Polyhydroxyalkanoate Synthase Research Articles

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

Engineering Pseudomonas chlororaphis HT66 for the Biosynthesis of Copolymers Containing 3-Hydroxybutyrate and Medium-Chain-Length 3-Hydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are promising alternatives to petroleum-based plastics, owing to their biodegradability and superior material properties. Here, the controllable biosynthesis of scl-co-mcl PHA containing 3-hydroxybutyrate (3HB) and mcl 3-hydroxyalkanoates was achieved in Pseudomonas chlororaphis HT66. First, key genes involved in fatty acid β-oxidation, the de novo fatty acid biosynthesis pathway, and the phaC1-phaZ-phaC2 operon were deleted to develop a chassis strain. Subsequently, an acetoacetyl-CoA reductase gene phaB and a PHA synthase gene phaC with broad substrate specificity were heterologously expressed for producing and polymerizing the 3HB monomer with mcl 3-hydroxyalkanoates under the assistance of native β-ketothiolase gene phaA. Furthermore, the monomer composition of scl-co-mcl PHA was regulated by adjusting the amount of glucose and dodecanoic acid supplemented. Notably, the cell dry weight and scl-co-mcl PHA content reached 14.2 g/L and 60.1 wt %, respectively, when the engineered strain HT11Δ::phaCB was cultured in King's B medium containing 5 g/L glucose and 5 g/L dodecanoic acid. These results demonstrated that P. chlororaphis can be a platform for producing scl-co-mcl PHA and has the potential for industrial application.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production

BackgroundAmong the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation.ResultsThe N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB−4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4.ConclusionThis research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Phenotypic and genotypic characterization of Marinobacterium weihaiense sp. nov. and Marinobacterium marinum sp. nov., isolated from marine sediment, and genomic properties of the genus Marinobacterium.

In this study, two novel bacterial strains were isolated from coastal sediment of Weihai, China. The two strains were Gram-stain-negative and facultatively aerobic, designated 3-1745T and A346T. Based on phenotypic, genetic and phylogenetic properties, strains 3-1745T and A346T represent two novel species of the genus Marinobacterium. The results of genome analysis revealed many central carbohydrate metabolism pathways such as gluconeogenesis, pyruvate oxidation, tricyclic acid cycle, pentose phosphate pathway and PRPP biosynthesis in the genus Marinobacterium. The ability of strains 3-1745T and A346T to utilize volatile fatty acids was experimentally confirmed. Polyhydroxyalkanoate synthases (PhaA, PhaB and PhaC) for the synthesis of polyhydroxyalkanoates were prevalent in the genus Marinobacterium. Multiple BGCs (biosynthetic gene clusters) including betalactone, ectoine, ranthipeptide, redox-cofactor, RiPPs (ribosomally synthesized post-translationally modified peptides) and T3PKS (polyketide synthases) in the genome of the genus Marinobacterium were found. Additional genome analyses suggested that the genus Marinobacterium contained diverse potential mechanisms of salt tolerance and mainly utilized oligosaccharides. This is the first report on broad genomic analyses of the genus Marinobacterium with the description of two novel species and potential ecological and biotechnological implications.

Toward the production of block copolymers in microbial cells: achievements and perspectives.

The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaCAR, enabled the direct synthesis of PHA-PHA type block copolymers in microbial cells. PhaCAR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaCAR can synthesize poly(D-lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. KEY POINTS: • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties.

Production of polyhydroxyalkanoates using sewage and cheese whey

Recently, polyhydroxyalkanoates (PHAs) have been produced using raw sewage in our laboratory; however, the production concentrations are low. Therefore, this study aimed to enhance PHA production by applying different strategies. PHA production was higher in sewage-containing medium than in mineral salt medium and was enhanced 22-fold after glucose supplementation. A relatively high degree of glucose consumption (83.6 ± 1.59 %) was also achieved. Bacteria incubated with cheese whey diluted with sewage showed higher PHA production than bacteria incubated with cheese whey diluted with distilled water did. The expression of the PHA synthase gene (phaC) was evaluated via real-time polymerase chain reaction using low- and high-carbon-containing sewage. Relatively higher phaC expression levels were observed in high-carbon-containing sewage but at lower nitrogen concentrations. The characteristics of the produced PHA were comparable to those of standard PHA. Therefore, this study revealed that the bacterium Bacillus sp. CYR1 can produce PHA from low- or high-carbon-containing wastewater.

Application of PHA surface binding proteins of alkali-tolerant Bacillus as surfactants.

Amphiphilic protein has lipophilic and hydrophilic domains, displaying the potential for development as a biosurfactant. The polyhydroxyalkanoate (PHA) surface binding protein derived from Bacillus is a type of protein that has not been studied for its emulsifying properties. In this study, PHA granule-associated protein (PhaP), PHA regulatory protein (PhaQ), and PHA synthase subunit (PhaR) derived from an alkali-tolerant PHA-producing Bacillus cereus HBL-AI were found and heterologously expressed in E. coli and purified to investigate their application as biosurfactants. It showed that the emulsification ability and stability of three amphiphilic proteins were higher than those of widely used chemical surfactants in diesel oil, vegetable oil, and lubricating oil. In particular, the PhaQ protein studied for the first time can form a stable emulsion layer in vegetable oil at a lower concentration (50µg/mL), which greatly reduced the amount of protein used in emulsification. This clearly demonstrated that the PHA-binding protein of HBL-AI can be well applied as an environmentally friendly biosurfactants.

Bioplastic (poly-3-hydroxybutyrate)-producing Massilia endophytica sp. nov., isolated from Cannabis sativa L. ‘Cheungsam’

A rod-shaped, motile, Gram-negative bacterial strain named DM-R-R2A-13T was isolated from the plant Cannabis sativa L. ‘Cheungsam’. The phylogenetic analysis of the 16S rRNA gene sequence revealed that strain DM-R-R2A-13T belongs to the family Oxalobacteraceae and is closely related to members of the genus Massilia, with Massilia flava (97.58% sequence similarity) and Massilia armeniaca (97.37% sequence similarity) being the closest members. The digital DNA-DNA hybridization (dDDH) values between strain DM-R-R2A-13T and Massilia flava CGMCC 1.10685T and Massilia armeniaca ZMN-3Twere 22.2% and 23.3%, while the average nucleotide identity (ANI) values were 78.85% and 79.63%, respectively. The DNA G+C content was measured to be 64.6 mol%. Moreover, the bacterium was found to contain polyhydroxyalkanoate (PHA) granules based on transmission electron microscopy, indicating its potential to produce bioplastic. Genome annotation revealed the presence of PHA synthase genes (phaC, phaR, phaP, and phaZ), and the biopolymer was identified as poly-3-hydroxybutyrate (PHB) based on nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) analyses. Using maltose as a carbon source, the strain produced PHB of up to 58.06% of its dry cell weight. Based on the phenotypic, chemotaxonomic, and phylogenetic characteristics, it has been determined that DM-R-R2A-13T represents a novel species belonging to the genus Massilia. As such, the name Massilia endophytica sp. nov. is proposed for this newly identified species. The type strain is DM-R-R2A-13T (= KCTC 92072T = GDMCC 1.2920T).

(R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent l-valine depletion caused by dominant l-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell.Key points(R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha(R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identifiedThe engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HBGraphical

Economical synthesis of γ-cyclodextrin catalyzed by oriented cyclodextrin glycosyltransferase displayed on bacterial polyhydroxyalkanoate nanogranules

BackgroundThe advantages of γ-cyclodextrin (γ-CD) include its high solubility, ability to form inclusion complexes with various poorly water-soluble molecules, and favorable toxicological profile; thus, γ-CD is an attractive functional excipient widely used in many industrial settings. Unfortunately, the high cost of γ-CD caused by the low activity and stability of γ-cyclodextrin glycosyltransferase (γ-CGTase) has hampered large-scale production and application.ResultsThis study reports the in vivo one-step production of immobilized γ-CGTase decorated on the surface of polyhydroxyalkanoate (PHA) nanogranules by the N-terminal fusion of γ-CGTase to PHA synthase via a designed linker. The immobilized γ-CGTase-PHA nanogranules showed outstanding cyclization activity of 61.25 ± 3.94 U/mg (γ-CGTase protein) and hydrolysis activity of 36,273.99 ± 1892.49 U/mg, 44.74% and 18.83% higher than that of free γ-CGTase, respectively. The nanogranules also exhibited wider optimal pH (cyclization activity 7.0–9.0, hydrolysis activity 10.0–11.0) and temperature (55–60 °C) ranges and remarkable thermo- and pH-stability, expanding its utility to adapt to wider and more severe reaction conditions than the free enzyme. A high yield of CDs (22.73%) converted from starch and a high ratio (90.86%) of γ-CD in the catalysate were achieved at pH 9.0 and 50 °C for 10 h with 1 mmol/L K+, Ca2+, and Mg2+ added to the reaction system. Moreover, γ-CGTase-PHA beads can be used at least eight times, retaining 82.04% of its initial hydrolysis activity and 75.73% of its initial cyclization activity.ConclusionsThis study provides a promising nanobiocatalyst for the cost-efficient production of γ-CD, which could greatly facilitate process control and economize the production cost.

Genome-centric polyhydroxyalkanoate reconciliation reveals nutrient enriched growth dependent biosynthesis in Bacillus cereus IBA1

Microbiological polyhydroxyalkanoates (PHAs) are rooted as the most promising bio-replacements of synthetic polymers. Inherent properties of these PHAs further expand their applicability in numerous industrial, environmental, and clinical sectors. To propel these, a new environmental, endotoxin free gram-positive bacterium i.e., Bacillus cereus IBA1 was identified to harbor advantageous PHA producer characteristics through high-throughput omics mining approaches. Unlike traditional fermentations, nutrient enriched strategy was used to enhance PHA granular concentrations by ∼2.3 folds to 2.78 ± 0.19 g/L. Additionally, this study is the first to confirm an underlying growth dependent PHA biogenesis through exploring PHA granule associated operons which harbour constitutively expressing PHA synthase (phaC) coupled with differentially expressing PHA synthase subunit (phaR) and regulatory protein (phaP, phaQ) amid different growth phases. Moreover, the feasibility of this promising microbial phenomenon could propel next-generation biopolymers, and increase industrial applicability of PHAs, thereby significantly contributing to the sustainable development.

Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol

BackgroundThis study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions.ResultsA newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH4Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate.ConclusionCupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

Ability of converting sugarcane bagasse hydrolysate into polyhydroxybutyrate (PHB) by bacteria isolated from stressed environmental soils

We isolated 109 potential polyhydroxybutyrate (PHB)-producing bacteria from various soil sources. Using Sudan Black B staining, lipid granules were observed in 65 isolates. UV spectrophotometry-based quantification revealed that 14 isolates effectively produced PHB, and all isolates carried the phaC gene encoding polyhydroxyalkanoate synthase required for PHB polymerization. After cultivation at 30 °C for 72 h in E2 medium containing 3.5% (w/v) glucose as the carbon source and 0.354% (w/v) NH4Y as the nitrogen source, the highest PHB content (%-CDW) and yield (g-PHB/g-glucose) with values of 20.4% and 0.28 and 19.7% and 0.07 were obtained for Priestia megaterium KKR5 and Burkholderia cepacia ASL22, respectively. TEM analysis revealed PHB granules with diameters of 0.2–0.7 μm inside KKR5 and ASL22 cells. We achieved the bioconversion of sugarcane bagasse hydrolysate (BGH) into PHB by these strains via separate hydrolysis and fermentation. H3PO4 steam-pretreated BGH generated reducing sugars containing glucose and xylose without toxic chemicals. Using the reducing sugars of BGH as the carbon source, ASL22 exhibited a higher PHB yield (0.22 g-PHB/g-reducing sugar) than KKR5 (0.12 g-PHB/g-reducing sugar). PHB from KKR5 and ASL22 had FTIR and NMR spectra that were similar to those of standard PHB. A GC/MS analysis confirmed the molecular structure of PHB as methyl hydroxybutyrate with a mass value of 43–103. By DSC analysis, the melting temperatures of PHB from KKR5 and ASL22 and standard PHB were 167.8, 168.5, and 176.6 °C, respectively. Thus, PHB-producing bacteria isolated from stressed soils successfully utilize a low-cost BGH substrate for PHB accumulation.

Real-time NMR analysis of polyhydroxyalkanoate synthase reaction that synthesizes block copolymer comprising glycolate and 3-hydroxybutyrate

The sequence-regulating polyhydroxyalkanoate (PHA) synthase PhaCAR spontaneously synthesizes the homo-random block copolymer, poly[3-hydroxybutyrate (3HB)]-b-poly[glycolate (GL)-ran-3HB]. In this study, a real-time in vitro chasing system was established using a high-resolution 800 MHz nuclear magnetic resonance (NMR) and 13C-labeled monomers to monitor the polymerization of GL-CoA and 3HB-CoA into this atypical copolymer. Consequently, PhaCAR initially consumed only 3HB-CoA and subsequently consumed both substrates. The structure of the nascent polymer was analyzed by extracting it with deuterated hexafluoro-isopropanol. In the primary reaction product, a 3HB-3HB dyad was detected, and GL-3HB linkages were subsequently formed. According to these results, the P(3HB) homopolymer segment is synthesized prior to the random copolymer segment. This is the first report of its kind which proposes the application of real-time NMR to a PHA synthase assay, paving the way for elucidating the mechanisms of PHA block copolymerization.

Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally diverse monomer units.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.

Complete genome sequence of Aquitalea pelogenes USM4 (JCM19919), a polyhydroxyalkanoate producer.

Polyhydroxyalkanoate (PHA) is a type of biopolymer produced by most bacteria and archaea, resembling thermoplastic with biodegradability and biocompatibility features. Here, we report the complete genome of a PHA producer, Aquitalea sp. USM4, isolated from Perak, Malaysia. This bacterium possessed a 4.2Mb circular chromosome and a 54,370bp plasmid. A total of 4067 predicted protein-coding sequences, 87 tRNA genes, and 25 rRNA operons were identified using PGAP. Based on ANI and dDDH analysis, the Aquitalea sp. USM4 is highly similar to Aquitalea pelogenes. We also identified genes, including acetyl-CoA (phaA), acetoacetyl-CoA (phaB), PHA synthase (phaC), enoyl-CoA hydratase (phaJ), and phasin (phaP), which play an important role in PHA production in Aquitalea sp. USM4. The heterologous expression of phaC1 from Aquitalea sp. USM4 in Cupriavidus necator PHB-4 was able to incorporate six different types of PHA monomers, which are 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), 3-hydroxyhexanoate (3HHx) and isocaproic acid (3H4MV) with suitable precursor substrates. This is the first complete genome sequence of the genus Aquitalea among the 22 genome sequences from 4 Aquitalea species listed in the GOLD database, which provides an insight into its genome evolution and molecular machinery responsible for PHA biosynthesis.

Genotypic and Phenotypic Detection of Polyhydroxyalkanoate Production in Bacterial Isolates from Food.

Polyhydroxyalkanoates (PHAs) are widely used in medical and potentially in other applications due to their biocompatibility and biodegradability. Understanding PHA biosynthetic pathways may lead to the detection of appropriate conditions (substrates) for producing a particular PHA type by a specific microbial strain. The aim of this study was to establish a method enabling potentially interesting PHA bacterial producers to be found. In the study, all four classes of PHA synthases and other genes involved in PHA formation (fabG, phaA, phaB, phaG, and phaJ) were detected by PCR in 64 bacterial collection strains and food isolates. Acinetobacter, Bacillus, Cupriavidus, Escherichia, Klebsiella, Lelliottia, Lysinibacillus, Mammaliicoccus, Oceanobacillus, Pantoea, Peribacillus, Priestia, Pseudomonas, Rahnella, Staphylococcus, and Stenotrophomonas genera were found among these strains. Fructose, glucose, sunflower oil, and propionic acid were utilized as carbon sources and PHA production was detected by Sudan black staining, Nile blue staining, and FTIR methods. The class I synthase and phaA genes were the most frequently found, indicating the strains' ability to synthesize PHA from carbohydrates. Among the tested bacterial strains, the Pseudomonas genus was identified as able to utilize all tested carbon sources. The Pseudomonas extremorientalis strain was determined as a prospect for biotechnology applications.

Unsterile production of a polyhydroxyalkanoate copolymer by Halomonas cupida J9

Microbial production of bioplastics polyhydroxyalkanoates (PHA) has opened new avenues to resolve “white pollution” caused by petroleum-based plastics. PHAs consisting of short- and medium-chain-length monomers, designated as SCL-co-MCL PHAs, exhibit much better thermal and mechanical properties than PHA homopolymers. In this study, a halophilic bacterium Halomonas cupida J9 was isolated from highly saline wastewater and proven to produce SCL-co-MCL PHA consisting of 3-hydroxybutyrate (3HB) and 3-hydroxydodecanoate (3HDD) from glucose and glycerol. Whole-genome sequencing and functional annotation suggest that H. cupida J9 may possess three putative PHA biosynthesis pathways and a class I PHA synthase (PhaCJ9). Interestingly, the purified His6-tagged PhaCJ9 from E. coli BL21 (DE3) showed polymerizing activity towards 3HDD-CoA and a phaCJ9-deficient mutant was unable to produce PHA, which indicated that a low-substrate-specificity PhaCJ9 was exclusively responsible for PHA polymerization in H. cupida J9. Docking simulation demonstrated higher binding affinity between 3HB-CoA and PhaCJ9 and identified the key residues involved in hydrogen bonds formation between 3-hydroxyacyl-CoA and PhaCJ9. Furthermore, His489 was identified by site-specific mutagenesis as the key residue for the interaction of 3HDD-CoA with PhaCJ9. Finally, PHA was produced by H. cupida J9 from glucose and glycerol in shake flasks and a 5-L fermentor under unsterile conditions. The open fermentation mode makes this strain a promising candidate for low-cost production of SCL-co-MCL PHAs. Especially, the low-specificity PhaCJ9 has great potential to be engineered for an enlarged substrate range to synthesize tailor-made novel SCL-co-MCL PHAs.

Continuous Supply of Non-Combustible Gas Mixture for Safe Autotrophic Culture to Produce Polyhydroxyalkanoate by Hydrogen-Oxidizing Bacteria.

Polyhydroxyalkanoates (PHAs) are eco-friendly plastics that are thermoplastic and biodegradable in nature. The hydrogen-oxidizing bacterium Ralstonia eutropha can biosynthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], the most common PHA, from carbon dioxide using hydrogen and oxygen as energy sources. In conventional autotrophic cultivation using R. eutropha, a gas mixture containing 75–80 vol% hydrogen is supplied; however, a gas mixture with such a high hydrogen content has a risk of explosion due to gas leakage. In this study, we aimed to develop an efficient cell culture system with a continuous supply of a non-combustible gas mixture (H2: O2: CO2: N2 = 3.8: 7.3: 13.0: 75.9) for safe autotrophic culture to produce P(3HB) by hydrogen-oxidizing bacteria, with a controlled hydrogen concentration under a lower explosive limit concentration. When the gas mixture was continuously supplied to the jar fermentor, the cell growth of R. eutropha H16 significantly improved compared to that in previous studies using flask cultures. Furthermore, an increased gas flow rate and agitation speed enhanced both cell growth and P(3HB) production. Nitrogen source deficiency promoted P(3HB) production, achieving up to 2.94 g/L P(3HB) and 89 wt% P(3HB) content in the cells after 144 h cultivation. R. eutropha NCIMB 11599, recombinant R. eutropha PHB-4, and Azohydromonas lata grew in a low-hydrogen-content gas mixture. R. eutropha H16 and recombinant R. eutropha PHB-4 expressing PHA synthase from Bacillus cereus YB-4 synthesized P(3HB) with a high weight-average molecular weight of 13.5–16.9 × 105. Thus, this autotrophic culture system is highly beneficial for PHA production from carbon dioxide using hydrogen-oxidizing bacteria as the risk of explosion is eliminated.

Polyhydroxyalkanoate (PHA) Biopolymer Synthesis by Marine Bacteria of the Malaysian Coral Triangle Region and Mining for PHA Synthase Genes.

Polyhydroxyalkanoate (PHA), a biodegradable and plastic-like biopolymer, has been receiving research and industrial attention due to severe plastic pollution, resource depletion, and global waste issues. This has spurred the isolation and characterisation of novel PHA-producing strains through cultivation and non-cultivation approaches, with a particular interest in genes encoding PHA synthesis pathways. Since sea sponges and sediment are marine benthic habitats known to be rich in microbial diversity, sponge tissues (Xestospongia muta and Aaptos aaptos) and sediment samples were collected in this study from Redang and Bidong islands located in the Malaysian Coral Triangle region. PHA synthase (phaC) genes were identified from sediment-associated bacterial strains using a cultivation approach and from sponge-associated bacterial metagenomes using a non-cultivation approach. In addition, phylogenetic diversity profiling was performed for the sponge-associated bacterial community using 16S ribosomal ribonucleic acid (16S rRNA) amplicon sequencing to screen for the potential presence of PHA-producer taxa. A total of three phaC genes from the bacterial metagenome of Aaptos and three phaC genes from sediment isolates (Sphingobacterium mizutaii UMTKB-6, Alcaligenes faecalis UMTKB-7, Acinetobacter calcoaceticus UMTKB-8) were identified. Produced PHA polymers were shown to be composed of 5C to nC monomers, with previously unreported PHA-producing ability of the S. mizutaii strain, as well as a 3-hydroxyvalerate-synthesising ability without precursor addition by the A. calcoaceticus strain.