Yasuharu Satoh

A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated 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 × 105, as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

Isolation and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil

A gram-positive bacterium (designated strain INT005) that accumulated polyhydroxyalkanoate (PHA) was isolated from gas field soil. From its morphological and physiological properties and the partial nucleotide sequence (about 500 bp) of its 16S rDNA, it was suggested that strain INT005 was similar to several species of the genus Bacillus. We confirmed that strain INT005 is a Bacillus sp. The PHA productivities of strain INT005 were higher than those of Bacillus megaterium and Ralstonia eutropha at 37-45 degrees C reported to date, and it was suggested that the PHA synthase of INT005 may exhibit moderate thermostability. The bacterium had the ability to produce poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate), and poly(3-hydroxybutyrate-co-6-hydroxyhexanoate-co-3-hydroxyhexanoate) from the appropriate carbon sources. The PHA synthase from INT005 showed similar substrate specificity to those of class I and III PHA synthases and strain INT005 produced PHAs with various monomer compositions. From the analysis of monomer composition and PHA accumulation in the presence of acrylic acid, it was suggested that de novo fatty acid synthesis and beta-oxidation are involved in the PHA synthesis of Bacillus sp. INT005. Since Bacillus sp. INT005 could synthesize PHA even at 45 degrees C and PHAs with various monomer compositions, and only one report on the cloning of the synthesis-related genes from a Bacillus species (B. megaterium) has been published;Bacillus sp. INT005 is thought to be very valuable source of PHA synthesis-related genes.

Structure of bacterial cellulose synthase subunit D octamer with four inner passageways

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 His6 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.

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and NADPH regeneration in Vitro.

We established a novel enzyme-catalyzed poly(3-hydroxybutyrate) [P(3HB)] synthesis system capable of recycling CoA on the basis of the P(3HB) biosynthetic pathway in Ralstonia eutropha. The system includes purified beta-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC), acetyl-CoA synthetase (Acs) and glucose dehydrogenase (GDH). In this system, acetyl-CoA was synthesized from acetate and CoA by Acs and ATP, and then two molecules of acetyl-CoA were condensed by PhaA to synthesize acetoacetyl-CoA, which was converted to (R)-3-hydroxybutyryl-CoA (3HBCoA) by PhaB and NADPH. The 3HBCoA was polymerized by PhaC and converted to P(3HB). In this system, the CoA molecules that were released during the condensation and polymerization reactions catalyzed by PhaA and PhaC, respectively, were reused successfully for the synthesis of acetyl-CoA. In addition, NADPH, which was consumed in the reduction of acetoacetyl-CoA, was regenerated by the action of GDH. In this system, the yield of P(3HB) synthesized from acetate as the substrate was 5.6 mg in a 5-ml reaction mixture, and the weight-average molecular weight and polydispersity were 6.64 x 10(6) and 1.36, respectively. Furthermore, CoA was reused at least 26 times, and NADPH was also regenerated at least 26 times during 24 h of reaction.

Structural characterization of the Acetobacter xylinum endo-β-1,4-glucanase CMCax required for cellulose biosynthesis

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis‐related endo‐β‐1,47‐glucanase (CMCax; EC 3.2.1.4) from the cellulose‐producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65‐Å resolution. CMCax falls into the glycoside hydrolase family 8 (GH‐8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH‐8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH‐8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing‐end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane‐anchored endoglucanases that are required for cellulose biosynthesis. Proteins 2006.

Polyhydroxyalkanoate synthase from Bacillus sp. INT005 is composed of PhaC and PhaR

A polyhydroxyalkanoate (PHA) biosynthesis gene locus from Bacillus sp. INT005 strain, which had been isolated from a gas field, was cloned and analyzed at the molecular level. We found that a 3.8-kbp DraI-digested fragment of genomic DNA of Bacillus sp. INT005 conferred PHA-producing ability to Escherichia coli, which was PHA-negative. The DNA fragment contained three genes, phaR, -B and -C. The activity of 3-ketoacyl-CoA reductase with NADPH was detected in the lysate from recombinant E. coli carrying the phaB gene. Although PHA synthase activity could be detected in the extract from E. coli carrying phaR, -B and -C genes, no such activity could be detected in that from E. coli carrying only the phaC gene. However, the mixture of the crude extracts of E. coli expressing phaR or phaC revealed very high PHA synthase activity. Furthermore, when His-tagged PhaC was purified by Ni-affinity chromatography from the mixture of crude extracts containing His-tagged PhaC or native PhaR, the eluate contained His-tagged PhaC and native PhaR. On the other hand, PhaR did not bind to the column directly. This purified PhaC with PhaR had 160-fold higher specific activity of PHA synthase than that without PhaR. In addition, the kinetics of the purified PhaC with PhaR revealed a lag phase that preceded the linear phase. It has been known that class III PHA synthase is composed of two different subunits, PhaC and PhaE, and phaC and phaE genes are directly linked in the genomes. Furthermore, the PHA synthase has no lag phase. We hence concluded that the PHA synthase of Bacillus sp. INT005 consists of PhaC and PhaR, and has characteristics different from class III PHA synthase.

Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol

The hydroxylation of tyrosine is an important reaction in the biosynthesis of many natural products. The use of bacteria for this reaction has not been very successful due to either the over-oxidation to ortho-quinone when using tyrosinases from bacteria or plants, or the lack of the native cofactor, tetrahydrobiopterin (BH4), needed for the activity of tyrosine hydroxylases (TH). Here, we demonstrate that an Escherichia coli cofactor, tetrahydromonapterin (MH4), can be used as an alternative cofactor for TH in presence of the BH4 regeneration pathway, and tyrosine hydroxylation is performed without over-oxidation. We used this platform for biosynthesis of one of the most powerful antioxidants, hydroxytyrosol. An endogenous aromatic aldehyde oxidase was identified and knocked out to prevent formation of the side product, and this resulted in nearly exclusive production of hydroxytyrosol in engineered E. coli. Finally, hydroxytyrosol production from a simple sugar as a sole carbon source was demonstrated.

A method of cell-sheet preparation using collagenase digestion of salmon atelocollagen fibrillar gel.

We prepared a cell sheet by using collagenase treatment to digest salmon atelocollagen fibrillar gel (SAC gel) on which human periodontal ligament (HPDL) cells had been cultured. The SAC gel was found to be digested completely within 2 h at a concentration of 50 U of collagenase per mg of collagen. The SAC gel on which HPDL cells were cultured for 10 d was treated with collagenase, resulting in the formation of a detached and shrunken cell sheet. Immunostaining results showed that the cytoskeleton and fibronectin matrix level of the cell sheet were maintained after collagenase treatment. In addition, collagenase treatment had almost no effect on the activities of HPDL cells.

Engineering of a Tyrosol-Producing Pathway, Utilizing Simple Sugar and the Central Metabolic Tyrosine, in Escherichia coli

Metabolic engineering was applied to the development of Escherichia coli capable of synthesizing tyrosol (2-(4-hydroxyphenyl)ethanol), an attractive phenolic compound with great industrial value, from glucose, a renewable carbon source. In this strain, tyrosine, which was supplied not only from the culture medium but also from the central metabolism, was converted into tyrosol via three steps: decarboxylation, amine oxidation, and reduction. The engineered strain synthesized both tyrosol and 4-hydroxyphenylacetate (4HPA), but disruption of the endogenous phenylacetaldehyde dehydrogenase gene shut off 4HPA production and improved the production of tyrosol as a sole product. The engineered mutant strain was capable of producing 0.5 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation.