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.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2⌬ and homozygous chs3⌬ null mutants.Like Saccharomyces cerevisiae, Candida albicans harbors three chitin synthase genes, designated CHS1, CHS2, and CHS3 (2, 6, 13). In S. cerevisiae, it was demonstrated by gene disruption experiments that chitin synthase 1 (Chs1p) is involved in the repair of damaged chitin, Chs2p is required for primary septum formation, and Chs3p is responsible for all other chitin syntheses (5,12,14). More recently, Kollar et al. reported that CHS3 also contributes to the formation of linkage between chitin and ␤-1,3-glucan in S. cerevisiae (10). In order to gain more insights into the physiological roles of the chitin synthases of C. albicans, we have disrupted both CHS2 and CHS3 in C. albicans by means of the URA blaster protocol (1).The homozygous chs2⌬ null mutant and the homozygous chs3⌬ null mutant strains of C. albicans were obtained by transforming CAI-4 cells (ura3⌬::imm34/ura3⌬::imm34) with DNA fragments containing either CHS2 in which the hisG-URA3-hisG cassette was inserted at the unique XhoI site or CHS3 in which the 0.8-kb NcoI-ClaI region was replaced by the hisG-URA3-hisG cassette by the lithium acetate method (9). These DNA fragments were successfully integrated into one of the diploid CHS2 or CHS3 alleles, respectively, and the URA3 gene was efficiently eliminated by 5-fluoroorotic acid (5-FOA) selection (11) (Fig. 1). Then these DNA fragments were again transfected into cells in which one of the diploid CHS2 or CHS3 alleles was already flanked by the hisG sequence. Although the second allele of the CHS2 locus was efficiently targeted by the same DNA fragment used to disrupt the first allele, the remaining CHS3 allele was not easily disrupted by transfection of the same DNA fragment. Therefore, we constructed another plasmid in which the hisG-URA3-hisG cassette was inserted at the NcoI site of CHS3. We assumed that use of this DNA for the second round of transfection would increase the efficiency of homologous recombination between the transfected DNA and the remaining intact CHS3 allele because the 0.8-kb NcoI-ClaI region of CHS3 was missing in the already targeted CHS3 locus. As expected, in 3 of 24 uracil auxotrophs, both of the CHS3 alleles were found to be flanked by the hisG sequence after 5-FOA selection, resulting in the homozygous chs3⌬ null mutation (Fig. 1).Cells lacking functional CHS3 grew in a rich medium such as YPD (1% peptone, 2% yeast extract, and 2% dextrose), but their growth was somewhat slower than that of cells missing CHS2 or the parental strain CAI-4 (the doubling times for CAI-4, the homozygous chs2⌬ null mutant, and the homozygous chs3⌬ null mutant were about 70, 72, and 90 min, respectively)...

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Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (l-cysteine (Cys), l-histidine, and l-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans.S ome microorganisms biosynthesize these PUFAs through PUFAs ynthases composed of four subunits with multiple catalytic domains.These PUFAsynthases each create aspecific PUFAwithout undesirable byproducts,even though the multiple catalytic domains in eachl arge subunit are very similar.H owever,t he detailed biosynthetic pathwaysa nd mechanisms for controlling final-product profiles are still obscure.I nt his study,t he FabA-type dehydratase domain (DH FabA )i nt he C-subunit and the polyketide synthase-type dehydratase domain (DH PKS )i nt he B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays.Furthermore,invitro analysis with truncated recombinant enzymes and C 4 -t oC 8 -acyl ACP substrates showed that ARA and EPAs ynthases utilized two types of DH domains,D H PKS and DH FabA ,d epending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.