Synthase PhaC Research Articles

High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains

Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533-4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.

Metabolic engineering of Escherichia coli for poly(3-hydroxypropionate) production from glycerol and glucose.

A new poly(3-hydroxypropionate) (P3HP) biosynthetic pathway employing β-alanine as an intermediate from an inexpensive carbon source was developed in recombinant Escherichia coli. After a series of systematic optimization, the genes for L-aspartate decarboxylase and its maturation factor (panD and panM, from E. coli), β-alanine-pyruvate transaminase (pp0596, from Pseudomonas putida), 3-hydroxy acid dehydrogenase and 3-hydroxypropionyl-CoA synthase (ydfG and prpE respectively, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Cupriavidus necator) were cloned and expressed in E. coli. Under shake-flask conditions, the recombinant strain produced 0.5g P3HP l(-1) from glycerol and glucose, up to 10.2% of CDW. Though the content of P3HP was low, this pathway has some advantages over other reported pathways, such as being redox neutral, does not require any coenzyme, and can use a wide range of carbon sources.

Influence of the operon structure on poly(3-hydroxypropionate) synthesis in Shimwellia blattae.

Glycerol has become a cheap and abundant carbon source due to biodiesel production at a large scale, and it is available for several biotechnological applications. We recently established poly(3-hydroxypropionate) [poly(3HP)] synthesis in a recombinant Shimwellia blattae strain (Heinrich et al. Appl Environ Microbiol 79:3582-3589, 2013). The major drawbacks of the current strains are (i) low poly(3HP) yields, (ii) low plasmid stability and (iii) insufficient conversion rates. In this study, we demonstrated the influence of alterations of the operon structure, consisting of 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate:coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2 and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16. It was shown that S. blattae ATCC33430/pBBR1MCS-2::dhaT::pct::aldD::phaC1 synthesized up to 14.5 % (wtPHA/wtCDW) in a 2-L fed-batch fermentation process. Furthermore, we overcame the problem of plasmid losses during the fermentation period by engineering a carbon source-dependent plasmid addiction system in a triose phosphate isomerase knockout mutant. An assumed poly(3-hydroxyalkanoic acid) degrading activity of the lipase/esterase YbfF could not be confirmed.

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

From waste to plastic: synthesis of poly(3-hydroxypropionate) in Shimwellia blattae.

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homo-oligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wild-type cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His6 and PhaM-His6 showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed.

Biosynthesis of poly(3-hydroxypropionate) from glycerol by recombinant Escherichia coli

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, a P3HP biosynthetic pathway from glycerol was constructed in recombinant Escherichia coli. The genes for glycerol dehydratase and its reactivating factor (dhaB123 and gdrAB, from Klebsiella pneumoniae), propionaldehyde dehydrogenase (pduP, from Salmonella typhimurium), and polyhydroxyalkanoate synthase (phaC1, from Cupriavidus necator) were cloned and expressed in E. coli. After culture condition optimization, the final engineered strain accumulated 10.1g/L P3HP (46.4% of the cell dry weight) using glycerol and glucose as cosubstrates in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without addition of any expensive precursor.

Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity

The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.

Pseudomonas aeruginosa P-5 균주로부터 3-Hydroxyvalerate와 Medium-chain-length 3-hydroxyalkanoates로 구성된 공중합체의 생합성

A bacterial strain capable of synthesizing polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units was isolated from activated sludge using the enrichment culture technique. The organism, identified as Pseudomonas aeruginosa P-5, produced polyesters consisting of 3-hydroxyvalerate and medium-chain-length (MCL) 3-hydroxyalkanoate monomer units when C- odd alkanoic acids such as nonanoic acid and heptanoic acid were fed as the sole carbon source. Solvent fractionation experiments using chloroform and hexane revealed that the 3-hydroxyalkanoate monomer units in these polyesters were copolymerized. The molar concentration of 3-hydroxyvalerate in the polyesters produced were significantly elevated up to 26 mol% by adding 1.0 g/L valeric acid as the cosubstrate. These copolyesters were sticky with low degrees of crystallinity. The PHA synthase genes were cloned, and the deduced amino acid sequences were determined. P. aeruginosa P-5 possessed genes encoding MCL-PHA synthases (PhaC1 and PhaC2) but lacked the short-chain-length PHA synthase gene, suggesting that the MCL-PHA synthases from P. aeruginosa P-5 are uniquely active for polymerizing (R)-3-hydroxyvaleryl-CoA as well as MCL (R)-3-hydroxyacyl-CoAs.

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Biosynthetic pathway for poly(3-Hydroxypropionate) in recombinant Escherichia coli

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

Comparative genomics study of polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 revealed extensive horizontal gene transfer events and co-evolutionary relationships

BackgroundHalophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far.ResultsThe draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment.ConclusionsThe accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.

Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16

A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16

A large number of polypeptides are attached to poly(3-hydroxybutyrate) (PHB) granules of Ralstonia eutropha, such as PHB synthase (PhaC1), several PHB depolymerases (PhaZs) and phasins (PhaPs), the regulator protein PhaR(Reu), and possibly others. In this study we used the bacterial adenylate cyclase-based two-hybrid assay to investigate interactions between known PHB granule-associated proteins (PGAPs) and to screen for new PGAPs. The utility of the system was tested by the in vivo verification of previously postulated interactions of the PHB synthase subunits of R. eutropha (PhaC1 homo-oligomerization) and of Bacillus megaterium (PhaC(Bmeg)-PhaR(Bmeg) hetero-oligomerization). Nine proteins (PhaA, PhaB1, PhaC1, PhaP1-PhaP4, PhaZ1 and PhaR), with established functions in PHB metabolism of R. eutropha, were tested for interaction in all combinations. While no significant interaction was detected between the PHB synthase PhaC1 and any of the other eight tested Pha proteins, strong interactions were found between all phasin proteins, in particular between PhaP2 and PhaP4. When PhaP2 was used as bait in a two-hybrid screening experiment with a genomic library of R. eutropha, the B1934 gene product was identified in 24 out of 53 isolated clones. B1934 encodes a hypothetical protein (15.7 kDa) with similarity to phasins of PHB-accumulating bacteria. A fusion protein of eYfp and the B1934 gene product colocalized with PHB granules, confirming that B1934 represents a new phasin (PhaP5). PhaP5 was not essential for PHB granule formation, but overexpression of PhaP5 increased the number of cells with PHB granules at the cell poles.

Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2Ps of Pseudomonas stutzeri via site-specific mutation for efficient production of PHA copolymers

The site-specific mutagenesis for PHA synthase PhaC2(Ps1317) from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2 (Ps1317) gene (phaC2 (Ps) QKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2 (Ps1317), the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2(Ps1317) activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2 (Ps1317) termed phaC2 (Ps) QKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.

Pathway engineering results the altered polyhydroxyalkanoates composition in recombinant Escherichia coli

To efficiently produce short-chain-length-medium-chain-length polyhydroxyalkanoates copolymer from substrate mixture containing sugars and/or fatty acids, fadA gene mutant was constructed in Escherichia coli DH5α phosphotransferase system (PTS) disrupted strain. Plasmids pCJY02, pBHR68 and pBHR71 were separately introduced into E. coli DH5α (ΔptsG, ΔFadA) by transformation, then the recombinants were cultivated in the medium containing glucose and/or decanoate as carbon resource, respectively. When cultivated in the medium containing decanoate, only pCJY02-harboring recombinant was able to accumulate SCL-MCL PHAs consisting of 3HB, 3HHx, 3HO and 3HD with mol ratios: 43.2:12.8:10.3:33.6. The copolymer content was 1.90wt% with 2.69gL(-1) cell dry weight. When cultivated in the medium containing both decanoate and glucose, the recombinant was found to utilize the mixture of glucose and fatty acids and accumulate SCL-MCL PHAs copolymer consisting of 3HB, 3HHx, 3HO and 3HD with mol ratios: 83.4:4.0:5.6:7.0. About 4.90gL(-1) cell dry weight was harvested and total PHAs content was 7.3wt% of CDW. This result indicated that the low-substrate-specificity PHA synthase PhaC2(Ps) endued hosts with the capability of synthesizing PHA copolymers, and the monomer composition of the synthesized PHA could be modulated by controlling the addition of carbon sources and by modifying metabolic pathways in the hosts.

In Vivo Enzyme Immobilization by Inclusion Body Display

A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged alpha-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 mum, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.

Conversion of Glycerol to Poly(3-Hydroxypropionate) in Recombinant Escherichia coli

We have developed the conversion of glycerol into thermoplastic poly(3-hydroxypropionate) [poly(3HP)]. For this, the genes for glycerol dehydratase (dhaB1) of Clostridium butyricum, propionaldehyde dehydrogenase (pduP) of Salmonella enterica serovar Typhimurium LT2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha were expressed in recombinant Escherichia coli. Poly(3HP) was accumulated up to 11.98% (wt/wt [cell dry weight]) in a two-step, fed-batch fermentation. The present study shows an interesting application to engineer a poly(3HP) synthesis pathway in bacteria.

Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents

Bioengineered bacterial polyester inclusions have the potential to be used as a vaccine delivery system. The biopolyester beads were engineered to display a fusion protein of the polyester synthase PhaC and the two key antigens involved in immune response to the infectious agent that causes tuberculosis, Mycobacterium tuberculosis, notably antigen 85A (Ag85A) and the 6-kDa early secreted antigenic target (ESAT-6) from Mycobacterium tuberculosis. Polyester beads displaying the respective fusion protein at a high density were successfully produced (henceforth called Ag85A-ESAT-6 beads) by recombinant Escherichia coli. The ability of the Ag85A-ESAT-6 beads to enhance mouse immunity to the displayed antigens was investigated. The beads were not toxic to the animals, as determined by weight gain and absence of lesions at the inoculation site in immunized animals. In vivo injection of the Ag85A-ESAT-6 beads in mice induced significant humoral and cell-mediated immune responses to both Ag85A and ESAT-6. Vaccination with Ag85A-ESAT-6 beads was efficient at stimulating immunity on their own, and this ability was enhanced by administration of the beads in an oil-in-water emulsion. In addition, vaccination with the Ag85A-ESAT-6 beads induced significantly stronger humoral and cell-mediated immune responses than vaccination with an equivalent dose of the fusion protein Ag85A-ESAT-6 alone. The immune response induced by the beads was of a mixed Th1/Th2 nature, as assessed from the induction of the cytokine gamma interferon (Th1 immune response) and increased levels of immunoglobulin G1 (Th2 immune response). Hence, engineered biopolyester beads displaying foreign antigens represent a new class of versatile, safe, and biocompatible vaccines.