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The use of coated nitrogen fertilizers increases nitrogen use efficiency by reducing the nutrient’s loss to the environment through ammonia volatilization, leaching, and denitrification. Biodegradable polymers are the preferred coating materials because they are consumed and eventually mineralized by microbes, which minimizes pollution. In this study, two biodegradable hydrophobic polymers, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) and poly(ε-caprolactone) (PCL), were used to coat ammonium nitrate (NH4NO3) granules using a heat-press technique that, for the first time, eliminated the need for organic solvents at the coating stage. With a polymer coating weight content of 8.6%, the release of nitrate ions (NO3–) into water from PCL-coated fertilizers was very fast (approximately 90% in 1 week), whereas there was no release of NO3–from the PHBH-coated fertilizers until after 10 weeks. The NO3–release rate, the water vapor permeability, and the Young’s modulus of the polymer coatings were regulated by blending PHBH and PCL (80:20 weight ratio). The controlled release of NO3–was also maintained in biologically active soil; biodegradation of the coatings resulted in no burst release of NO3–over the time period tested. These results demonstrated that PHBH, PCL, or their blend, when applied to the surface of NH4NO3granules using a heat-press technique, could effectively control nutrient release rate.

Sequence-regulating polyhydroxyalkanoate synthase PhaCARis a chimeric enzyme comprising PhaCs from Aeromonas caviaeand Ralstonia eutropha(Cupriavidus necator). It spontaneously synthesizes a short-chain-length (SCL, ≤C5) 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 PhaCARwas performed to increase its activity toward a medium-chain-length (MCL, C6–12) monomer, 3-hydroxyhexanoyl (3HHx)-coenzyme A (CoA). Random mutagenesis and selection based on P(3HB-co-3HHx) production in Escherichia colifound 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 PhaCARby 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 PhaCARis characterized by the capacity to incorporate unusual glycolate (GL) units and spontaneously synthesize block copolymers. The GL and 3-hydroxybutyrate (3HB) copolymer synthesized by PhaCARis 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 PhaCARfor the first time. The coenzyme A (CoA) ligase from Pseudomonas oleovorans(AlkK) serves as a simple 3HHx-CoA supplying route in Escherichia colifrom 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, PhaCARsynthesized 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.Graphical AbstractA novel terpolymer P(GL-co-3HB-co-3HHx) was produced using PCT and AlkK for monomer supply and PhaCARfor polymerization in Escherichia coli.

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 PHBHTMKaneka 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 CO2fixation 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.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCARwas engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCARwere 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.

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 vivometabolic intermediate analysis and the relevant in vitroenzymatic 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 coliJW1375 (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 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).

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 vitroanalysis 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. coliwas below the detection limit, while that in C. glutamicumwas 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.

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. coliLS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coliLS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflAgene 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.

P[(R)-lactate-co-(R)-3-hydroxybutyrate] [P(LA-co-3HB)] was produced in engineered Escherichia coliusing 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.

Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonassp. 61−3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) P(3HB) homopolymer and P(3HB-co-3-hydroxyalkanoate) P(3HB-co-3HA) copolymer productions in the recombinants of Escherichia coli.The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.

Based on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61‐3 strains that synthesize random copolyesters consisting of (R)‐3‐hydroxybutyrate (3HB) and (R)‐medium‐chain‐length 3‐hydroxyalkanoate (mcl‐3HA) units, P(3HB‐co‐3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB‐co‐3HA) copolyester were very similar to those of low‐density polyethylene. We carried out the molecular cloning and characterization of a PhaGPs encoding (R)‐3‐hydroxyacyl‐acyl carrier protein coenzyme A transferase of Pseudomonas sp 61‐3. It was concluded that the PhaGPs gene product is involved in providing mcl‐3HA‐CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1Ps gene encoding PHA synthase from Pseudomonas sp 61‐3 was performed in the PhbCRe negative mutant (PHB−4) of Ralstonia eutropha. The recombinant PHB−4 strain successfully produced PHA copolyesters consisting of 3HB and mcl‐3HA units of 6–12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95–97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbABRe genes from R eutropha encoding β‐ketothiolase and NADPH‐dependent acetoacetyl‐coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe.© 2002 Society of Chemical Industry

Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacumcv. 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.

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

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

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

We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillussp. B29. Purified recombinant AzrB and AzrC were homodimers with 23 kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 °C (AzrB) and 55 °C (AzrC), and retained activities up to 55 °C (AzrB) and 50 °C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.

The chemically modified thanatins with the methyl group (CH3), ethyl group (C2H5), and normal-octyl group (C8H17) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteusthan wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.