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 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.

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.

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity.

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha.

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.

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 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

In vivo effects of isolated implantation of salmon-derived crosslinked atelocollagen sponge into an osteochondral defect.

We have developed crosslinked salmon-derived atelocollagen (SC) sponge, which has a denaturation temperature of 47°C. Sixty-four knees of 32 mature rabbits were randomly divided into 4 groups after creating an osteochondral defect in the femoral trochlea. Defects in Groups I, II, and III were filled with the crosslinked SC sponge, the crosslinked porcine collagen (PC) sponge, and the non-crosslinked PC sponge, respectively. In Group IV, defects were left untreated as the control. At 12 weeks after implantation, the histological score showed that Group I was significantly greater than Groups III (P = 0.0196) and IV (P = 0.0021). In addition, gene expression of type-2 collagen, aggrecan, and SOX9 was the greatest in Group I at 12 weeks. The fundamental in vivo properties of the crosslinked SC sponge showed that this is a promising biomaterial, specifically as a scaffold for cartilage tissue engineering.

Development of tissue-engineered human periodontal ligament constructs with intrinsic angiogenic potential.

One approach to treat periodontal diseases is grafting of tissue-engineered periodontal ligaments. Therefore, periodontal ligaments were constructed by layering cell sheets. A cell sheet was prepared by enzymatic digestion of salmon collagen gel on which human periodontal ligament fibroblasts (HPLFs) were co-cultured with or without human umbilical vein endothelial cells (HUVECs). Three cell sheets were layered and then cultured in angiogenic media, in which the HUVECs were found to form capillary-like structures when co-cultured on the HPLFs. The layered HPLFs sheets with HUVEC co-culture (PL-EC construct) demonstrated longer survival, higher alkaline phosphatase activities and lower osteocalcin production than layered HPLFs sheets without HUVEC co-culture (PL construct). Hematoxylin-eosin and Masson's trichrome staining of histological sections showed that cell density, mass and extracellular matrix deposition of the PL-EC construct were higher than those of the PL construct. Furthermore, CD31 immunostaining revealed the formation of capillary-like structures throughout the PL-EC construct. In conclusion, we successfully developed tissue-engineered periodontal ligament constructs with intrinsic angiogenic potential using cell sheet engineering and HUVEC co-culture.

Development of novel covered stents using salmon collagen.

In this study, we newly developed self-expandable or balloon-expandable covered stents with a biodegradable salmon collagen (SC) film. The SC-covered stents were fabricated by placing a bare stent in a mixture of acidic SC solution and a fibrillogenesis-inducing buffer (pH 6.8) including a cross-linking agent (water-soluble carbodiimide), and subsequent incubation at 4 degrees C for 24 h and lyophilization. The stents obtained were completely covered with an SC film having a nanofibrous structure (fibril diameter, about 70 nm). On immersion in water, the film is converted to a gel with slight swelling. There was no rupture of the SC cover after mounting on a balloon catheter or after expansion. Preliminary implantation was conducted by placing the balloon-expandable covered stents in the common carotid arteries of beagles. One month after implantation, angiography showed that all stented arteries were patent with no significant neointimal thickening. In conclusion, SC is potentially useful as a cover material of endovascular stents to enhance patency.

Purification, crystallization and preliminary X-ray studies of AxCesD required for efficient cellulose biosynthesis in Acetobacter xylinum.

AxCesD protein required for bacterial cellulose biosynthesis in Acetobacter xylinum was overexpressed in E. coli, purified and crystallized. Single crystals of SeMet-substituted AxCesD were obtained by the sitting-drop vapor-diffusion method. The crystal belongs to the primitive trigonal space group P3 2, with unit-cell parameters a = b = 77.7 A, and c = 213.9 A. The asymmetric unit in the crystal was assumed to contain 8 protein molecules giving the Matthews coefficient (VM) of 2.54 A3 Da(-1). Se-MAD data were collected to 2.3 A resolution using synchrotron radiations.

Biological properties of crosslinked salmon collagen fibrillar gel as a scaffold for human umbilical vein endothelial cells.

Collagen derived from chum salmon (Oncorhynchus keta) was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) during collagen fibrillogenesis and applied to an in vitro cell culture to evaluate its potential use as a scaffold for vascular tissue engineering. Human umbilical vein endothelial cells (HUVEC) were cultured on the crosslinked salmon collagen fibrillar gel (EDC-SC gel), and their growth rates and production levels of cytokines, including platelet-derived growth factor-BB and von Willebrand factor, were measured. Comparison was also made with bovine collagen gel crosslinked with EDC (EDC-BC gel). The growth and cytokine production of the HUVEC cultured on the EDC-SC gel were higher than those on the EDC-BC gel. In addition, HUVEC were found to attach to the EDC-BC gel through alpha2beta1 integrin for native collagen, whereas they attached to the EDC-SC gel through alphavbeta3 integrin for denatured collagen as well as the alpha2beta1 integrin, indicating that HUVEC recognized denatured domains in the EDC-SC gel. In conclusion, the EDC-SC gel can be used as a scaffold to support HUVEC growth, although the integrin-mediated attachment manner differs between the two gels.

In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel.

Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.

Preparation and characterization of multilayered hydroxyapatite/silk fibroin film.

We prepared multilayered films consisting of silk fibroin (SF) and hydroxyapatite (HAp) by alternating lamination using untreated SF and HAp-deposited SF films. Untreated SF films were prepared from a regenerated SF solution by air drying. HAp-deposited SF films were prepared by soaking methanol-treated SF films containing >5 wt% CaCl2 in a simulated body fluid with the ion concentration 1.5-fold higher than that of the standard one. The multilayered HAp/SF films had HAp layers with approximate thicknesses of 3-5 microm and SF layers with thicknesses of 40-70 microm. The bonding strength between the SF and HAp layers was significantly affected by temperature and compression time under the lamination method. The optimal conditions for achieving the maximum T-peel strength and beta-sheet contents were determined to be 130 degrees C for 4 min. The Young's modulus of the multilayered films (133.4 MPa) was higher than that of the films consisting of SF alone (92.5 MPa) under swollen conditions. The biocompatibility of the HAp-deposited SF films was analyzed by culturing of osteoblasts (MC3T3-E1) on a film. The results indicate that HAp-deposited SF films and SF films show similar degrees of cell adhesion and alkaline phosphatase activities.

Structural characterization of the Acetobacter xylinum endo-beta-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-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A 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.

Activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen scaffold.

We investigated the effect of gamma-irradiation on the activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen (SAC) scaffolds. SAC-cultured samples were irradiated at doses of 10, 15, and 25 kGy. Gamma-irradiation had a significant impact on the activities of MC3T3-E1 cells. The proliferation rates and alkaline phosphatase activities of MC3T3-E1 cells increased with gamma-irradiation dose.

The effects of herring-roe lyophilized powder on lipid metabolism.

Herring-roe, which contains large amounts of docosahexaenoic acid and eicosapentaenoic acid, has anti-dyslipidemia effects. Here, we evaluated the effects of herring-roe on lipid metabolism in 33 adult subjects in a randomized, double-blind, placebo-controlled study. We divided the subjects into a test group that ingested herring-roe lyophilized powder (herring-roe powder) and a placebo group that ingested non-herring-roe powder, with each member of each group ingesting 15 g daily for 8 weeks. Hematological tests and body composition measurements were performed before and after 4, 6, and 8 weeks of the study period. Although no significant differences in low density lipoprotein were observed, high density lipoprotein was found to be increased in subjects who ingested herring-roe powder. In addition, the level of free fatty acid was significantly improved in the herring-roe powder group. These results suggest that ingestion of herring-roe could influence lipid metabolism.

Crystallization and preliminary crystallographic analysis of the cellulose biosynthesis-related protein CMCax from Acetobacter xylinum.

The cellulose biosynthesis-related protein CMCax from Acetobacter xylinum was overexpressed in Escherichia coli, purified and crystallized. Single crystals of selenomethionine (SeMet) substituted CMCax were obtained by the hanging-drop vapour-diffusion method at 293 K, primarily using polyethylene glycol 4000 as a precipitant. The crystals belong to the primitive hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 89.1, c = 94.2 A. The predicted Matthews coefficient (VM) value is 3.0 A3 Da(-1) for one CMCax monomer in the asymmetric unit. A single-wavelength anomalous dispersion (SAD) data set was collected to a resolution of 2.3 A using synchrotron radiation.

Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase.

We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.

Cloning of cellulose synthesis related genes from Acetobacter xylinum ATCC23769 and ATCC53582: comparison of cellulose synthetic ability between strains.

About 14.5 kb of DNA fragments from Acetobacter xylinum ATCC23769 and ATCC53582 were cloned, and their nucleotide sequences were determined. The sequenced DNA regions contained endo-beta-1,4-glucanase, cellulose complementing protein, cellulose synthase subunit AB, C, D and beta-glucosidase genes. The results from a homology search of deduced amino acid sequences between A. xylinum ATCC23769 and ATCC53582 showed that they were highly similar. However, the amount of cellulose production by ATCC53582 was 5 times larger than that of ATCC23769 during a 7-day incubation. In A. xylinum ATCC53582, synthesis of cellulose continued after glucose was consumed, suggesting that a metabolite of glucose, or a component of the medium other than glucose, may be a substrate of cellulose. On the other hand, cell growth of ATCC23769 was twice that of ATCC53582. Glucose is the energy source in A. xylinum as well as the substrate of cellulose synthesis, and the metabolic pathway of glucose in both strains may be different. These results suggest that the synthesis of cellulose and the growth of bacterial cells are contradictory.

Effects of irradiation on cementum matrix cytokins function during periodontal regeneration.

The influence of gamma-ray irradiation on a cementum-impregnated gelatine membrane (CGM) was analyzed with emphasis on its function during periodontal regeneration. In brief, proteins were extracted from gamma-ray irradiated cementum (gammaC). With the gammaC protein, sample cells (gingival fibroblasts, periodontal ligament cells, and alveolar bone cells) were co-cultured, and cytological parameters (cell attachment, cell differentiation and alkaline phosphatase activity) were analyzed. Additionally, kinetics of some gene expression was analysed using reverse transcript RT-PCR, which included osteoproteogerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) mRNA. BMP-2 and osteonectin were resistant to gamma-rays, and other cytokines involved in regeneration were decreased. Thus, the attachment activity of osteoblasts to gammaC protein was higher than that of non-irradiated cementum (control C). The expression of OPG/OCIF mRNA was lower in co-cultured cells with gammaC protein than those with in control C protein. Together the results imply that some cytokine in intact cementum prevents the attachment (differentiation) of bone cells onto the root surface, which may explain why the introduction of CGM following gingival flap surgery induces new cementum, new ligament and new bone formation, but CGM irradiated with gamma-rays for clinical use causes ankylosis.

Novel biomaterial from reinforced salmon collagen gel prepared by fibril formation and cross-linking.

The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature (Td) of the SC solution was found to be 18.6 degrees C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4 degrees C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM (f-50 gel). In particular, the melting temperature of the f-50 gel was 47 degrees C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f-50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation.