Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.     

Biosynthesis of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) copolyesters with a high molar fraction of 3-hydroxyvalerate by an insect-symbiotic <Emphasis Type="Italic">Burkholderia</Emphasis> sp. IS-01

Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).     

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct _Cp ) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1 _Ps6-19 and Pct _Cp was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.     

Biosynthesis and characterization of copolymer poly(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) from saponified <Emphasis Type="Italic">Jatropha curcas</Emphasis> oil by <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polymers with promising application in the formulation of plastic materials. PHAs are produced by numerous bacteria as energy/carbon storage materials from various substrates, including sugars and plant oils. Since these substrates compete as food sources, their use as raw material for industrial-scale production of PHA is limited. Therefore, efforts have been focused on seeking alternative sources for bacterial production of PHA. One substrate that seems to have great potential is the seed oil of Jatropha curcas plant. Among other favorable properties, J. curcas seed oil is non-edible, widely available, and can be cheaply produced. In this study, Pseudomonas oleovorans (ATCC 29347) was grown in a mineral salt medium supplemented with saponified J. curcas seed oil as the only carbon source under batch fermentation. Optimum PHA yield of 26.06% cell dry weight was achieved after 72 h. The PHA had a melting point (T _m) between 150 and 160°C. Results of polymer analyses by gas chromatography/mass spectrometry (GC/MS) identified only the methyl 3-hydroxybutanoate monomeric unit. However, electrospray ionization–time of flight mass spectroscopy (ESI–TOF MS) confirmed that the PHA was a copolymer with the characteristic HB/HV peaks at m/z 1155.49 (HB) and 1,169, 1,184–1,194 (HV). The data were further supported by¹H and ¹³C NMR analysis. Polymer analysis by gel permeation chromatography (GPC) indicated a peak molecular weight (MP) of 179,797, molecular weight (M _W) of 166,838, weight number average mass (M _n) of 131,847, and polydispersity (M _w/M _n) of 1.3. The data from this study indicate that J. curcas seed oil can be used as a substrate to produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV).     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Site-Directed Mutagenesis of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate)

The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJ_Ah). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJ_Ah. The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJ_Ah, while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ _Ah was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaC_Ah) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJ_Ah play an important role in determining the acyl chain length substrate specificity. The mutated PhaJ_Ah (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2<Subscript>Ps</Subscript> of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> 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.     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).     

Role of (R)-specific enoyl coenzyme A hydratases of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp in the production of polyhydroxyalkanoates

Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the β-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7–11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10–11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa has produced PHA with more hydroxy octanoate and decanoate (6–17% increase), respectively, when PhaJ1 and PhaJ4 have been over-expressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.     

Expression of PHA polymerase genes of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

<Emphasis Type="Italic">rpoS</Emphasis> Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H₂O₂ and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Expression and characterization of (<Emphasis Type="Italic">R</Emphasis>)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4 _Pa from Pseudomonas aeruginosa, termed phaJ4 _Pp, was identified. The gene products of phaJ1 _Pp, which was identified previously, and phaJ4 _Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4_Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4_Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1_Pp was suggested to be different from that of PhaJ1_Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity.     

Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of <Emphasis Type="Italic">Solemya velum</Emphasis>: <Emphasis Type="Italic">cbbLSQO</Emphasis>

Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, 3 mol min^–1 mg protein ^–1, and a K _CO2 of 40.3 M. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3 region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3 region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.