesses the capability for PHBV accumulation. The functional hydrolytic-acidogenic microorganisms, such as Clostridium sensu stricto and Bacteroides sp. were accumulated. The functional genes encoding hydrolysis, carbohydrates metabolism, VFAs generation were enriched. This study offered a possible strategy for VFAs production and verified the feasibility of sludge hydrolysate as a high-quality carbon substrate for PHAs fermentation.

Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux

The haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

Low Crystallinity of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Bioproduction by Hot Spring Cyanobacterium Cyanosarcina sp. AARL T020.

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from cyanobacteria is an environmentally friendly biodegradable polymer. The low yield of PHBV’s production is the main hindrance to its sustainable production, and the manipulation of PHBV production processes could potentially overcome this obstacle. The present research investigated evolutionarily divergent cyanobacteria obtained from local environments of Thailand. Among the strains tested, Cyanosarcina sp. AARL T020, a hot spring cyanobacterium, showed a high rate of PHBV accumulation with a fascinating 3-hydroxyvalerate mole fraction. A two-stage cultivation strategy with sole organic carbon supplementation was successful in maximizing cyanobacterial PHBV production. The use of an optimized medium in the first stage of cultivation provided a 4.9-fold increase in biomass production. Subsequently, the addition of levulinic acid in the second stage of cultivation can induce significant biomass and PHBV production. With this strategy, the final biomass production and PHBV productivity were increased by 6.5 and 73.2 fold, respectively. The GC-MS, FTIR, and NMR analyses confirmed that the obtained PHBV consisted of two subunits of 3-hydroxyvaryrate and 3-hydroxybutyrate. Interestingly, the cyanobacterial PHBV contained a very high 3-hydroxyvalerate mole fraction (94%) exhibiting a low degree of crystallinity and expanding in processability window, which could be applied to polymers for desirable advanced applications.

Deletion of the pps-like gene activates the cryptic phaC genes in Haloferax mediterranei.

Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes, phaC, phaC1, phaC2, and phaC3. In the wild-type strain, except phaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene named pps-like significantly enhanced PHBV accumulation probably resulted from the upregulation of pha cluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence of pps-like gene deletion on the cryptic phaC genes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the ΔEPSΔpps-like geneΔphaC mutant. Sequential knockout of the cryptic phaC genes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in ΔEPSΔpps-like geneΔphaC mutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression of phaC2 did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator of phaC1 gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in ΔEPSΔpps-like geneΔphaC mutant and deletion of pps-like gene released the negative effect from phaC1 expression and thereby restored PHBV accumulating ability in ΔphaC mutant. KEY POINTS: • Cryptic phaC genes were activated by pps-like gene deletion. • PPS-like protein probably regulated phaC1 expression by binding to its promoter. • Both PhaC1 and PhaC3 formed active PHA synthase with PhaE.

Nitrous Oxide Abatement Coupled with Biopolymer Production As a Model GHG Biorefinery for Cost-Effective Climate Change Mitigation.

N2O represents ∼6% of the global greenhouse gas emission inventory and the most important O3-depleting substance emitted in this 21st century. Despite its environmental relevance, little attention has been given to cost-effective and environmentally friendly N2O abatement methods. Here we examined, the potential of a bubble column (BCR) and an internal loop airlift (ALR) bioreactors of 2.3 L for the abatement of N2O from a nitric acid plant emission. The process was based on the biological reduction of N2O by Paracoccus denitrificans using methanol as a carbon/electron source. Two nitrogen limiting strategies were also tested for the coproduction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) coupled with N2O reduction. High N2O removal efficiencies (REs) (≈87%) together with a low PHBV cell accumulation were observed in both bioreactors in excess of nitrogen. However, PHBV contents of 38-64% were recorded under N limiting conditions along with N2O-REs of ≈57% and ≈84% in the ALR and BCR, respectively. Fluorescence in situ hybridization analyses showed that P. denitrificans was dominant (>50%) after 6 months of experimentation. The successful abatement of N2O concomitant with PHBV accumulation confirmed the potential of integrating biorefinery concepts into biological gas treatment for a cost-effective GHG mitigation.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) under photoautotrophy and heterotrophy by non-heterocystous N2-fixing cyanobacterium

The photoautotrophically grown cyanobacterium Oscillatoria okeni TISTR 8549 was found to produce bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This PHBV production occurred under nitrogen deprivation (–N) that yielded PHBV accumulation of 14±4% (w/w DW) in which 3-hydroxyvalerate accounted for 5.5mol%. The heterotrophically grown (–N condition with acetate supplementation) cells under light showed no increase of PHBV storage, but under dark condition these cells increased PHBV accumulation to 42±8% (w/w DW) with 6.5mol% of 3-hydroxyvalerate. Compared to poly-3-hydroxybutyrate (PHB), the PHBV from O. okeni had a lower melting temperature by 5–7°C, a higher % elongation at break by 4–7times and a greater Young’s elastic modulus by 2.3–2.5times.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41–29.1mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0mol% in the biopolymer, indicating promising potentials of commercialization in the future.

Propionyl coenzyme A (propionyl-CoA) carboxylase in Haloferax mediterranei: Indispensability for propionyl-CoA assimilation and impacts on global metabolism.

Propionyl coenzyme A (propionyl-CoA) is an important intermediate during the biosynthesis and catabolism of intracellular carbon storage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in haloarchaea. However, the haloarchaeal propionyl-CoA carboxylase (PCC) and its physiological significance remain unclear. In this study, we identified a PCC that catalyzed propionyl-CoA carboxylation with an acetyl-CoA carboxylation side activity in Haloferax mediterranei. Gene knockout/complementation demonstrated that the PCC enzyme consisted of a fusion protein of a biotin carboxylase and a biotin-carboxyl carrier protein (PccA [HFX_2490]), a carboxyltransferase component (PccB [HFX_2478]), and an essential small subunit (PccX [HFX_2479]). Knockout of pccBX led to an inability to utilize propionate and a higher intracellular propionyl-CoA level, indicating that the PCC enzyme is indispensable for propionyl-CoA utilization. Interestingly, H. mediterranei DBX (pccBX-deleted strain) displayed multiple phenotypic changes, including retarded cell growth, decreased glucose consumption, impaired PHBV biosynthesis, and wrinkled cells. A propionyl-CoA concentration equivalent to the concentration that accumulated in DBX cells was demonstrated to inhibit succinyl-CoA synthetase of the tricarboxylic acid cycle in vitro. Genome-wide microarray analysis showed that many genes for glycolysis, pyruvate oxidation, PHBV accumulation, electron transport, and stress responses were affected in DBX. This study not only identified the haloarchaeal PCC for the metabolism of propionyl-CoA, an important intermediate in haloarchaea, but also demonstrated that impaired propionyl-CoA metabolism affected global metabolism in H. mediterranei.

Characterization of genes for chitin catabolism in Haloferax mediterranei

Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion.

Biosynthesis and Thermal Properties of PHBV Produced from Levulinic Acid by Ralstonia eutropha

Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0–40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2–6 g L−1 LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L−1 ammonium chloride and 2 g L−1 casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L−1 with a PHBV concentration of 12.61 g L−1 (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.

Multiple Propionyl Coenzyme A-Supplying Pathways for Production of the Bioplastic Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) in Haloferax mediterranei

Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ΔphaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction.