Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalystmediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 ؋ 10 5 , as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.lactate coenzyme A ͉ polyhydroxyalkanoate synthase ͉ substrate specificity ͉ CoA transferase ͉ enzyme engineering T he current polymer materials in common use are nearly all derived from petrochemical sources, and the industry is a significant contributor to greenhouse gas emissions, particularly during the processes of production and incineration of plastics. At present, the development of nonpetrochemical sources for plastic has focused on renewable resources, such as sugars, plant oils, and even CO 2 to replace diminishing supplies of fossil fuel. Polylactate (PLA) is a representative bio-based polyester, which is chemically synthesized by ring-opening polymerization of a cyclic diester (lactide) of lactic acid (LA), produced by microbial fermentation (the left portion in Fig. 1) (1, 2). By introducing variations in molecular weight and crystallinity, PLA is turned into highly valuable materials for biomedical, food, and generalpurpose applications, as described in numerous patents. Thus, PLA combines inexpensive large-scale fermentation with chemical processing capacity to produce a value-added polymer product. However, as the chemo-process of PLA can be carried out via harmful metal catalysts with high reaction velocities, it often leaves chemical residues that are subject to health and safety concerns. The paradigm shift from the chemo-process to the bio-process for PLA production is thus preferable to overcome this problem.The complete biosynthesis of PLA is an enormous challenge for both academic research and industry. For this purpose, a ''LA-polymerizing enzyme,'' which can function as an alternative to a metal catalyst, would be desired to establish the bio-process, as shown in Fig. 1. The simplest strategy would be the discovery of a PLA-producing micro-organism, but this approach has not succeeded yet. Thus, we focused on the microbial biosynthetic...

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His 6 tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65  Å , outer diameter: ∼90  Å , and inner diameter: ∼25  Å ) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

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This paper describes the synthesis of systematic sets of figure-eightand tadpole-shaped amphiphilic block copolyethers (BCPs) consisting of poly(decyl glycidyl ether) and poly[2-(2-(2-methoxyethoxy)ethoxy)ethyl glycidyl ether], together with the corresponding cyclic counterparts, via combination of the t-Bu-P 4 -catalyzed ring-opening polymerization (ROP) and click cyclization. The clickable linear BCP precursors, with precisely controlled azido and ethynyl group placements as well as a fixed molecular weight and monomer composition (degree of polymerization for each block was adjusted to be around 50), were prepared by the t-Bu-P 4catalyzed ROP with the aid of functional initiators and terminators. The click cyclization of the precursors under highly diluted conditions produced a series of cyclic, figure-eight-, and tadpole-shaped BCPs with narrow molecular weight distributions of less than 1.06. Preliminary studies of the BCPs self-assembly in water revealed the significant variation in their cloud points depending on the BCP architecture, though there were small architectural effects on their critical micelle concentration and morphology of the aggregates.

Nano-sized metal particles are attracting much interest in industrial and biomedical applications due to the recent progress and development of nanotechnology, and the surface-modifications by appropriate polymers are key techniques to stably express their characteristics. Herein, we applied cyclic poly(ethylene glycol) (c-PEG), having no chemical inhomogeneity, to provide a polymer topology-dependent stabilization for the surface-modification of gold nanoparticles (AuNPs) through physisorption. By simply mixing c-PEG, but not linear counterparts, enables AuNPs to maintain dispersibility through freezing, lyophilization, or heating. Surprisingly, c-PEG endowed AuNPs with even better dispersion stability than thiolated PEG (HS–PEG–OMe). The stronger affinity of c-PEG was confirmed by DLS, ζ-potential, and FT-IR. Furthermore, the c-PEG system exhibited prolonged blood circulation and enhanced tumor accumulation in mice. Our data suggests that c-PEG induces physisorption on AuNPs, supplying sufficient stability toward bio-medical applications, and would be an alternative approach to the gold–sulfur chemisorption.

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Alkali metal carboxylates were discovered as efficient and simple catalysts for the ring-opening polymerization of cyclic esters that are alternatives to conventional metal-based catalysts and organocatalysts. In our system using an alcohol initiator and this simple catalyst, biodegradable and biocompatible aliphatic polyesters, such as poly(lactide), poly(ε-caprolactone), poly(δ-valerolactone), and poly(trimethylene carbonate), were obtained with predictive molecular weights ranging from 3500 to 22 600 and narrow dispersities. A kinetic experiment for the ROP of L-lactide confirmed the controlled/living nature of the present ROP system, which allowed the precise synthesis of end-functionalized polyesters as well as multihydroxyl-containing polyesters, including α,ω-hydroxy telechelic and starshaped polyesters. Furthermore, a block copolymer containing the poly(L-lactide) segment was successfully synthesized using a macroinitiator possessing a hydroxyl group at the chain end. The tunability of the alkali metal carboxylates by the appropriate choice of the alkyl moiety and countercation enables not only control of the polymerization behavior but also expansion of the scope of the applicable monomers. Given the low cost, easy handling, and low toxicity of the alkali metal carboxylates, the present ROP system can be highly promising for both laboratory-and industrial-scale polyester productions.

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.