Recent Advances in Polymers Bearing Activated Esters for the Synthesis of Glycopolymers by Postpolymerization Modification.

Glycopolymers are functional polymers with saccharide moieties on their side chains and are attractive candidates for biomaterials. Postpolymerization modification can be employed for the synthesis of glycopolymers. Activated esters are useful in various fields, including polymer chemistry and biochemistry, because of their high reactivity and ease of reaction. In particular, the formation of amide bonds caused by the reaction of activated esters with amino groups is of high synthetic chemical value owing to its high selectivity. It has been employed in the synthesis of various functional polymers, including glycopolymers. This paper reviews the recent advances in polymers bearing activated esters for the synthesis of glycopolymers by postpolymerization modification. The development of polymers bearing hydrophobic and hydrophilic activated esters is described. Although water-soluble activated esters are generally unstable and hydrolyzed in water, novel polymer backbones bearing water-soluble activated esters are stable and useful for postpolymerization modification for synthesizing glycopolymers in water. Dual postpolymerization modification can be employed to modify polymer side chains using two different molecules. Thiolactone and glycine propargyl esters on the polymer backbone are described as activated esters for dual postpolymerization modification.

Cellulose-fueled microbial fuel cells equipped with a bipolar membrane using hydrogen phosphate as the final electron acceptor.

OBJECTIVES: A bipolar membrane microbial fuel cell (bMFC) is used to generate electricity using cellulose in phosphate buffer solution as fuel, and the mechanism of electricity generation is elucidated from five reference experiments. RESULTS: The bMFC was operated for 20 days using cellulose as fuel and Cellulomonas fimi. In the first reference experiment, no microorganism was used. In the second experiment, a cation-exchange membrane was used instead of a bipolar membrane. In the third experiment, the bipolar membrane was used in the opposite orientation as in the main experiment. In the fourth experiment, D2O was used instead of H2O in the cathode chamber. In the final experiment, the tris-maleate buffer was used instead of a phosphate buffer. Sufficient power generation did not occur in either reference experiment. CONCLUSIONS: The bMFC continuously generated electricity for 20 days, and elucidated H+ and OH- react in bipolar membrane, where the counter cation of dihydrogen phosphate served as the final electron acceptor.

Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates the detection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4 °C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.

Production of glycerate from glucose using engineered Escherichiacoli.

In this study, glycerate was produced from glucose using engineered Escherichia coli BW25113. Plasmid pSR3 carrying gpd1 and gpp2 encoding two isoforms of glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae and plasmid pLB2 carrying aldO encoding alditol oxidase from Streptomyces violaceoruber were introduced into E. coli to enable the production of glycerate from glucose via glycerol. Disruptions of garK and glxK genes in the E. coli genome were performed to minimize the consumption of glycerate produced. As a result, E. coli carrying these plasmids could produce nearly three times higher concentration of glycerate (0.50 ± 0.01 g/L) from 10 g/L glucose compared to E. coli EG_2 (0.14 ± 0.02 g/L). In M9 medium, disruption of garK and glxK resulted in an impaired growth rate with low production of glycerate, while supplementation of 0.5 g/L casamino acids and 0.5 g/L manganese sulfate to the medium replenished the growth rate and elevated the glycerate titer. Further disruption of glpF, encoding a glycerol transporter, increased the glycerate production to 0.80 ± 0.00 g/L. MR2 medium improved the glycerate production titers and specific productivities of E. coli EG_4, EG_5, and EG_6. Upscale production of glycerate was carried out in a jar fermentor with MR2 medium using E. coli EG_6, resulting in an improvement in glycerate production up to 2.37 ± 0.46 g/L with specific productivity at 0.34 ± 0.11 g-glycerate/g-cells. These results indicate that E. coli is an appropriate host for glycerate production from glucose.

The first report of enzymatic transglycosylation catalyzed by family GH84 N-acetyl-ß-d-glucosaminidase using a sugar oxazoline derivative as a glycosyl donor.

O-Glycosylated N-acetyl-ß-d-glucosamine-selective N-acetyl-ß-d-glucosaminidase (O-GlcNAcase), belonging to glycoside hydrolase family 84 (GH84), is known as a retaining glycosidase with the possibility of enzymatic transglycosylation. However, no enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase has been reported. Here, enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase was first reported. The enzymatic transglycosylation catalyzed by the GH84 O-GlcNAcase from Bacteroides thetaiotaomicron (BtGH84 O-GlcNAcase) was attained using 1,2-oxazoline derivative of N-acetyl-d-glucosamine (GlcNAc oxazoline) as a glycosyl donor substrate. The ß-linked N-acetyl-d-glucosamine (GlcNAc) derivative was enzymatically synthesized using N-(2-hydroxyethyl)acrylamide as an acceptor substrate. Interestingly, the ß1,6-linked disaccharide derivative of GlcNAc was also obtained in the case of using the GlcNAc derivative with a triazole-linked acrylamide group as an acceptor substrate. Additionally, a one-pot chemo-enzymatic transglycosylation starting from unprotected GlcNAc through GlcNAc oxazoline successfully showed through the combination with the direct synthesis of GlcNAc oxazoline in water and the enzymatic transglycosylation.

Production of R- and S-1,2-propanediol in engineered Lactococcus lactis.

1,2-propanediol (1,2-PDO) is a versatile chemical used in multiple manufacturing processes. To date, some engineered and non-engineered microbes, such as Escherichia coli, Lactobacillus buchneri, and Clostridium thermosaccharolyticum, have been used to produce 1,2-PDO. In this study, we demonstrated the production of R- and S-1,2-PDO using engineered Lactococcus lactis. The L- and D-lactic acid-producing L. lactis strains NZ9000 and AH1 were transformed with the plasmid pNZ8048-ppy harboring pct, pduP, and yahK genes for 1,2-PDO biosynthesis, resulting in L. lactis LL1 and LL2, respectively. These engineered L. lactis produced S- and R-1,2-PDO at concentrations of 0.69 and 0.50 g/L with 94.4 and 78.0% ee optical purities, respectively, from 1% glucose after 72 h of cultivation. Both 1% mannitol and 1% gluconate were added instead of glucose to the culture of L. lactis LL1 to supply NADH and NADPH to the 1,2-PDO production pathway, resulting in 75% enhancement of S-1,2-PDO production. Production of S-1,2-PDO from 5% mannitol and 5% gluconate was demonstrated using L. lactis LL1 with a pH-stat approach. This resulted in S-1,2-PDO production at a concentration of 1.88 g/L after 96 h of cultivation. To our knowledge, this is the first report on the production of R- and S-1,2-PDO using engineered lactic acid bacteria.

Microbial fuel cells using α-amylase-displaying Escherichia coli with starch as fuel.

Escherichia coli JM109 (pGV3-SBA) can assimilate starch by fusing the starch-digesting enzyme α-amylase from Streptococcus bovis NRIC1535 to an OprI' lipoprotein anchor on the cell membrane. This study shows microbial fuel cells (MFCs) development using this recombinant type of E. coli and starch as fuel. We observed the current generation of MFCs with E. coli JM109 (pGV3-SBA) for 120 h. During this period, it consumed 7.1 g/L of starch. A mediator in the form of anthraquinone-2,6-disulfonic acid disodium salt at 0.2, 0.4, and 0.8 mM was added to the MFCs. The highest maximum-current density (271 mA/m2) and maximum-power density (29.3 mW/m2) performances occurred in the 0.4 mM mediator solution. Coulomb yields were calculated as 3.4%, 3.0%, and 3.5% in 1.0, 5.0, and 10.0 g/L of initial starch, respectively. The concentrations of acetic acid, succinic acid, fumaric acid, and ethanol as metabolites were determined. In particular, 38.3 mM of ethanol was produced from 7.1 g/L of starch. This study suggests the use of recombinant E. coli which can assimilate starch present in starch-fueled MFCs. Moreover, it proposes the possibility of gene recombination technology for using wide variety of biomass as fuel and improving MFC's performance.

Chemo-enzymatic synthesis of Lacto-N-biose I catalyzed by ß-1,3-galactosidase from Bacillus circulans using 4,6-dimethoxy-1,3,5-triazin-2-yl ß-galactopyranoside as a glycosyl donor.

Chemo-enzymatic synthesis of lacto-N-biose I (LNB) catalyzed by ß-1,3-galactosidase from Bacillus circulans (BgaC) has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl ß-galactopyranoside (DMT-ß-Gal) and GlcNAc as the donor and acceptor substrates, respectively. BgaC transferred the Gal moiety to the acceptor, giving rise to LNB. The maximum yield of LNB was obtained at the acceptor : donor substrate ratio of 1:30.

Utility of Thermal Cross-Linking in Stabilizing Hydrogels with Beta-Tricalcium Phosphate and/or Epigallocatechin Gallate for Use in Bone Regeneration Therapy.

ß-tricalcium phosphate (ß-TCP) granules are commonly used materials in dentistry or orthopedic surgery. However, further improvements are required to raise the operability and bone-forming ability of ß-TCP granules in a clinical setting. Recently, we developed epigallocatechin gallate (EGCG)-modified gelatin sponges as a novel biomaterial for bone regeneration. However, there is no study on using the above material for preparing hydrogel incorporating ß-TCP granules. Here, we demonstrate that vacuum heating treatment induced thermal cross-linking in gelatin sponges modified with EGCG and incorporating ß-TCP granules (vhEc-GS-ß) so that the hydrogels prepared from vhEc-GS-ß showed high stability, ß-TCP granule retention, operability, and cytocompatibility. Additionally, microcomputed tomography morphometry revealed that the hydrogels from vhEc-GS-ß had significantly higher bone-forming ability than ß-TCP alone. Tartrate-resistant acid phosphatase staining demonstrated that the number of osteoclasts increased at three weeks in defects treated with the hydrogels from vhEc-GS-ß compared with that around ß-TCP alone. The overall results indicate that thermal cross-linking treatment for the preparation of sponges (precursor of hydrogels) can be a promising process to enhance the bone-forming ability. This insight should provide a basis for the development of novel materials with good operativity and bone-forming ability for bone regenerative medicine.

Gas Permeability of Mold during Freezing Process Alters the Pore Distribution of Gelatin Sponge and Its Bone-Forming Ability.

Freeze-drying, also known as lyophilization, is widely used in the preparation of porous biomaterials. Nevertheless, limited information is known regarding the effect of gas permeability on molds to obtain porous materials. We demonstrated that the different levels of gas permeability of molds remarkably altered the pore distribution of prepared gelatin sponges and distinct bone formation at critical-sized bone defects of the rat calvaria. Three types of molds were prepared: silicon tube (ST), which has high gas permeability; ST covered with polyvinylidene chloride (PVDC) film, which has low gas permeability, at the lateral side (STPL); and ST covered with PVDC at both the lateral and bottom sides (STPLB). The cross sections or curved surfaces of the sponges were evaluated using scanning electron microscopy and quantitative image analysis. The gelatin sponge prepared using ST mold demonstrated wider pore size and spatial distribution and larger average pore diameter (149.2 µm) compared with that prepared using STPL and STPLB. The sponges using ST demonstrated significantly poor bone formation and bone mineral density after 3 weeks. The results suggest that the gas permeability of molds critically alters the pore size and spatial pore distribution of prepared sponges during the freeze-drying process, which probably causes distinct bone formation.

Engineering Escherichia coli for Direct Production of 1,2-Propanediol and 1,3-Propanediol from Starch.

Diols are versatile chemicals used for multiple manufacturing products. In some previous studies, Escherichia coli has been engineered to produce 1,2-propanediol (1,2-PDO) and 1,3-propanediol (1,3-PDO) from glucose. However, there are no reports on the direct production of these diols from starch instead of glucose as a substrate. In this study, we directly produced 1,2-PDO and 1,3-PDO from starch using E. coli engineered for expressing a heterologous α-amylase, along with the expression of 1,2-PDO and 1,3-PDO synthetic genes. For this, the recombinant plasmids, pVUB3-SBA harboring amyA gene for α-amylase production, pSR5 harboring pct, pduP, and yahK genes for 1,2-PDO production, and pSR8 harboring gpd1-gpp2, dhaB123, gdrAB, and dhaT genes for 1,3-PDO production, were constructed. Subsequently, E. coli BW25113 (ΔpflA) and BW25113 strains were transformed with pVUB3-SBA, pSR5, and/or pSR8. Using these transformants, direct production of 1,2-PDO and 1,3-PDO from starch was demonstrated under microaerobic condition. As a result, the maximum production titers of 1,2-PDO and 1,3-PDO from 1% glucose as a sole carbon source were 13 mg/L and 150 mg/L, respectively. The maximum production titers from 1% starch were similar levels (30 mg/L 1,2-PDO and 120 mg/L 1,3-PDO). These data indicate that starch can be an alternative carbon source for the production of 1,2-PDO and 1,3-PDO in engineered E. coli. This technology could simplify the upstream process of diol bioproduction.

Itaconic acid derivatives: structure, function, biosynthesis, and perspectives.

Itaconic acid possessing a vinylidene group, which is mainly produced by fungi, is used as a biobased platform chemical and shows distinctive bioactivities. On the other hand, some fungi and lichens produce itaconic acid derivatives possessing itaconic acid skeleton, and the number of the derivatives is currently more than seventy. Based on the molecular structures, they can be categorized into two groups, alkylitaconic acids and α-methylene-γ-butyrolactones. Interestingly, some itaconic acid derivatives show versatile functions such as antimicrobial, anti-inflammatory, antitumor, and plant growth-regulating activities. The vinylidene group of itaconic acid derivatives likely participates in these functions. It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first biosynthesized from acyl-CoA and oxaloacetic acid. Some modifying enzymes such as hydroxylase and dehydratase are likely involved in the further modification after biosynthesis of their precursors. This contributes to the diversity of itaconic acid derivatives. In this review, we summarize their structures, functions, and biosynthetic pathways together with a discussion of a strategy for the industrial use. KEY POINTS: • Itaconic acid derivatives can be categorized into alkylitaconic acids and α-methylene-γ-butyrolactones. • The vinylidene group of itaconic acid derivatives likely participates in their versatile function. • It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first synthesized from acyl-CoA and oxaloacetic acid.

One-pot chemoenzymatic synthesis of glycopolymers from unprotected sugars via glycosidase-catalysed glycosylation using triazinyl glycosides.

Glycopolymers were successfully synthesised from unprotected sugars in aqueous media via a one-pot chemoenzymatic process of three reactions; the direct synthesis of 4,6-dimethoxy-1,3,5-triazin-2-yl glycosides from unprotected sugars, a glycosidase-catalysed glycosylation using the triazinyl glycoside to afford glycomonomers and a radical polymerisation. The resulting glycopolymers exhibited specific interactions with the corresponding lectin as glycoclusters.

Correction: Sano, M., et al. Microbial Screening Based on the Mizoroki-Heck Reaction Permits Exploration of Hydroxyhexylitaconic-Acid-Producing Fungi in Soils. Microorganisms 2020, 8, 648.

The authors wish to make the following correction to this paper [...].

Augmentation of Bone Regeneration by Depletion of Stress-Induced Senescent Cells Using Catechin and Senolytics.

Despite advances in bone regenerative medicine, the relationship between stress-induced premature senescence (SIPS) in cells and bone regeneration remains largely unknown. Herein, we demonstrated that the implantation of a lipopolysaccharide (LPS) sustained-release gelatin sponge (LS-G) increases the number of SIPS cells and that the elimination of these cells promotes bone formation in critical-sized bone defects in the rat calvaria. Histological (hematoxylin-eosin and SA-ß-gal) and immunohistological (p16 and p21 for analyzing cellular senescence and 4-HNE for oxidation) staining was used to identify SIPS cells and elucidate the underlying mechanism. Bone formation in defects were analyzed using microcomputed tomography, one and four weeks after surgery. Parallel to LS-G implantation, local epigallocatechin gallate (EGCG) administration, and systemic senolytic (dasatinib and quercetin: D+Q) administration were used to eliminate SIPS cells. After LS-G implantation, SA-ß-gal-, p16-, and p21-positive cells (SIPS cells) accumulated in the defects. However, treatment with LS-G+EGCG and LS-G+D+Q resulted in lower numbers of SIPS cells than that with LS-G in the defects, resulting in an augmentation of newly formed bone. We demonstrated that SIPS cells induced by sustained stimulation by LPS may play a deleterious role in bone formation. Controlling these cell numbers is a promising strategy to increase bone regeneration.

Biobased Poly(itaconic Acid-co-10-Hydroxyhexylitaconic Acid)s: Synthesis and Thermal Characterization.

Renewable vinyl compounds itaconic acid (IA) and its derivative 10-hydroxyhexylitaconic acid (10-HHIA) are naturally produced by fungi from biomass. This provides the opportunity to develop new biobased polyvinyls from IA and 10-HHIA monomers. In this study, we copolymerized these monomers at different ratios through free radical aqueous polymerization with potassium peroxodisulfate as an initiator, resulting in poly(IA-co-10-HHIA)s with different monomer compositions. We characterized the thermal properties of the polymers by thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). The nuclear magnetic resonance analysis and the gel permeation chromatography showed that the polymerization conversion, yield, and the molecular weights (weight-averaged Mw and number-averaged Mn) of the synthesized poly(IA-co-10-HHIA)s decreased with increasing 10-HHIA content. It is suggested that the hydroxyhexyl group of 10-HHIA inhibited the polymerization. The TGA results indicated that the poly(IA-co-10-HHIA)s continuously decomposed as temperature increased. The FT-IR analysis suggested that the formation of the hydrogen bonds between the carboxyl groups of IA and 10-HHIA in the polymer chains was promoted by heating and consequently the polymer dehydration occurred. To the best of our knowledge, this is the first time that biobased polyvinyls were synthesized using naturally occurring IA derivatives.

Microbial Screening Based on the Mizoroki-Heck Reaction Permits Exploration of Hydroxyhexylitaconic-Acid-Producing Fungi in Soils.

Recently, we developed a unique microbial screening method based on the Mizoroki-Heck reaction for itaconic acid (IA)-producing fungi. This method revealed that 37 out of 240 fungal strains isolated from soils produce vinyl compounds, including IA. In this study, we further characterized these compounds in order to verify that the screening method permits the isolation of fungi that produce other vinyl compounds, excluding IA. HPLC analysis showed that 11 out of 37 isolated strains produced IA, similar to Aspergillus terreus S12-1. Surprisingly, the other 8 isolated strains produced two vinyl compounds with HPLC retention times different from that of IA. From these strains, the vinyl compounds of Aspergillus niger S17-5 were characterized. Mass spectrometric and NMR analyses showed that they were identical to 8-hydroxyhexylitaconic acid (8-HHIA) and 9-HHIA. This finding showed that 8-HHIA- and 9-HHIA-producing fungi, as well as IA-producing fungi, are ubiquitously found in soils. Neither 8-HHIA nor 9-HHIA showed antibacterial or anti-inflammatory activities. Interestingly, 8-HHIA and 9-HHIA showed cytotoxicity against the human cervical cancer cell line (HeLa) and human diploid cell line (MRC-5), and MRC-5 only, respectively, compared to IA at the same concentration. This study indicates that the screening method could easily discover fungi producing 8-HHIA and 9-HHIA in soils.

Synthesis and Aggregation Behavior of Temperature- and pH-Responsive Glycopolymers as Sugar-Displaying Conjugates.

Stimuli-responsive polymers have attracted significant interest in the fields of advanced materials and biomaterials. Herein, temperature- and pH-responsive glycopolymers, which are composed of N-isopropylacrylamide, methacrylic acid, and an acrylamide derivative bearing a lactose moiety, were synthesized via radical copolymerization. The series of resulting glycopolymers had different degrees of substitution of the lactose moieties, were responsive to temperatures between 26.6 °C and 47.6 °C, and formed aggregates above the lower critical solution temperature limit in mild acidic aqueous media (pH 4-6). The temperature-responsive behavior was dependent on the prevailing pH conditions, as no aggregation was observed in neutral and basic aqueous media (pH > 7). The aggregates had saccharide moieties on the surface in aqueous media. The number of saccharide moieties on the surface depended on the saccharide-containing unit ratio in the glycopolymer. The ratio was determined via enzymatic hydrolysis of the lactose moieties using ß-galactosidase and the subsequent detection of the released galactose.

Effect of the Application of a Dehydrothermal Treatment on the Structure and the Mechanical Properties of Collagen Film.

Dehydrothermal (DHT) treatment was used to improve the properties of collagen casings because of its non-cytotoxicity. Understanding the effects of DHT treatment on the structure and mechanical properties of collagen films is beneficial to developing satisfying collagen casings. Herein, DHT treatment with various temperatures (85-145 °C) and timescales (1-7 days) were investigated. It was clarified that the chemical crosslinking covalent bond between collagen molecules was formed after the DHT treatment. Crosslinking density increased with increasing DHT treatment temperatures, contributing to the increase of tensile strength up to over three times of that of the untreated collagen film. The increased crosslinking density was also found when increasing the DHT treatment time, and the maximum was obtained in 3 days. Further DHT treatment time did not change the crosslinking density. The damage in the triple helix structure and the self-assembly of collagen molecules were observed from IR and SAXS. The extent of denaturation increased with increasing DHT treatment temperature and time, although the effect of the DHT treatment time on the denaturation was more moderate. When the DHT treatment temperature was as high as 145 °C or the DHT treatment time exceeded 5 days, serious denaturation occurs, leading to the deterioration of mechanical properties.

Aqueous One-pot Synthesis of Glycopolymers by Glycosidase-catalyzed Glycomonomer Synthesis Using 4,6-Dimetoxy Triazinyl Glycoside Followed by Radical Polymerization.

Glycopolymers have attracted increased attention as functional polymeric materials, and simple methods for synthesizing glycopolymers remain needed. This paper reports the aqueous one-pot and chemoenzymatic synthesis of four types of glycopolymers via two reactions: the ß-galactosidase-catalyzed glycomonomer synthesis using 4,6-dimetoxy triazinyl ß-D-galactopyranoside and hydroxy group-containing (meth)acrylamide and (meth)acrylate derivatives as the activated glycosyl donor substrate and as the glycomonomer precursors, respectively, followed by radical copolymerization of the resulting glycomonomer and excess glycomonomer precursor without isolating the glycomonomers. The resulting glycopolymers bearing galactose moieties exhibited specific and strong interactions with the lectin peanut agglutinin as glycoclusters.