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Duckweed is a rapid-growing plant with a high starch and low lignin content. The duckweed was saccharified via dual enzymatic treatment using amylase and cellulase complex. The duckweed-derived glucose was utilized for polyhydroxybutyrate (PHB) production in engineered Escherichia coli. In addition, the low concentration supplementation of the duckweed extract promoted cell growth compared to the analytical grade glucose., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

This study synthesized poly(3-hydroxypropionate) [P(3HP)]-containing polyhydroxyalkanoate (PHA) block copolymers, P(3HP)-b-P[2-hydroxybutyrate (2HB)] and P(3HP)-b-P(D-lactate) (PDLA), using Escherichia coli. The cells expressing an evolved sequence-regulating PHA synthase, PhaC AR NDFH, and propionyl-CoA transferase were cultured with the supplementation of the corresponding monomer precursors in the medium. The block structure of P(3HP)-b-PDLA was confirmed by proton nuclear magnetic resonance analysis and solvent fractionation. The molecular weights of the polymers were in the range of 0.8-2.8 × 10 5 . The solvent-cast polymer films were subjected to isothermal treatment to promote phase separation and crystallization and were subsequently melt-quenched to produce an amorphous phase. The melt-quenched P(3HP)-b-P(2HB) film exhibited a high elongation at break (1153%), resulting in a toughness of 181 MJ/m 3 . The solvent-cast film of P(3HP)-b-65 mol% PDLA exhibited partial elastic deformation, in which the P(3HP) phase functioned as a soft segment. The melt-quenching of the polymer resulted in embrittlement presumably due to the high lactate fraction. Overall, the P(3HP)-based block copolymers exhibited several mechanical properties depending on the higher-order structure of the polymer and the properties of the P(2-hydroxyalkanoate) segments. This study findings show that P(3HP)-b-P(2HB) and P(3HP)-b-PDLA can function excellently if their microstructures are properly controlled., (© 2024 The Author(s). Biopolymers published by Wiley Periodicals LLC.)

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC AR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC AR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB- co -6HHx) synthesis in Escherichia coli . As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB- co -6HHx) decreased as the 6HHx fraction increased.

The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaC AR , enabled the direct synthesis of PHA-PHA type block copolymers in microbial cells. PhaC AR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaC AR can synthesize poly(D-lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. KEY POINTS: • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties., (© 2024. The Author(s).)

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaC AR , a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaC AR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent L-valine depletion caused by dominant L-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent D-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent L-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaC AR + , ldhA Cd -hadA Cd + encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaC AR . The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaC AR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. KEY POINTS: (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB., (© 2023. The Author(s).)

The sequence-regulating polyhydroxyalkanoate (PHA) synthase PhaC AR spontaneously synthesizes the homo-random block copolymer, poly[3-hydroxybutyrate (3HB)]-b-poly[glycolate (GL)-ran-3HB]. In this study, a real-time in vitro chasing system was established using a high-resolution 800 MHz nuclear magnetic resonance (NMR) and 13 C-labeled monomers to monitor the polymerization of GL-CoA and 3HB-CoA into this atypical copolymer. Consequently, PhaC AR initially consumed only 3HB-CoA and subsequently consumed both substrates. The structure of the nascent polymer was analyzed by extracting it with deuterated hexafluoro-isopropanol. In the primary reaction product, a 3HB-3HB dyad was detected, and GL-3HB linkages were subsequently formed. According to these results, the P(3HB) homopolymer segment is synthesized prior to the random copolymer segment. This is the first report of its kind which proposes the application of real-time NMR to a PHA synthase assay, paving the way for elucidating the mechanisms of PHA block copolymerization., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaC AR , a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaC AR synthesizes both a block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)], and a random copolymer, poly(3HB-co-3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaC AR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaC AR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C 3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested., Results: Recombinant Escherichia coli harboring PhaC AR , CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaC AR . 3HB-based binary copolymers P(3HB-co-LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB-co-LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)-b-P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB-co-LMC HA)-b-P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis., Conclusion: PhaC AR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaC AR . The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)-b-P(2HB) and P(3HB-co-LMC HA)-b-P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study's results provide insights into the mechanism of sequence regulation by PhaC AR and pave the way for designing PHA block copolymers., (© 2022. The Author(s).)

Sequence-regulating polyhydroxyalkanoate synthase PhaC AR is a chimeric enzyme comprising PhaCs from Aeromonas caviae and Ralstonia eutropha ( Cupriavidus necator ). It spontaneously synthesizes a short-chain-length (SCL, ≤C 5 ) block copolymer poly(2-hydroxybutyrate)- b -poly(3-hydroxybutyrate) [P(2HB)- b -P(3HB)] from a mixture of monomer substrates. In this study, directed evolution of PhaC AR was performed to increase its activity toward a medium-chain-length (MCL, C 6-12 ) monomer, 3-hydroxyhexanoyl (3HHx)-coenzyme A (CoA). Random mutagenesis and selection based on P(3HB- co -3HHx) production in Escherichia coli found that beneficial mutations N149D and F314L increase the 3HHx fraction. The site-directed saturation mutagenesis at position 314, which is adjacent to the catalytic center C315, demonstrated that F314H synthesizes the P(3HHx) homopolymer. The F314H mutant exhibited increased activity toward 3HHx-CoA compared with the parent enzyme, whereas the activity toward 3HB-CoA decreased. The predicted tertiary structure of PhaC AR by AlphaFold2 provided insight into the mechanism of the beneficial mutations. In addition, this finding enabled the synthesis of a new PHA block copolymer, P(3HHx)- b -P(2HB). Solvent fractionation indicated the presence of a covalent linkage between the polymer segments. This novel MCL-SCL block copolymer considerably expands the range of the molecular design of PHA block copolymers.

Chimeric polyhydroxyalkanoate synthase PhaCAR is characterized by the capacity to incorporate unusual glycolate (GL) units and spontaneously synthesize block copolymers. The GL and 3-hydroxybutyrate (3HB) copolymer synthesized by PhaCAR is a random-homo block copolymer, poly(GL-ran-3HB)-b-poly(3HB). In the present study, medium-chain-length 3-hydroxyhexanoate (3HHx) units were incorporated into this copolymer using PhaCAR for the first time. The coenzyme A (CoA) ligase from Pseudomonas oleovorans (AlkK) serves as a simple 3HHx-CoA supplying route in Escherichia coli from exogenously supplemented 3HHx. NMR analyses of the obtained polymers revealed that 3HHx units were randomly connected to 3HB units, whereas GL units were heterogeneously distributed. Therefore, the polymer is composed of 2 segments: P(3HB-co-3HHx) and P(GL-co-3HB-co-3HHx). The thermal and mechanical properties of the terpolymer indicate no contiguous P(3HB) segments in the material, consistent with the NMR results. Therefore, PhaCAR synthesized the novel block copolymer P(3HB-co-3HHx)-b-P(GL-co-3HB-co-3HHx), which is the first block polyhydroxyalkanoate copolymer comprising 2 copolymer segments., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The first polyhydroxyalkanoate (PHA) block copolymer poly(2-hydroxybutyrate-b-3-hydroxybutyrate) [P(2HB-b-3HB)] was previously synthesized using engineered Escherichia coli expressing a chimeric PHA synthase PhaC AR with monomer sequence-regulating capacity. In the present study, the physical properties of the block copolymer and its relevant random copolymer P(2HB-ran-3HB) were evaluated. Stress-strain tests on the P(88 mol% 2HB-b-3HB) film showed an increasing stress value during elongation up to 393%. In addition, the block copolymer film exhibited slow contraction behavior after elongation, indicating that P(2HB-b-3HB) is an elastomer-like material. In contrast, the P(92 mol% 2HB-ran-3HB) film, which was stretched up to 692% with nearly constant stress, was stretchable but not elastic. The differential scanning calorimetry and wide-angle X-ray diffraction analyses indicated that the P(2HB-b-3HB) contained the amorphous P(2HB) phase and the crystalline P(3HB) phase, whereas P(2HB-ran-3HB) was wholly amorphous. Therefore, the elasticity of P(2HB-b-3HB) can be attributed to the presence of the crystalline P(3HB) phase and a noncovalent crosslinked structure by the crystals. These results show the potential of block PHAs as elastic materials., (© 2021. The Author(s).)

Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL- co -3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1 H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL- ran -3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL- ran -3HB)- b -P(3HB)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Arai, Sakakibara, Mareschal, Ooi, Zinn and Matsumoto.)

The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaC AR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaC AR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaC AR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaC AR , LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes β-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaC AR ; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaC AR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the β-oxidation weakened strain of Pseudomonas putida KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic acid (C10) to an unusual PHA monomer precursor, such as 10-undecenoic acid (C11:1) or phenylvaleric acid (PhVA). Obtained PHA content was in the range of 10 to 25 wt%. When different ratios of C10 and C11:1 were fed to P. putida , the produced PHA had a slightly higher molar ratio in favor of C11:1-based 3-hydroxy-10-undecenoate. However, in case of PhVA a significantly lower incorporation of 3-hydroxy-5-phenylvalerate over 3-hydroxydecanoate took place when compared to the ratio of their precursors in the feed medium. A result that is explained by a less efficient uptake of PhVA compared to C10 and a 24% lower yield of polymer from the aromatic fatty acid ( y P H A - M P h V A = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight M n ¯ two times lower than the PHA produced from C10 alone. Detection of products from PhVA metabolism in the culture supernatant showed that uptaken PhVA was not entirely converted into PHA, thus explaining the difference in the yield polymer from substrate. It was concluded that PhVA or its related metabolites increased the chain transfer rate during PHA biosynthesis in P. putida KTQQ20, resulting in a reduction of the polymer molecular weight., (Copyright © 2019 Hanik, Utsunomia, Arai, Matsumoto and Zinn.)

For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1 Ps STQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1 Ps STQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), β-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [(R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E. coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1 H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C 3 ), 74.9 mol% 3HB (C 4 ), and 5.4 mol% MCL-3HA units of C 8 and C 10 . Furthermore, the recombinant E. coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C 3 , C 8 , C 10 , and C 12 . 13 C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid)., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Poly(2-hydroxybutyrate) [P(2HB)] is an artificial polyhydroxyalkanoate (PHA) synthesized using engineered 2-hydroxyalkanoate-polymerizing PHA synthase. In the present study, the effect of temperature on P(2HB) synthesis was investigated. Recombinant Escherichia coli harboring PHA synthetic genes were cultivated with 2HB and 3-hydroxybutyrate (3HB) supplementation at varied temperatures ranging from 24 to 36°C for the synthesis of P(2HB) and natural PHA P(3HB), respectively. P(2HB) production and its molecular weight increased considerably at a threshold temperature of 32-34°C. The trend was not observed during the synthesis of P(3HB). Notably, the threshold temperature was close to the glass transition temperature ( T g ) of P(2HB) (30°C), while the T g of P(3HB) (4°C) was much lower than the cultivation temperature. The results suggest that thermal motion of the polymer chains influenced the production and molecular weight of the obtained polymer. According to the results, the production and molecular weight of PHA drastically changes at the threshold temperature, which is linked to the T g of the polymer. The hypothesis should be applicable to PHAs in general, and potentially explains the inability to biosynthesize high-molecular-weight polylactate homopolymer with a T g of 60°C.

Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is produced in engineered Escherichia coli harboring the genes encoding an LA-polymerizing enzyme (LPE) and monomer-supplying enzymes. In this study, high cell-density fed-batch jar fermentation was developed using xylose and/or glucose as the carbon source. Fed-batch fermentation was initially performed with 20 g/L sugar during the batch phase for 24 h, and subsequent sugar feeding from 24 to 86 h. The feeding rate was increased in a stepwise manner. When xylose alone was used for cultivation, the cells produced the polymer at 11.6 g/L, which was higher than the 4.3 g/L obtained using glucose as the sole carbon source. However, in the first 24 h the growth in the glucose culture was greater than in the xylose culture. Based on these results, glucose was used for cell growth (at the initial stage) and xylose was used for polymer production (at the feeding stage). As expected, in the glucose/xylose switching fermentation method, polymer production was significantly enhanced, eventually reaching 26.7 g/L. The enhanced polymer production obtained by using xylose was presumably due to overflow metabolism. In fact, during xylose feeding, acetic acid excretion was greater than that in case of the glucose grown culture, suggesting the channeling of the metabolic flux from acetyl-CoA towards polymer production over into the tricarboxylic acid cycle in the xylose-fed cultures. Therefore, this sequential glucose/xylose feed strategy is potentially useful for production of acetyl-CoA derived compounds in E. coli., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1 Ps STQK) 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 PhaC1 Ps STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1 Ps STQK 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.

In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1 Ps (S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1 Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1 Ps (S325T/Q481K). Among them, the cells expressing PhaC1 Ps (S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1 Ps (ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1 Ps (ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1 Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB)., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Lignocellulose-utilizing biorefinery is a promising strategy for the sustainable production of value-added products such as bio-based polymers. Simultaneous consumption of glucose and xylose in Escherichia coli was achieved by overexpression of the gene encoding Mlc, a multiple regulator of glucose and xylose uptake. This catabolite derepression gave the enhancement in the production of poly (15 mol% lactate-co-3-hydroxybutyrate), up to 65% from 50% (wild-type strain) in the cellular contents, of the Mlc-overexpressing strain of E. coli on a mixture of glucose and xylose as carbon sources. Microscopic analysis indicated that the Mlc-overexpressing strain showed the enlargement of cell volume in the presence and absence of polymer production, consequently making an expanded volumetric space available for enhanced polymer accumulation. The enhanced polymer production by the catabolite derepression was also reproducible using the biomass, Miscanthus×giganteus (hybrid Miscanthus), which was cultivated in the farm of Hokkaido University., (Copyright © 2018 The Society for Biotechnology, Japan. All rights reserved.)

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.

Glycolate (GL)-based polyhydroxyalkanoate (PHA), P[GL- co -3-hydroxybutyrate (3HB)], was characterized with respect to its physical properties and hydrolytic degradability. The copolymers were produced from GL and xylose in recombinant Escherichia coli JW1375 (Δ ldhA ) expressing an engineered PHA synthase and monomer supplying enzymes. The GL molar ratio in the copolymer was regulated in the range of 0 to 16 mol % dependent on the concentration of GL supplemented in the medium. Unlike P(3HB) homopolymers which are rigid and opaque, the transparency and elasticity of P(GL- co -3HB) films could be tuned dependent on the GL molar ratio. For example, Young's modulus of the films varied in the range of 1620 to 54 MPa. The hydrothermal treatment of P(GL- co -3HB)s resulted in the generation of water-soluble oligomers, and their concentration was positively correlated with the GL molar ratio in the polymer, indicating that the GL units in the polymer chain promoted the hydrolytic degradation of the polymer. The results of this study demonstrate that the GL molar ratio is a potent determinant for regulating the elasticity and hydrolytic degradability of P(GL- co -3HB).

d-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of d-LA and d-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. coli, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, ΔompF and ΔompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOs due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the ΔmngA, ΔargT, ΔmacA, ΔcitA and ΔcpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.

Recently, we have succeeded in establishing the microbial platform for the secretion of lactate (LA)-based oligomers (D-LAOs), which consist of D-LA and d-3-hydroxybutyrate (d-3HB). The secretory production of D-LAOs was substantially enhanced by the supplementation of diethylene glycol (DEG), which resulted in the generation of DEG-capped oligomers at the carboxyl terminal (referred as D-LAOs-DEG). The microbial D-LAOs should be key compounds for the synthesis of lactide, an important intermediate for polylactides (PLAs) production, eliminating the costly chemo-oligomerization step in the PLA production process. Therefore, in order to demonstrate a proof-of-concept, here, we attempted to convert the D-LAOs-DEG into lactide via metal-catalyzed thermal depolymerization. As a result, D-LAOs-DEG containing 68 mol% LA were successfully converted into lactide, revealing that the DEG bound to D-LAOs-DEG does not inhibit the conversion into lactide. However, the lactide yield (4%) was considerably lower than that of synthetic LA homo-oligomers (33%). We presumed that 3HB units in the polymer chain blocked the lactide formation, and therefore, we investigated the LA enrichment in the oligomers. As the results, the combination of an LA-overproducing Escherichia coli mutant (Δdld and ΔpflA) with the use of xylose as a carbon source exhibited synergistic effect to increase LA fraction in the oligomers up to 89 mol%. The LA-enriched D-LAOs-DEG were converted into lactide with greater yield (18%). These results demonstrated that a greener shortcut route for PLA production can be created by using the microbial D-LAOs secretion system., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (ΔpdhR, ΔcspG, ΔyneJ, ΔchbR, ΔyiaU, ΔcreB, ΔygfI and ΔnanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA , phaB , and phaC (collectively referred to as phaABC ). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC -overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.

Consolidated bioprocessing of lignocellulose is an attractive strategy for the sustainable production of petroleum-based alternatives. One of the underutilized sources of carbon in lignocellulose is the hemicellulosic fraction which largely consists of the polysaccharide xylan. In this study, Escherichia coli JW0885 (pyruvate formate lyase activator protein mutant, pflA(-)) was engineered to express recombinant xylanases and polyhydroxyalkanoate (PHA)-producing enzymes for the biosynthesis of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] from xylan as a consolidated bioprocess. The results show that E. coli JW0885 was capable of producing P(LA-co-3HB) when xylan was the only feedstock and different feeding and growth parameters were examined in order to improve upon initial yields. The highest yields of P(LA-co-3HB) copolymer obtained in this study occurred when xylan was added during mid-exponential growth after cells had been grown at high shaking-speeds (290 rpm). The results showed an inverse relationship between total PHA production and LA-monomer incorporation into the copolymer. Proton nuclear magnetic resonance ((1)H NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses corroborate that the polymers produced maintain physical properties characteristic of LA-incorporating PHB-based copolymers. The present study achieves the first ever engineering of a consolidated bioprocessing bacterial system for the production of a bioplastic from a hemicelluosic feedstock., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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.

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.

Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly(lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers.

A new approach at the transcriptional level was applied to lactate-based polyester production. Four σ factor disruptants, ΔrpoN, ΔrpoS, ΔfliA and ΔfecI, of Escherichia coli were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, ΔrpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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.

The production of bacterial polyesters, polyhydroxyalkanoates (PHAs), has been improved by several rational approaches such as overexpression and/or engineering of the enzymes directly related to PHA biosynthetic pathways. In this study, a new approach at transcription level has been applied to a new category of the copolymer of lactate (LA) and 3-hydroxybutyrate (3HB), P(LA-co-3HB). When the 4 disrupting mutants of sigma factors in Escherichia coli, rpoN, rpoS, fliA, fecI, were used as platforms for production of P(LA-co-3HB), increases in the production level and LA fraction of the copolymer were observed for the mutant strain with rpoN disruption. These positive impacts on the polymer production were caused in an "indirect manner" via changes in the multiple genes governed by RpoN. A genome-wide engineering by sigma factors would be a versatile approach for the production of value-added products of interest and available for combination with the other beneficial tools.

The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.

Engineering of microorganisms to directly utilize plant biomass as a feedstock for the biosynthesis of value-added products such as bioplastics is the aim of consolidated bioprocessing. In previous research we successfully engineered E. coli LS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coli LS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflA gene resulted in increased content of LA repeating units in the copolymers by over 3-fold compared with the wild type; however, this increase was offset by reduced yields in cell mass. Additionally, when acetate was used as a feedstock LA monomer incorporation reached 18.5 (mol%), which suggests that acetate can be used as a feedstock for the production of P(LA-co-HA) copolymers by E. coli.

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.

The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-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.

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.

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].

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.

Direct polyhydroxybutyrate (PHB) production from starch was for the first time achieved using engineered Corynebacterium glutamicum expressing PHB biosynthetic genes and displaying α-amylase on its cell surface. The engineered strain accumulated 6.4 wt% PHB from starch which was higher than that obtained from glucose (4.9 wt%)., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

(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 β-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.

The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D: -lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.

Poly[3-hydroxybutyrate-co-3-hydroxyvalerate(3HV)] was produced in recombinant Escherichia coli LS5218 from ruthenium-catalyzed cellulose hydrolysate and propionate. The strain was found to be resistant to 5-hydroxymethylfurfural (5-HMF), which is a major inhibitory byproduct generated in the cellulose hydrolysis reaction. The 3HV fraction was successfully regulated in the range of 5.6-40 mol%., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)