Polyhydroxyalkanoate Synthase Research Articles

A Novel Nucleic Lateral Flow Assay for Screening phaR-Containing Bacillus spp.

Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.

Synergy of valine and threonine supplementation on poly(2-hydroxybutyrate-block-3-hydroxybutyrate) synthesis in engineered Escherichia coli expressing chimeric polyhydroxyalkanoate synthase

The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes β-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E.coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.

Production of P(3HB-co-4HB) copolymer with high 4HB molar fraction by Burkholderia contaminans Kad1 PHA synthase

Burkholderia sp. has been reported to synthesize polyhydroxyalkanoates (PHAs) with different monomers such as 3-hydroxybutyrate and 3-hydroxyvalerate. In this study, the PHA synthase gene of B. contaminans Kad1 (phaCBcon) was successfully cloned and characterized. Heterologous expression of phaCBcon conferred PHA synthesis to the mutant Cupriavidus necator PHB¯4, confirming that phaCBcon encoded a functional active protein. The transformant C. necator PHB¯4 (Cn-Kad1) harboring phaCBcon was able to synthesize PHA when grown on sodium-4-hydroxybutyrate (sodium-4-HB) as the sole carbon source in a two-stage cultivation. NMR results confirmed the presence of poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB). Cn-Kad1 has the ability to produce a broad range of copolymer with a molecular weight (Mw) in the range of 8.0 × 105 to 10 × 105 Dalton. However, the number average molecular weight (Mn) decreased from 5.0 × 105 to 4.7 × 105 Da, when the sodium-4-HB concentration increased from 0.1 to 0.4 M. Higher concentration of sodium-4-HB, resulted in higher 4HB compositions in the polymer and consequently gave lower values for both the melting temperature (Tm) and the glass transition temperature (Tg). Interestingly, Cn-Kad1 accumulated P(3HB-co-4HB) with 4HB monomer up to 51 mol% from sodium-4-HB, while, the wild type B. contaminans Kad1 has limited ability to accumulate PHA containing 4HB monomer.

Increased Production and Molecular Weight of Artificial Polyhydroxyalkanoate Poly(2-hydroxybutyrate) Above the Glass Transition Temperature Threshold

Poly(2-hydroxybutyrate) [P(2HB)] is an artificial polyhydroxyalkanoate (PHA) synthesized using engineered 2-hydroxyalkanoate-polymerizing PHA synthase. In the present study, the effect of temperature on P(2HB) synthesis was investigated. Recombinant Escherichia coli harboring PHA synthetic genes were cultivated with 2HB and 3-hydroxybutyrate (3HB) supplementation at varied temperatures ranging from 24 to 36°C for the synthesis of P(2HB) and natural PHA P(3HB), respectively. P(2HB) production and its molecular weight increased considerably at a threshold temperature of 32–34°C. The trend was not observed during the synthesis of P(3HB). Notably, the threshold temperature was close to the glass transition temperature (Tg) of P(2HB) (30°C), while the Tg of P(3HB) (4°C) was much lower than the cultivation temperature. The results suggest that thermal motion of the polymer chains influenced the production and molecular weight of the obtained polymer. According to the results, the production and molecular weight of PHA drastically changes at the threshold temperature, which is linked to the Tg of the polymer. The hypothesis should be applicable to PHAs in general, and potentially explains the inability to biosynthesize high-molecular-weight polylactate homopolymer with a Tg of 60°C.

In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

Extremophiles - Platform Strains for Sustainable Production of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are biodegradable polyesters, which are produced by various bacteria including numerous halophiles. Employment of halophilic strain for PHA production brings numerous benefits such as robustness of the process against contamination by ubiquitous mesophiles or possibility to isolate polymer from bacterial biomass via hypotonic lysis. In this work, we screened three moderate halophiles – Halomonas halophila, Halomonas organivorans and Halomonas salina for the presence of phaC gene encoding for PHA synthase and, subsequently, we have investigated their PHA production potential on various sugars. Among tested strains, H. organivorans demonstrated the extraordinary capacity of PHA production in particular on galactose and mannose since on these saccharides PHA content in dried bacterial cells reached 83 and 90 wt. % on mannose and galactose, respectively. Therefore, H. organivoras can be considered being promising PHA producing strain in particular suitable for the valorization of lignocellulose materials rich in galactomannans such as spent coffee grounds.

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.

Polyester as Antigen Carrier toward Particulate Vaccines.

Spherical polyhydroxyalkanoate (PHA) inclusions are naturally self-assembled inside bacteria. These PHA beads are shell-core structures composed of a hydrophobic PHA core surrounded by proteins, such as PHA synthase (PhaC). PhaC is covalently attached and serves as an anchor protein for foreign protein vaccine candidate antigens. PHA beads displaying single and multiple antigens showed enhanced immunological properties when compared to respective soluble vaccines. This review highlights the unique design space of the PHA bead-based vaccines toward the development of safe and synthetic particulate vaccines. The PHA bead technology will be compared with chemically synthesized polyesters, such as polylactic acids, formulated to deliver vaccine antigens. The performance of PHA bead vaccine candidates to induce specific immune responses and protective immunity against bacterial and viral pathogens in animal trials will be summarized. We propose that the PHA bead technology offers a versatile vaccine platform for design and cost-effective manufacture of synthetic multivalent vaccines.

Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment

Abstract. Tan WA, Wijaya I, Purwadaria T. 2019. Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment. Biodiversitas 20: 1309-1315. Polyhydroxyalkanoates (PHA) are potential alternates to conventional synthetic plastics. PHA production in bacteria involves PHA synthase gene encoded by phaC. In this study, we isolated PHA-producing bacteria from the coastline and 1 mile from the coastline of three beaches in Indonesia. Further phaC detection and characterization of PHA production were conducted. The isolates were subjected to phylogenetic analysis based on 16S rDNA. Red Nile staining on minimal agar revealed that twenty-three isolates showed orange fluorescent, which indicated that they accumulated PHA in their cells. PCR detection showed the presence of PHA synthase class I-encoding gene phaC in twelve isolates. One representative amplicon was sequenced to verify its identity, in which it shared 86% similarity with the PHA synthase class I-encoding gene from an uncultured bacterium. Interestingly, the production of PHA in isolate ST.PA.75, which was closely related to Vibrio sp., was 2.1-fold higher than that in the Ralstonia eutropha JMP134 control. Three isolates showed similarity with bacterial genera and/or species for which PHA producing phenotypes had never been described before TP.SWC.20, which was closely related to Microbacterium arborescens, as well as TP.SWC.33 and TP.SWC.85, which were similar to Psychrobacter spp. Phylogenetic analysis showed that the PHA producing isolates were clustered into three phyla: γ-Proteobacteria, Actinomycetes, and Bacilli. A majority of the isolates (75%) were related to γ-Proteobacteria. In this study, we uncovered diverse novel promising strains for use in the production of PHA as a more environmentally-friendly alternative to hydrocarbon-based plastics.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-mediated de novo synthesis of glycolate-based polyhydroxyalkanoate in Escherichia coli

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1PsSTQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E.coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1PsSTQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R.rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E.coli but also in photosynthetic organisms.

Molecular mechanism of acetoacetyl-CoA enhanced kinetics for increased bioplastic production from Cupriavidus necator 428

Polyhydroxyalkanoates are gaining importance due to their biodegradable nature and close analogy to plastics. Polyhydroxybutyrate (PHB) is the most widely used bioplastic from polyalkanoate family, which is produced by a legion of bacterial species via phbCAB operon encoding β-ketothiolase (PhaA), NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC). Augmentation in the activity of these enzymes is promising for increased PHB production which is achieved by enzyme engineering strategies including non-structural and structural approaches. Our study is deployed on directed evolution-based experimentally reported mutants of PhaB enzyme with increased efficiency due to impact on critical structural factors. We have analyzed and compared the native PhaB with two of its variants Q47L and T173S in complex with their cofactor i.e. NADPH as well as the substrate i.e. acetoacetyl-CoA, via long range molecular dynamics simulations. Interaction profile, MMPBSA, essential dynamics, and free energy landscape analysis revealed that the enzyme efficiency is critically affected by cofactor interactions. It was also observed that mutants have higher equilibrium constant with lesser but optimal affinity for substrate and cofactor than the wild type, which might be the reason for increased efficiency of the mutants via enhanced substrate and cofactor exchange rate. Our study provides insights into the cofactor and substrate binding affinities to PhaB enzyme at atomistic level, which will facilitate designing of highly efficient PhaB enzymes for increased PHB production. Communicated by Ramaswamy H. Sarma

Biosynthesis and characterization of co and ter-polyesters of polyhydroxyalkanoates containing high monomeric fractions of 4-hydroxybutyrate and 5-hydroxyvalerate via a novel PHA synthase

Polyhydroxyalkanoates (PHAs) are a group of microbial polyesters that are widely studied due to their versatile physicochemical and mechanical properties. The biosynthesis of PHAs is governed by the enzyme PHA synthase. We have previously reported the discovery of a novel wide substrate specificity PHA synthase isolated from a mangrove environment. In this study, we report the biosynthesis, via the novel PHA synthase, of various PHA co- and terpolymers containing 4-hydroxybutyrate (4HB) and 5-hydroxyvalerate (5HV) monomers, which are lipase degradable. The biosynthesized polymers have varying 4HB and 5HV contents between 5 and 70 mol % depending on the concentration of precursor compounds used. All the polymers exhibit low melting temperature (Tm) and crystallinity and elastomeric behaviour due to high 4HB and 5HV monomer contents. Supplementation of crude palm kernel oil (CPKO) as carbon source with appropriate precursors enabled the biosynthesis of interesting terpolymers and copolymers containing 3-hydroxyhexanoate (3HHx) along with 5HV and/or 4HB monomers. Lipase degradation assays indicated that co- and ter-polymers containing 4HB and 5HV monomers were successfully hydrolysed by various lipases, but addition of 3HHx monomers in the polymer chain reduced its degradability. These properties of the polymers suggest potential uses as biomaterials.

Microbial production of poly (glycolate-co-lactate-co-3-hydroxybutyrate) from glucose and xylose by Escherichia coli

Escherichia coli was metabolically engineered to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate) using glucose and xylose as carbon sources. The combinatorial biosynthetic route was constructed by the overexpression of a series of enzymes including D-tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, aldehyde dehydrogenase, propionyl-CoA transferase, β-ketothiolase, acetoacetyl-CoA reductase, and polyhydroxyalkanoate synthase. Overexpression of polyhydroxyalkanoate granule associated protein significantly improved biopolymer synthesis, and the recombinant strain reached 3.73 g/L cell dry weight with 38.72% (W/W) biopolymer content. A co-culture engineering strategy was developed to produce biopolymer from a mixture of glucose and xylose, achieving 4.01 g/L cell dry weight containing 21.54% (W/W) biopolymer. The results of this work offer an approach for simultaneously utilizing glucose and xylose and indicate the potential for future biopolymer production from lignocellulosic biomass.

Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose

Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), β-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [(R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E.coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7mol% LA (C3), 74.9mol% 3HB (C4), and 5.4mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E.coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).

Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01

Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.

Effect of reducing the activity of respiratory chain on biosynthesis of poly(3-hydroxybutyrate-co-lactate) in Escherichia coli

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates (PHA) family and possesses promising properties including biocompatibility and biodegradability. In this study, we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha, the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli. The polymer content was 83.9% (W/W), and the molar percentage of lactate reached 1.6%. On this basis, in order to accumulate lactate, we reduced the activity of respiratory chain by deleting the ubiX gene, which is involved in the synthesis of coenzyme Q8. Moreover, we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation. With these manipulations, the molar percentage of lactate in the polymer was improved to 14.1%, with an 81.7% (W/W) of polymer content. The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer, and it contributed a new approach to change the content of monomer components in the polymer.

Enhancement of lactate fraction in poly(lactate-co-3-hydroxybutyrate) synthesized by Escherichia coli harboring the D-lactate dehydrogenase gene from Lactobacillus acetotolerans HT.

For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its β-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

Physiological characterization of poly-β-hydroxybutyrate accumulation in the moderately thermophilic hydrogen-oxidizing bacterium Hydrogenophilus thermoluteolus TH-1

Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing microorganism that has the highest growth rate among autotrophs. Genomic analysis revealed that this strain comprises the complete gene set for poly-β-hydroxybutyrate (PHB) synthesis, i.e., three copies of acetyl-CoA acetyltransferase and polyhydroxyalkanoate synthase and one copy of acetoacetyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase/3-hydroxybutyryl-CoA epimerase. An investigation on PHB accumulation in strain TH-1 demonstrated that PHB accumulation was induced by nitrogen limitation under autotrophic as well as heterotrophic conditions. This strain accumulated up to 430.4±14.3mgL-1 PHB during a 3-h incubation under nitrogen-limited heterotrophic conditions. The highest PHB accumulation rates under autotrophic and heterotrophic conditions were 38.6% (w/w) of the dry cells after a 6-h induction and 53.8% after 3h, respectively. Although PHB granules started to accumulate after 15min of nitrogen limitation under heterotrophic conditions, a drastic decrease of PHB was observed after 9h of induction.

PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.