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With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) using Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Designing sustainable biobased and/or biodegradable plastics opens up opportunities to achieve a low-carbon society and overcome plastic pollution. Bioplastics manufactured from renewable resources are being designed to feature a minimal carbon footprint and complete biodegradability/compostability. Among them, naturally occurring polyhydroxyalkanoates (PHAs) have currently received increasing attention from academia and industry. A symbolic state-of-the-art PHA industry is a rapidly growing market of PHBHTM Kaneka polymers that display excellent marine biodegradability. From an academic perspective, there have been several major breakthroughs in the PHA research field starting with the pioneering works of genetically engineered platforms for the production of artificial PHAs. The discovery of a lactate-polymerizing enzyme enabled us to produce lactate-based PHAs in one-pot microbial systems, whereas polylactide and other relevant copolymers are currently synthesized via biological and chemical processes. This proof-of-concept has been implemented in practical integrated bioprocesses for carbon-neutral polymer production starting from renewable raw bioresources. Challengingly, the photosynthetic machinery RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), has also been applied to synthesize glycolate-based copolymers as a CO2 fixation model in the current project. Such game-changing technologies contribute to realizing a circular bioeconomy through the utilization of CO2. This review presents the current progress in evolving microbial polymerization systems, including the direct secretion of polymerized products and the creation of sequence-regulated polyesters, which have been considered nearly impossible biological events to date. Polyhydroxyalkanoates (PHAs) are biobased and biodegradable materials. The artificial PHAs, such as lactate-based polymers, synthesized by engineered platforms expand the range of physical properties. The artificial polymers with superior properties are produced mainly from CO2-derived biomass using microbial platform with engineered enzymes. The oligomers can be secreted from cells and derivatized into high-molecular-weight polymers through assembling with other segments. The review summaries recent advances in the biosynthesis and biodegradation of artificial PHAs and oligomers.

The biodegradable polyester poly-(

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). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C3), 74.9 mol% 3HB (C4), and 5.4 mol% MCL-3HA units of C8 and C10. 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 C3, C8, C10, and C12. 13C-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).

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.

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 (

Glycolate (GL)-based polyhydroxyalkanoate (PHA), P[GL

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 Cβ 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.

A polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], was biosynthesized from biphenyl as the sole carbon source using Alcaligenes (currently Achromobacter) denitrificans A41. This strain is capable of degrading polychlorinated biphenyls (PCBs) and biphenyl. This proof-of-concept of the conversion of aromatic chemicals such as the environmental pollutant PCBs/biphenyl to eco-friendly products such as biodegradable polyester PHA was inspired by the uncovering of two genes encoding PHA synthases in the A. denitrificans A41 genome. When the carbon/nitrogen (C/N) ratio was set at 21, the cellular P(3HB-co-3HV) content in strain A41 reached its highest value of 10.1%. A two-step cultivation protocol improved the accumulation of P(3HB-co-3HV) by up to 26.2% of the dry cell weight, consisting of 13.0 mol% 3HV when grown on minimum salt medium without nitrogen sources. The highest cellular content (47.6%) was obtained through the two-step cultivation of strain A41 on biphenyl as the sole carbon source. The purified copolymer had ultra-high molecular weight (weight-average molecular weight of 3.5 × 106), as revealed through gel-permeation chromatography. Based on the genomic information related to both polymer synthesis and biphenyl degradation, we finally proposed a metabolic pathway for the production of P(3HB-co-3HV) associated with the degradation of biphenyl by strain A41. This is, as it were, a metabolic link between PHA synthesis and biphenyl degradation.

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.

A 74-year-old man visited our hospital complaining of abdominal pain. An abdominal computed tomography scan showed multiple wall thickness of the small bowel. Capsule endoscopy was recommended for further evaluation, and patency capsule examination was performed. Eighteen hours after patency capsule ingestion, he experienced small bowel perforation with severe peritonitis caused by intestinal pressure rising because of the patency capsule trapped in his terminal ileum. An ileocolic resection was performed, including the removal of the sclerotic ileum as an emergency surgery. A pathological examination showed transmural inflammation and multiple ulcers with perforation of the small intestine, consistent with Crohn’s disease. Here, we report a rare and valuable case of novel tag-less AgileTM patency capsule (Given Imaging Ltd., Yoqneam, Israel) retention leading to small bowel perforation.

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.

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.

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.

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.

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.

Poly[( R )-lactate- co -( R )-3-hydroxybutyrate] [P(LA- co -3HB)] is a biobased polyester with flexible properties efficiently synthesized by engineered Escherichia coli . Here, we aimed at optimizing the monomeric composition of the copolymer in terms of its flexibility, and elucidating structural features contributing to their mechanical properties. The LA fraction was successfully regulated in the range of 6–66 mol% by combination of metabolic and enzyme engineering approaches. The copolymers with higher LA fraction showed decreasing melting point from 160 to 125 °C, but increasing glass transition temperature from 7 to 27 °C. Crystallinity of the as-cast film, that was mainly attributed to the crystallization of 3HB unit, also decreased with the increasing LA fraction. Owing to the combined effect of these parameters, the highest elongation to break (approximately 400%), which is comparable to that of polyethylene, was obtained for the copolymers with middle range LA fraction (33 mol%). The high flexibility was maintained in most of the copolymers. Notably, P(21mol%LA- co -3HB) retained its high elongation at break (about 300%) even after storage under ambient condition for 5 months. These results demonstrate that introduction of LA units into the polymer chain effectively and stably inhibited the crystallization of 3HB units.

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.

Establishment of the regeneratable whole-cell catalyst platform for the production of biobased polymeric materials is a typical topic of synthetic biology. In this commentary, discovery story of a “lactate-polymerizing enzyme” (LPE) and LPE-based achievements for creating a new variety of polyesters with incorporated unnatural monomers are presented. Besides the importance of microbial platform itself is discussed referring to the “ballooning”-Escherichia coli. Open image in new window

Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate- co -3-hydroxybutyrate) [P(LA- co -3HB)] in engineered Escherichia coli . The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA- co -3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA- co -3HB) production, when the removal of acetic acid was satisfactorily accomplished.

Here, we report the one-step secretory production of D-lactate (LA)-based oligomers (D-LAOs) by engineered Escherichia coli from glucose. Notably, D-LAOs are spontaneously secreted into the medium without the introduction of exogenous exporters. This study was originated from the finding that a small amount of oligo[LA-co-3-hydroxybutyrate(3HB)] was secreted by the cells expressing the D-specific LA-polymerizing enzyme (LPE). To increase D-LAOs production, we attempted to increase the frequency of chain transfer (CT) reaction of LPE using CT agents, which bear alcoholic moiety. As the result, addition of diethylene glycol into the culture medium was found to be particularly effective. The highest yield of D-LAOs production of 8.3 ± 1.5 g L−1 from 20 g L−1 glucose was 57% of the theoretical maximum. Furthermore, we demonstrated the covalent conjugation of diethylene glycol at the carboxyl terminal of D-LAOs by NMR analyses. The secreted D-LAOs have the potential to be used as precursors of lactide, enablin...

The biodegradable polyester 3-hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo-oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)-deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end-capped forms may be used as physiologically active substances and building blocks for polymeric materials.

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.

Most plastics, materials, fuels and other organic chemicals are presently derived from fossil fuel feedstocks. Due to the finite nature and foreseeable depletion potential of these raw materials, concerted efforts are being explored to find sustainable alternatives to the fossil fuel feedstock-derived products. Among these, bioplastics and oligomers derived from fermentation of the renewable plant biomass are promising candidates to replace fossil-fuel-derived plastics. Bioplastics are a class of storage polymers synthesized by microorganisms. Natural plastics can also be produced via a bio-chemo process that combines fermentative production of monomers or oligomers, followed by a chemical synthesis process to produce a variety of polymers. These polymers, particularly polyhydroxyalkanoates (PHAs ) represent futuristic biomaterials owing to their biodegradability and biocompatibility. Furthermore, PHAs have physicochemical properties that are similar to petrochemical-based plastics hence their potential replacement. Designing efficient processes holds the key towards their adoption. This chapter discusses opportunities and challenges regarding the production of lactic acid (LA)-based polymers and related oligomers that can act as precursors for catalytic synthesis of polylactic acid (PLA ). It covers crucial steps of their production using genetically modified organisms and engineered enzymes as well as providing future developments.

Given their ubiquity in modern society, the development of biodegradable and renewably sourced plastics is essential for the creation of an environmentally sustainable society. One of the drawbacks for currently available biodegradable plastics such as poly(l-lactic acid) (PLLA) and polyhydroxyalkanoates (PHAs) is that it is difficult to simultaneously achieve mechanical flexibility and certain crystallization behavior in these materials, which limits their use as replacements for established petroleum-based plastics such as isotactic polypropylene (iPP). Here, we report the synthesis and characterization of a new biodegradable plastic, poly(3-hydroxy-2-methylbutyrate) [P(3H2MB)], which is a member of the bacterial PHA family whose members include an α-methylated monomer unit. Biosynthesis of P(3H2MB) was achieved using recombinant Escherichia coli expressing an engineered pathway. Biosynthesized P(3H2MB) exhibited the highest melting temperature (197 °C) among the biosynthesized PHAs and improved thermal resistance. It also exhibited improved crystallization behavior and mechanical flexibility nearly equal to those of iPP. The primary nucleation rate of P(3H2MB) was faster than that of P(3HB), and the spherulite morphology of P(3H2MB) was much finer than that of P(3HB). This crystal morphology may result in more rapid crystallization behavior, increased transparency, and enhanced mechanical properties. The superior physical properties of P(3H2MB) have the potential to open new avenues for the production of high-performance biodegradable plastics for replacing petroleum-based bulk commodity plastics. The biosynthesis and characterization of a new biodegradable plastic, poly(3-hydroxy-2-methylbutyrate) [P(3H2MB)], which is a member of the bacterial polyhydroxyalkanoate (PHA) family whose members include an α-methylated monomer unit, were investigated. Biosynthesized P(3H2MB) exhibited the highest melting temperature (197 °C) among the biosynthesized PHAs and improved thermal resistance. It also exhibited improved crystallization behavior and mechanical flexibility nearly equal to those of isotactic polypropylene (iPP). The superior physical properties of P(3H2MB) have the potential to open new avenues for the production of high-performance biodegradable plastics for replacing petroleum-based bulk commodity plastics.

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.

Poly[(R)-lactate-co-(R)-2-hydroxybutyrate], which is biosynthesized as a random co-polyester by microbial organisms, with various ratios of lactic acid (LA) to hydroxybutyrate monomers has been isothermally crystallized to investigate its thermal properties and crystal structure changes. Differential scanning calorimetry on the isothermally crystallized samples detected endothermic peaks due to the melting of the crystals at all LA unit ratios. Melting temperatures of the copolymers increased from ca. 107 °C–146 °C as the LA unit ratio increased. Glass transition temperatures also increased from 24 °C to 44 °C with increasing LA unit ratio. A melting temperature was observed even at LA unit ratios around 50%. Wide-angle X-ray measurements also revealed that co-crystallization occurred at all samples, including at LA unit ratios of ca. 50%. Despite of “jumping” phenomena many co-crystallizable random copolymers having, the lattice constants changed “linearly” from resembling poly[(R)-lactate] to poly[(R)-2-hydroxybutyrate] depending on the LA unit ratio. This unique co-crystallization behavior is discussed in detail.

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 (ΔldhA) 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...

P[ d -lactate (LA)-co-3-hydroxybutyrate (3HB)] is an artificial polyhydroxyalkanoate (PHA) containing unusual d -LA units. In this study, the P( d -LA-co-3HB)-degrading bacterial group in the soil was analyzed and the bacterial degradation of the d -LA clustering structure in the copolymer were evaluated by using chemically synthetic d -LA homo-oligomers. A total of 216 soil samples were screened on the basis of clear zone formation on agar plates containing emulsified P(64 mol% d -LA-co-3HB). The 16S rRNA analysis of the isolated bacteria resulted in the identification of eight Variovorax, three Acidovorax, and one Burkholderia strains, which are closely related to previously identified natural PHA-degrading bacteria. These bacteria nearly consumed the P( d -LA-co-3HB) emulsion in the liquid culture; however, a small amount of the d -LA fraction remained unconsumed, which should be attributable to the d -LA-clustering structure in the copolymer. Cultivation of the isolated bacteria with the d -LA homo-oligomers revealed that the oligomers with a degree of polymerization (DP) ranging from 10 to 30 were partly consumed by six Variovorax and one Acidovorax strains. In contrast, the oligomers with DP ranging from 20 to 60 were not consumed by the isolated bacteria. These results indicate that d -LA homo-oligomers with DP higher than approximately 20 are hardly degraded by the soil bacteria. Molecular dynamic simulation of the d -LA homo-oligomers indicated that the upper limit of DP is likely to be determined by the conformational structure of the oligomers in water. The information obtained in this study will be useful for the molecular design of biodegradable d -LA-containing polymers.