Inhalation of hydrogen gas mitigates sevoflurane-induced neuronal apoptosis in the neonatal cortex and is associated with changes in protein phosphorylation.

Inhalation of hydrogen (H2) gas is therapeutically effective for cerebrovascular diseases, neurodegenerative disorders, and neonatal brain disorders including pathologies induced by anesthetic gases. To understand the mechanisms underlying the protective effects of H2 on the brain, we investigated the molecular signals affected by H2 in sevoflurane-induced neuronal cell death. We confirmed that neural progenitor cells are susceptible to sevoflurane and undergo apoptosis in the retrosplenial cortex of neonatal mice. Co-administration of 1-8% H2 gas for 3 h to sevoflurane-exposed pups suppressed elevated caspase-3-mediated apoptotic cell death and concomitantly decreased c-Jun phosphorylation and activation of the c-Jun pathway, all of which are induced by oxidative stress. Anesthesia-induced increases in lipid peroxidation and oxidative DNA damage were alleviated by H2 inhalation. Phosphoproteome analysis revealed enriched clusters of differentially phosphorylated proteins in the sevoflurane-exposed neonatal brain that included proteins involved in neuronal development and synaptic signaling. H2 inhalation modified cellular transport pathways that depend on hyperphosphorylated proteins including microtubule-associated protein family. These modifications may be involved in the protective mechanisms of H2 against sevoflurane-induced neuronal cell death.

Enhanced production of γ-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation.

BACKGROUND: Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics. RESULTS: Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture. CONCLUSIONS: Our results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.

Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation.

Although inhibition of epidermal growth factor receptor (EGFR)-mediated cell signaling by the EGFR tyrosine kinase inhibitor gefitinib is highly effective against advanced non-small cell lung cancer, this drug might promote severe acute interstitial pneumonia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive anti-oxidant. Here, we show that treatment with H2 effectively protects the lungs of mice from severe damage caused by oral administration of gefitinib after intraperitoneal injection of naphthalene, the toxicity of which is related to oxidative stress. Drinking H2-rich water ad libitum mitigated naphthalene/gefitinib-induced weight loss and significantly improved survival, which was associated with a decrease in lung inflammation and inflammatory cytokines in the bronchoalveolar lavage fluid. Naphthalene decreased glutathione in the lung, increased malondialdehyde in the plasma, and increased 4-hydroxy-2-nonenal production in airway cells, all of which were mitigated by H2-rich water, indicating that the H2-rich water reverses cellular damage to the bronchial wall caused by oxidative stress. Finally, treatment with H2 did not interfere with the anti-tumor effects of gefitinib on a lung cancer cell line in vitro or on tumor-bearing mice in vivo. These results indicate that H2-rich water has the potential to improve quality of life during gefitinib therapy by mitigating lung injury without impairing anti-tumor activity.

Identification of CCL5/RANTES as a novel contraction-reducible myokine in mouse skeletal muscle.

Skeletal muscle is an endocrine organ that secretes several proteins, which are collectively termed myokines. Although many studies suggest that exercise regulates myokine secretion, the underlying mechanisms remain unclear and all the exercise-dependent myokines have not yet been identified. Therefore, in this study, we attempted to identify novel exercise-dependent myokines by using our recently developed in vitro contractile model. Differentiated C2C12 myotubes were cultured with or without electrical pulse stimulation (EPS) for 24 h to induce cell contraction, and the myokines secreted in conditioned medium were analyzed using a cytokine array. Although most myokine secretions were not affected by EPS, the secretion of Chemokine (C-C motif) ligand 5 (CCL5) (regulated on activation, normal T cell expressed and secreted (RANTES)) was significantly reduced by EPS. This was further confirmed by ELISA and quantitative PCR. Contraction-dependent calcium transients and activation of 5'-AMP activating protein kinase (AMPK) appears to be involved in this decrease, as the chelating Ca2+ by EGTA blocked contraction-dependent CCL5 reduction, whereas the pharmacological activation of AMPK significantly reduced it. However, Ccl5 gene expression was increased by AMPK activation, suggesting that AMPK-dependent CCL5 decrease occurred via post-transcriptional regulation. Finally, mouse experiments revealed that voluntary wheel-running exercise reduced serum CCL5 levels and Ccl5 gene expression in the fast-twitch muscles. Overall, our study provides the first evidence of an exercise-reducible myokine, CCL5, in the mouse skeletal muscle. Although further studies are required to understand the precise roles of the skeletal muscle cell contraction-induced decrease in CCL5, this decrease may explain some exercise-dependent physiological changes such as those in immune responses.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

BACKGROUND: L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. RESULTS: Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. CONCLUSIONS: Metabolome analysis identified potential bottlenecks in D-glucose and L-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of D-glucose and L-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.

Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine.

Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.

Skeletal muscle cell contraction reduces a novel myokine, chemokine (C-X-C motif) ligand 10 (CXCL10): potential roles in exercise-regulated angiogenesis.

Accumulating evidence indicates that skeletal muscle secrets proteins referred to as myokines and that exercise contributes to their regulation. In this study, we propose that chemokine (C-X-C motif) ligand 10 (CXCL10) functions as a novel myokine. Initially, we stimulated differentiated C2C12 myotubes with or without electrical pulse stimulation (EPS) to identify novel myokines. Cytokine array analysis revealed that CXCL10 secretion was significantly reduced by EPS, which was further confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction analysis. Treadmill experiments in mice identified significant reduction of Cxcl10 gene expression in the soleus muscle. Additionally, contraction-dependent p38 MAPK activation appeared to be involved in this reduction. Furthermore, C2C12 conditioned medium obtained after applying EPS could induce survival of MSS31, a vascular endothelial cell model, which was partially attenuated by the addition of recombinant CXCL10. Overall, our findings suggest CXCL10 as a novel exercise-reducible myokine, to control endothelial cell viability.

Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process.

The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2 g L-1 sucrose, 8.5 g L-1 glucose, and 4.5 g L-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9 g L-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5 g L-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Differences in glucose yield of residues from among varieties of rice, wheat, and sorghum after dilute acid pretreatment.

Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 °C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.

Engineering cell factories for producing building block chemicals for bio-polymer synthesis.

Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.

FudC, a protein primarily responsible for furfural detoxification in Corynebacterium glutamicum.

Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

Natural variation in the glucose content of dilute sulfuric acid-pretreated rice straw liquid hydrolysates: implications for bioethanol production.

Rice straw is a promising resource for bioethanol production. Because the glucose content of pretreatment liquid hydrolysates is highly correlated with ethanol yield, the selection of appropriate rice cultivars is essential. The glucose content in liquid hydrolysates of pretreated rice straws of 208 diverse cultivars was evaluated in natural field in 2013 and 2014 using a novel high-throughput system. The glucose content of the rice straw samples varied across cultivars and was affected by environmental factors such as temperature and solar radiation. Several high-quality cultivars exhibiting high glucose content in both years were identified. The results of this study can aid in development of novel rice cultivars suitable as both feedstocks for bioethanol production and cooking.

Comparison of osteoradionecrosis of the jaw after superselective intra-arterial chemoradiotherapy versus conventional concurrent chemoradiotherapy of oral cancer.

PURPOSE: The purpose of the present study was to compare the incidence of osteoradionecrosis between superselective intra-arterial chemoradiotherapy and intravenous chemoradiotherapy and to verify the risk factors for osteoradionecrosis. MATERIALS AND METHODS: Of the 79 patients with oral cancer, 40 were treated with intra-venous chemoradiotherapy and 39 were treated with superselective intra-arterial chemoradiotherapy. The incidence of, and risk factors for, osteoradionecrosis were evaluated using statistical analysis. RESULTS: Of the 79 patients, 4 (10%) of 40 in the intravenous chemoradiotherapy and 7 (17.9%) of 39 in the superselective intra-arterial chemoradiotherapy group developed osteoradionecrosis of the jaw. No significant difference was found between the 2 groups. Although the chemoradiotherapy methods, anatomic tumor location, smoking behavior, alcohol consumption, condition of teeth, teeth extraction before radiation, and progression of dental caries were considered predisposing factors for the occurrence of osteoradionecrosis, only progressive dental caries resulted in a significant difference for osteoradionecrosis. CONCLUSIONS: The present study is the first report comparing the incidence of osteoradionecrosis between superselective intra-arterial chemoradiotherapy and intravenous chemoradiotherapy. The administration methods of anticancer drugs were not related to the incidence of osteoradionecrosis in our study. From our study, dental caries is the most important risk factor for osteoradionecrosis; therefore, a radiation caries prevention program is crucial to control osteoradionecrosis.

Two-step production of D-lactate from mixed sugars by growing and resting cells of metabolically engineered Lactobacillus plantarum.

To develop cost-effective systems for D-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on D-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, D-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the D-lactate production rate. The D-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l D-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l D-lactate with the yield of 0.93 g/g. In total, 141.9 g/l D-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of D-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of D-lactate.

Long-Term Survival of a Patient With Anaplastic Thyroid Carcinoma Treated With Hypofractionated Radiotherapy: A Case Report.

Anaplastic thyroid carcinoma, a rare type of primary thyroid cancer, is one of the most aggressive neoplasms with a poor prognosis. Many cases are in the advanced stage at the time of the initial visit, and curative treatment is impossible. Because of the highly radioresistant nature of anaplastic thyroid carcinoma, this condition cannot be properly controlled with conventional radiotherapy. Herein, we report the case of a patient with anaplastic thyroid carcinoma who underwent hypofractionated radiotherapy, attained a complete response, and is still alive more than 10 years after treatment with no evidence of disease. To overcome the high radioresistance of anaplastic thyroid carcinoma, we administered 50 Gy in 10 fractions three times a week. Furthermore, we administered paclitaxel and carboplatin sequentially before and after radiotherapy. Consequently, the patient completed treatment and reached a complete response. He is still alive more than 10 years after treatment with no evidence of disease or severe adverse events. Hypofractionated radiation therapy may provide good control of locally advanced anaplastic thyroid carcinoma.

A thermophilic gram-negative nitrate-reducing bacterium, Calditerrivibrio nitroreducens, exhibiting electricity generation capability.

To exploit the potential diversity of thermophilic exoelectrogens, two-chamber microbial fuel cells (MFCs) were inoculated with thermophilic anaerobic digester sludge and operated at 55 °C without supplementing with exogenous redox mediator. The MFC generated a maximum power density of 823 mW m(-2) after 200 h of operation. Molecular phylogenetic analyses suggested that the microbial population on the anode was dominated by a species closely related to a thermophilic nitrate-reducing bacterium Calditerrivibrio nitroreducens, for which a strain (Yu37-1) has been isolated in pure culture. Thus, a pure culture of the C. nitroreducens strain Yu37-1 was inoculated into MFC to examine the electricity generation capability. Without an exogenous mediator, MFCs stably produced electricity with a maximum power density of 272 mW m(-2) for >400 h of operation. The MFC current recovered to the original level within few hours after medium replacement, suggesting that the electricity generation was caused by the anodic microorganisms. Cyclic voltammetry indicated that redox systems (E3 and Ec) with similar potentials (-0.14 and -0.17 V) made the main contributions to the exoelectrogenic activities of the sludge-derived consortium and C. nitroreducens Yu37-1, respectively. This study undertook the bioelectrochemical characterization of C. nitroreducens as the first example of a thermophilic Gram-negative exoelectrogen.

Effect of different metabolic pathways on itaconic acid production in engineered Corynebacterium glutamicum.

Itaconic acid (IA), a C5-dicarboxylic acid, is a potential bio-based building block for the polymer industry. There are three pathways for IA production from natural IA producers; however, most of the engineered strains were used for IA production by heterologous expression of cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus. In this study, IA was produced by an engineered Corynebacterium glutamicum ATCC 13032 expressing two different types of genes from two distinct pathways. The first involves the mammalian immunoresponsive gene1 (Irg1) derived from Mus musculus. The second (termed here the trans-pathway) involves two genes from the natural IA producer Ustilago maydis which are aconitate-delta-isomerase (Adi1) and trans-aconitate decarboxylase (Tad1) genes. The constructed strains developing the two distinct IA production pathways: C. glutamicum ATCC 13032 pCH-Irg1opt and C. glutamicum ATCC 13032 pCH-Tad1optadi1opt were used for production of IA from different carbon sources. The results reflect the possibility for IA production from C. glutamicum expressing the trans-pathway (Adi1/Tad1 genes) and cis-pathway (Irg1 gene) other than the well-known cis-pathway that depends mainly on cadA gene from A. terreus. The developed strain expressing trans-pathway from U. maydis; however, proved to be better at IA production with high titers of 12.25, 11.34, and 11.02 g/L, and a molar yield of 0.22, 0.42, and 0.43 mol/mol from glucose, maltose, and sucrose, respectively, via fed-batch fermentation. The present study suggests that trans-pathway is better than cis-pathway for IA production in engineered C. glutamicum.

Enhanced production of itaconic acid from enzymatic hydrolysate of lignocellulosic biomass by recombinant Corynebacteriumglutamicum.

Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A. terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C. glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.

An analysis of sugary endosperm in sorghum: Characterization of mutant phenotypes depending on alleles of the corresponding starch debranching enzyme.

Sorghum is the fifth most important cereal crop. Here we performed molecular genetic analyses of the 'SUGARY FETERITA' (SUF) variety, which shows typical sugary endosperm traits (e.g., wrinkled seeds, accumulation of soluble sugars, and distorted starch). Positional mapping indicated that the corresponding gene was located on the long arm of chromosome 7. Within the candidate region of 3.4 Mb, a sorghum ortholog for maize Su1 (SbSu) encoding a starch debranching enzyme ISA1 was found. Sequencing analysis of SbSu in SUF uncovered nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region, containing substitutions of highly conserved amino acids. Complementation of the rice sugary-1 (osisa1) mutant line with the SbSu gene recovered the sugary endosperm phenotype. Additionally, analyzing mutants obtained from an EMS-induced mutant panel revealed novel alleles with phenotypes showing less severe wrinkles and higher Brix scores. These results suggested that SbSu was the corresponding gene for the sugary endosperm. Expression profiles of starch synthesis genes during the grain-filling stage demonstrated that a loss-of-function of SbSu affects the expression of most starch synthesis genes and revealed the fine-tuned gene regulation in the starch synthetic pathway in sorghum. Haplotype analysis using 187 diverse accessions from a sorghum panel revealed the haplotype of SUF showing severe phenotype had not been used among the landraces and modern varieties. Thus, weak alleles (showing sweet and less severe wrinkles), such as in the abovementioned EMS-induced mutants, are more valuable for grain sorghum breeding. Our study suggests that more moderate alleles (e.g. produced by genome editing) should be beneficial for improving grain sorghum.