Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases

Eleven laboratory-evolved polyhydroxyalkanoate (PHA) synthases which originated from Pseudomonas sp. 61-3 enzyme (PhaC1(Ps)), together with the wild-type enzyme, were applied for PHA synthesis from fructose using Ralstonia eutropha PHB(-)4 as a host strain. The evolved PhaC1(Ps) mutants had amino acid substitution(s) at position 325 and/or position 481. In these mutants, serine-325 (S325) was replaced by cysteine (C) or threonine (T), while glutamine-481 (Q481) was replaced by lysine (K), methionine (M) or arginine (R). All recombinant strains harboring the genes of the evolved PhaC1(Ps) mutants produced a significantly increased amount of PHA (55-68 wt.-%) compared with the one harboring the wild-type gene (49 wt.-%). Particularly, those evolved PhaC1(Ps) mutants having multiple amino acid substitutions showed higher activities for PHA synthesis. Characterization of the PHA by NMR spectroscopy revealed that they were copolymers consisting of (R)-3-hydroxybutyrate (98-99 mol-%) and medium-chain-length comonomers (1-2 mol-%). This study also confirmed that amino acid substitution at position 481 in PhaC1(Ps) led to an increasing molecular weight of PHA. The number-average molecular weight (Mn) of PHA (Mn = 240,000) synthesized by the evolved PhaC1(Ps) (Q481K) mutant was 4.6-fold greater than that (Mn = 52,000) synthesized by the wild-type enzyme.     

Evaluating the ability of polyhydroxyalkanoate synthase mutants to produce P(3HB-co-3HA) from soybean oil

Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaC1(Ps)) is able to synthesize P(3HB-co-3HA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.     

Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coli

Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme. [diagram in text].     

Variation in copolymer composition and molecular weight of polyhydroxyalkanoate generated by saturation mutagenesis of Aeromonas caviae PHA synthase

Amino acid substitutions at two residues downstream from the active-site histidine of polyhydroxyalkanoate (PHA) synthases are effective for changing the composition and the molecular weight of PHA. In this study, saturation mutagenesis at the position Ala505 was applied to PHA synthase (PhaCAc) from Aeromonas caviae to investigate the effects on the composition and the molecular weight of PHA synthesized in Ralstonia eutropha. The copolymer composition and molecular weight of PHA were varied by association with amino acid substitutions. There was a strong relationship between copolymer composition and PHA synthase activity of the cells. This finding will serve as a rationale for producing tailor-made PHAs.     

Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1)

The biosynthesis of the potent environmental carcinogen aflatoxin B(1) is initiated by norsolorinic acid synthase (NorS), a complex of an iterative type I polyketide synthase and a specialized yeast-like pair of fatty acid synthases. NorS has been partially purified from Aspergillus parasiticus, has been found to have a mass of approximately 1.4 x 10(6) Da, and carries out the synthesis of norsolorinic acid in the presence of acetylCoA, malonylCoA, and NADPH where hexanoylCoA is not a free intermediate. The N-acetylcysteamine thioester of hexanoic acid can substitute for the catalytic functions of HexA/B to initiate norsolorinic acid synthesis by the complex in the presence of only malonylCoA. An alpha(2)beta(2)gamma(2) stoichiometry is proposed for NorS in keeping with its estimated mass and the observed dimeric or higher-order quarternary structures of PKS and FAS enzymes.     

Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) synthase (PhaC) from Wautersia eutropha was expressed in a wide range of production level in Escherichia coli XL1-Blue cells and its effects on PhaC activity, poly[(R)-3-hydroxybutyrate] [P(3HB)] production and its molecular weights were investigated. The production level of PhaC was controlled both by the amount of chemical inducer (isopropyl-β-d-thiogalactopyranoside, IPTG) added into the medium and the use of different copy number of plasmids. In a flask experiment, as PhaC production level in the cells increased, the PhaC activity also increased in the range of low PhaC concentration. However, PhaC activity did not further increase in the range of high PhaC concentration, probably due to the formation of inclusion body in the cells. The molecular weight of P(3HB) was found to decrease with increasing PhaC activity. This trend was also verified in high cell density cultivation using 10-l jar fermentor. Furthermore, we demonstrated that the use of low copy number plasmid and appropriate induction of PhaC expression were effective in achieving both high productivity and high molecular weight of P(3HB).     

New concept for a toxicity assay based on multiple indexes from the wave shape of damped metabolic oscillation induced in living yeast cells (part I): characterization of the phenomenon

The damped glycolytic oscillation phenomenon occurring in starved cells of the yeast Saccharomyces cerevisiae (NBRC 0565) was characterization for application to a toxicity bioassay. S. cerevisiae was grown under semi-anaerobic conditions. The transient oscillations were observed photometrically as the time course of the fluorescent intensity of reduced pyridine nucleotide resulting from instantaneous addition of glucose to a cell suspension. In this study, simple and reproducible conditions inducing damped oscillations were obtained by modifying a literature method. For estimation of the wave shapes of the damped oscillations we used six indexes. To investigate the total reproducibility as the averaged relative standard deviation (RSD_av) for the six indexes obtained from the wave shapes, the damped oscillations were induced under the optimum conditions and the RSD_av values were calculated as 14% in a buffer cell suspension (n = 62) and 22% in a water cell suspension (n = 78). Finally, the effects of glucose concentration on the six indexes were examined, and all the indexes changed when the glucose concentration was changed. Excellent correlations were obtained between the index of oscillation-state time and the concentration of glucose in a buffer cell suspension (r = 0.9985, 0.5–250 mmol L⁻¹, 10 points) and in a water cell suspension (r = 0.9989, 2.5 μmol L⁻¹–250 mmol L⁻¹, 12 points), respectively. Figure Characterization of damped glycolytic oscillation, (a) typical shape, and (b) its estimation Electronic supplementary material The online version of this article (doi:)contains supplementary material, which is available to authorized users.