Miwa Yamada

A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 × 105, as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

Microbial Production of Lactate-Enriched Poly[(R)-lactate-co-(R)-3-hydroxybutyrate] with Novel Thermal Properties

Considerable enrichment of the lactate (LA) fraction in Poly[(R)-LA-co-(R)-3-hydroxybutyrate (3HB)] has been achieved, reaching up to 47 mol % from the previous 6 mol % [S. Taguchi et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (45), 17323-17327], when recombinant Escherichia coli W3110, harboring the LA-polymerizing enzyme gene together with three genes for supplying monomers, lactyl-coenzyme A (CoA), and 3-hydroxybutyryl-CoA, were grown on glucose under anaerobic conditions. The molecular weights of the copolymer were M(n) = 1.5 x 10(4) g/mol and M(w) = 2.0 x 10(4) g/mol, respectively. Notably, alkaline-hydrolyzed product analysis revealed that only the (R)-enantiomer of LA was incorporated in the copolymer. Furthermore, the triad sequence of LA-LA-LA was clearly detected in the polymer chain based on NMR analysis. A remarkably contrasting melting temperature was observed between the copolymer obtained here P[(R)-LA-co-(R)-3HB] (157 degrees C) and the chemically synthesized P[(S)-LA-co-(R)-3HB] (105 degrees C) with similar monomer composition.

Adjustable Mutations in Lactate (LA)-Polymerizing Enzyme for the Microbial Production of LA-Based Polyesters with Tailor-Made Monomer Composition

Lactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombinant Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaC1(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPE at position 392, 17 mutants produced P(LA-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaC1(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaC1(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol %. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.

Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli

In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Youngs modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers.

A New Pathway for Poly(3-hydroxybutyrate) Production in Escherichia coli and Corynebacterium glutamicum by Functional Expression of a New Acetoacetyl-coenzyme A Synthase

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).

Characterization of Site-Specific Mutations in a Short-Chain-Length/ Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

ABSTRACT Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

A new beneficial mutation in pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase for enhanced cellular content of 3-hydroxybutyrate-based PHA explored using its enzyme homolog as a mutation template.

A newly isolated mutation (Gln508Leu) and a combination of it with previously discovered beneficial mutations in polyhydroxyalkanoate synthase 1 from Pseudomonas sp. 61-3 were found to enhance the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoate)s in recombinant Escherichia coli.

Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]

The repressor protein PhaR, which is a component of poly[(R)-3-hydroxybutyrate] granules, functions as a repressor of the gene expression of the phasin PhaP and of PhaR itself. We used a quartz crystal microbalance to investigate the binding behavior by which PhaR in Ralstonia eutropha H16 targets DNAs and amorphous poly[(R)-3-hydroxybutyrate] thin films. Binding rate constants, dissociation rate constants, and dissociation constants of the binding of PhaR to DNA and to amorphous poly[(R)-3-hydroxybutyrate] suggested that PhaR bind to both in a similar manner. On the basis of the binding rate constant values, we proposed that the phaP gene would be derepressed in harmony with the ratio of the concentration of the target DNA to the concentration of amorphous poly[(R)-3-hydroxybutyrate] at the start of poly[(R)-3-hydroxybutyrate] synthesis in R. eutropha H16.

Novel acidophilic β-galactosidase with high activity at extremely acidic pH region from Teratosphaeria acidotherma AIU BGA-1.

A β-galactosidase exhibiting maximal activity at pH 1.0 was purified from Teratosphaeria acidotherma AIU BGA-1. The enzyme had a molecular mass of 180 kDa and consisted of two heterosubunits of 120 kDa and 66 kDa. The N-terminal amino acid sequence of the large subunit was found to be SPNLQDIVTVDGESY. These physicochemical properties differed from those of other microbial β-galactosidases. At pH values of 1.5 and pH 4.5, the enzyme exhibited its highest activity at temperatures of 70°C and 80°C, respectively. Thus, the enzyme exhibited the lowest optimal pH and highest optimal temperature among the microbial β-galactosidases thus reported. The enzyme retained more than 80% of its original activity in the pH range from 2.0 to 8.0 by incubation at 50°C for 30 min. The enzyme hydrolyzed 4-nitrophenyl-β-D-fucopyranoside, 2-nitrophenyl-β-D-galactopyranoside, and 4-nitrophenyl-β-D-galacto-pyranoside at relative reaction rates of 100, 59, and 24, respectively, at pH 1.5, and its affinity for β-D-galactopyranosides was higher than that for β-D-fucopyranosides. The enzyme also efficiently hydrolyzed lactose in milk and whey from yoghurt at pH 1.5.

Production of P(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) Terpolymers Using a Chimeric PHA Synthase in Recombinant Ralstonia eutropha and Pseudomonas putida

Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87–97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.