Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units.

Polyhydroxyalkanoates (PHAs), aliphatic polyesters synthesized by microorganisms, have gained considerable attention as biodegradable plastics. Recently, α-carbon-methylated PHAs have been shown to exhibit several interesting properties that differ from those of conventional PHAs, such as their crystallization behavior and material properties. This study investigated α-carbon methylated (S)- and (R)-3-hydroxy-2-methylpropionate (3H2MP) as new repeating units. 3H2MP units were homopolymerized or copolymerized with (R)-3-hydroxybutyrate (3HB) by manipulating the culture conditions of recombinant Escherichia coli LSBJ. Consequently, PHAs with 3H2MP units ranging from 5 to 100 mol % were synthesized by external addition of (R)- and (S)-enantiomers or the racemic form of 3H2MPNa. The (S)-3H2MP precursor supplemented into the culture medium was almost directly polymerized into PHA while maintaining its chirality. Therefore, a highly isotactic P(3H2MP) (R:S = 1:99) was synthesized, which displayed a melting temperature of 114-119 °C and a relatively high enthalpy of fusion (68 J/g). In contrast, in cultures supplemented with (R)-3H2MP, the precursor was racemized and polymerized into PHA, resulting in the synthesis of the amorphous polymer atactic P(3H2MP) (R:S = 40:60). However, racemization was not observed at a low concentration of the (R)-3H2MP precursor, thereby synthesizing P(3HB-co-8 mol % 3H2MP) with 100% (R)-3H2MP units. The thermogravimetric analysis revealed that the thermal degradation temperatures at 5% weight loss of P(3H2MP)s occurred at approximately 313 °C, independent of tacticity, which is substantially higher than that of P(3HB) (257 °C). This study demonstrates a new concept for controlling the physical properties of biosynthesized PHA by manipulating the polymers' tacticity using 3H2MP units.

Cell-growth phase-dependent promoter replacement approach for improved poly(lactate-co-3-hydroxybutyrate) production in Escherichia coli.

Escherichia coli is a useful platform for producing valuable materials through the implementation of synthetic gene(s) derived from other organisms. The production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] was carried out in E. coli using a set of five other species-derived genes: Pseudomonas sp. 61-3-derived phaC1STQK (for polymerization), Cupriavidus necator-derived phaAB (for 3HB-CoA generation), and Megasphaera elsdenii-derived pct (for LA-CoA generation) cloned into pTV118NpctphaC1ps(ST/QK)AB. Here, we aimed to optimize the expression level and timing of these genes to improve the production of P(LA-co-3HB) and to manipulate the LA fraction by replacing the promoters with various promoters in E. coli. Evaluation of the effects of 21 promoter replacement plasmids revealed that the phaC1STQK-AB operon is critical for the stationary phase for P(LA-co-3HB) production. Interestingly, the effects of the promoters depended on the composition of the medium. In glucose-supplemented LB medium, the dps promoter replacement plasmid resulted in the greatest effect, increasing the accumulation to 8.8 g/L and an LA fraction of 14.1 mol% of P(LA-co-3HB), compared to 2.7 g/L and 8.1 mol% with the original plasmid. In xylose-supplemented LB medium, the yliH promoter replacement plasmid resulted in the greatest effect, with production of 5.6 g/L and an LA fraction of 40.2 mol% compared to 3.6 g/L and 22.6 mol% with the original plasmid. These results suggest that the selection of an appropriate promoter for expression of the phaC1STQK-AB operon could improve the production and LA fraction of P(LA-co-3HB). Here, we propose that the selection of cell-growth phase-dependent promoters is a versatile biotechnological strategy for effective intracellular production of polymeric materials such as P(LA-co-3HB), in combination with the selection of sugar-based carbon sources.

Effect of introducing a disulfide bridge on the thermostability of microbial transglutaminase from Streptomyces mobaraensis.

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industry through cross-linking or posttranslational modification of proteins. In our previous paper, the activity-increased MTG mutants were obtained by means of rational mutagenesis and random mutagenesis coupled with the newly developed screening system. In addition, the improvement of heat resistance of MTG is needed to expand further its industrial applications. Here, a structure-based rational enzyme engineering approach was applied to improve the thermostability of MTG by introducing an artificial disulfide bridge. As a result of narrowing down candidates using a rational approach, we successfully engineered a disulfide bridge into the N-terminal region of MTG by substituting Thr-7 and Glu-58 with cysteine. The T7C/E58C mutant was observed to have a de novo disulfide bridge and showed an increased melting temperature (Tm value) of 4.3 °C with retained enzymatic activity. To address the benefit-gained reason, we focused on the Cß temperature factor of the amino-acid residues that might form a disulfide bridge in MTG. Introducing the disulfide bridge had no remarkable effect on the mutant aiming to stabilize the high temperature factor. On the other hand, the mutation was effective on the relatively stable region. The introduction of a disulfide bridge may therefore be effective to stabilize further the relatively stable part. This finding is considered to be useful for the rational design of mutants aiming at heat resistance of proteins.Key Points• Microbial transglutaminase (MTG) is used as a binder in the food industry.• MTG has the potential for use in the manufacturing of various commercial materials.• Enhanced thermostability was observed for the disulfide bridge mutant, T7C/G58C.

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.

In Vitro Analysis of d-Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate- co-3-hydroxybutyrate) Syntheses.

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1PsSTQK) efficiently produces poly(lactate- co-3-hydroxybutyrate) [P(LA- co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaC1PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1PsSTQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA- co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA- co-3HB) biosynthetic systems.

Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered Escherichia coli.

Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.

Microbial production of poly(lactate-co-3-hydroxybutyrate) from hybrid Miscanthus-derived sugars.

P[(R)-lactate-co-(R)-3-hydroxybutyrate] [P(LA-co-3HB)] was produced in engineered Escherichia coli using lignocellulose-derived hydrolysates from Miscanthus × giganteus (hybrid Miscanthus) and rice straw. Hybrid Miscanthus-derived hydrolysate exhibited no negative effect on polymer production, LA fraction, and molecular weight of the polymer, whereas rice straw-derived hydrolysate reduced LA fraction. These results revealed that P(LA-co-3HB) was successfully produced from hybrid Miscanthus-derived sugars.

Advances and needs for endotoxin-free production strains.

The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets.

Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism.

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Što N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.

Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter.

Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA + Δdld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA + Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the ΔpflA + Δdld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.

Modification of poly(lactate) via polymer blending with microbially produced poly[(R)-lactate-co-(R)-3-hydroxybutyrate] copolymers.

This study investigated the effect of polymer blending of microbially produced poly[(R)-lactate-co-(R)-3-hydroxybutyrate] copolymers (LAHB) with poly(lactate) (PLA) on their mechanical, thermal, and biodegradable properties. Blending of high lactate (LA) content and high molecular weight LAHB significantly improved the tensile elongation of PLA up to more than 250 % at optimal LAHB composition of 20-30 wt%. Temperature-modulated differential scanning calorimetry and dynamic mechanical analysis revealed that PLA and LAHB were immiscible but interacted with each other, as indicated by the mutual plasticization effect. Detailed morphological characterization using scanning probe microscopy, small-angle X-ray scattering, and solid-state NMR confirmed that PLA and LAHB formed a two-phase structure with a characteristic length scale as small as 20 nm. Because of mixing in this order, the polymer blends were optically transparent. The biological oxygen demand test of the polymer blends in seawater indicated an enhancement of PLA biodegradation during biodegradation of the polymer blends.

Complete sequence randomness of lactate-based copolymers (LAHBs) with varied lactate monomer fractions employing a series of propionyl-CoA transferases.

Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme.

Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34mol%) than that grown on glucose (26mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60mol% LA from xylose, while glucose gave a 47mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3gl(-1)) was remarkably higher than that of P(3HB) (4.1gl(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization.

Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with ß-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

One-pot microbial production, mechanical properties, and enzymatic degradation of isotactic P[(R)-2-hydroxybutyrate] and its copolymer with (R)-lactate.

P[(R)-2-hydroxybutyrate] [P((R)-2HB)] is an aliphatic polyester analogous to poly(lactic acid) (PLA). However, little has been known for its properties because of a high cost of commercially available chiral 2HB as a starting substance for chemical polymer synthesis. In this study, P[(R)-2HB] and P[(R)-2HB-co-(R)-lactate] [P((R)-2HB-co-(R)-LA)] with a new monomer combination were successfully synthesized in recombinant Escherichia coli LS5218 from less-expensive racemic 2HB using an R-specific polyester synthase. The cells expressing an engineered polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 and propionyl-CoA transferase from Megasphaera elsdenii were grown on LB medium containing 2HB and glucose in a shake flask and accumulated up to 17 wt % of P[(R)-2HB] with optical purity of >99.1%. In addition, the same cells cultured in a jar-fermentor produced P(86 mol % 2HB-co-LA) copolymer. Notably, the molecular weights (Mw) of P(2HB) (27000) and P(2HB-co-LA) (39000) were 2- and 3-fold higher than that of P(2HB) previously synthesized by chemical polycondensation. P(2HB) was processed into a transparent film by solvent-casting and it had flexible properties with elongation at break of 173%, which was contrast to the rigid PLA. Regarding mechanical properties, P(2HB-co-LA) was tougher but less stretchy than P(2HB). These results demonstrated that P(2HB) has useful properties and LA units in 2HB-based polymers can act as a controllable modulator of the material properties. In addition, P[(R)-2HB] was efficiently degraded by treatment of Novozym 42044 (lipase) but not Savinase 16L (protease), indicating that the degrading behavior of the polymer was similar to that of P[(R)-LA].

Biosynthetic polyesters consisting of 2-hydroxyalkanoic acids: current challenges and unresolved questions.

2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[(R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.

Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature.

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The (13)C and (1)H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10(5) Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA -LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence.

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase.

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by ß-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L(-1) (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L(-1), with a productivity of 0.22 g L(-1) h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of (13)C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.