Shuichi Karita

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose, and soluble polysaccharides

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate‐binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non‐crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid‐swollen cellulose is best fitted by the Langmuir two‐site model, suggesting that there are two CjCBM3 binding sites on acid‐swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.

Bacterial production and secretion of water-insoluble fuel compounds from cellulose without the supplementation of cellulases.

Achieving economic biofuel production from cellulosic biomass will require significant cost reductions. Enzymatic degradation of cellulosic biomass and distillation of water-soluble fuel compounds substantially increase the cost of biofuel production. Consolidated bioprocessing is a strategy to circumvent expensive biofuel production steps. Clostridium thermocellum is a promising bacterium for consolidated bioprocessing because it does not require the supplementation of lignocellulose-degrading enzymes. To produce water-insoluble fuel compounds, C. thermocellum was engineered to express a fatty acyl-acyl carrier protein reductase and an aldehyde-deformylating oxygenase from Synechococcus elongatus PCC 7942. Expression of the aldehyde-deformylating oxygenase gene was clearly detected, whereas only slight expression of the fatty acyl-acyl carrier protein reductase gene was detected. Cells expressing the fatty acyl-acyl carrier protein reductase and the aldehyde-deformylating oxygenase accumulated fatty aldehydes (higher alcohol precursors). After cultivation with cellulose, the higher alcohols, decanol and dodecanol, were detected in the organic solvent phase of the culture broth, indicating that the strain secreted the higher alcohols. These results suggest that the engineered C. thermocellum strain, expressing fatty acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase genes, directly produces and secretes higher alcohols from cellulose without the supplementation of cellulases. The higher alcohols can be collected by phase separation.

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials. Graphical abstract Substrate specificities of an endoglucanase, Cel5E from Clostridium thermocellum were affected by fusion of different type CBMs.

Sequence analysis and heterologous expression of the wool cuticle-degrading enzyme encoding genes in Fusarium oxysporum 26-1

Two protease-like proteins, KrtA and KrtC, were identified in Fusarium oxysporum 26-1. Genes coding these proteins, krtA and krtC, were isolated and characterized. Recombinant KrtA (rKrtA) and KrtC (rKrtC) were successfully expressed in Aspergillus oryzae and secreted. The combination of rKrtA and rKrtC completely removed the cuticle of wool fibers.

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the β-glucosidase gene

Efficient cellulosic biomass saccharification technologies are required to meet biorefinery standards. Biological simultaneous enzyme production and saccharification (BSES), which is glucose production from cellulosic biomass by Clostridium thermocellum, can be a reliable cellulose saccharification technology for biorefineries. However, the current BSES processes require purified β-glucosidase supplementation. In this study, recombinant bacteria expressing the β-glucosidase gene were developed and directly applied to BSES. The engineered Escherichia coli expressing the thermostable β-glucosidase gene from Thermoanaerobacter brockii exhibited 0.5xa0U/ml of β-glucosidase activities. The signal peptide sequence of lytF gene from Bacillus subtilis was the most appropriate for the β-glucosidase secretion from Brevibacillus choshinensis, and the broth exhibited 0.74xa0U/ml of β-glucosidase activities. The engineered E. coli and B. choshinensis expressing the thermostable β-glucosidase gene produced 47.4 g/L glucose and 49.4 g/L glucose, respectively. Glucose was produced by the hydrolysis of 100 g/L Avicel cellulose for 10 days through BSES, and the product yield was similar to that obtained through BSES with purified β-glucosidase supplementation. Our findings indicate that the direct supplementation of β-glucosidase using bacterial cells expressing β-glucosidase gene or their broth was applicable to BSES, suggesting the potential of this process as a cost-effective approach to cellulose saccharification.

Screening of yeast isolates from flowers for effective ethanol production

The use of alternative substrates to produce biofuel is a striking option nowadays. This study aimed to screen bioethanolproducing yeast strains. From different flowers, 65 yeasts were isolated. Twelve isolates assimilated glucose by liberation of CO 2 anaerobically. Out of these, only 5 yeast isolates fermented glucose in medium consisting of 0.8 g/L Mg2+ ions to produce 2.05 ± 0.03% ethanol. The selected five strains were identified as members of the genera Metschnikowia or Meyerozyma based on molecular characterization. Selected yeast strains were used for conversion of rice into bioethanol following dilute acid hydrolysis and fermentation. Consistent ethanol production was 1.80 ± 0.05% at days 2-4 by Metschnikowia cibodasensis Y34 and 2.20 ± 0.21% by Meyerozyma caribica Y42 at days 4-6 with a gradual decrease at the time of experiment termination (day 10). Metschnikowia cibodasensis Y34 and Meyerozyma caribica Y42 produced the highest ethanol at pH 3, i.e. 1.75 ± 0.14% at days 3 and 5 and 2.05 ± 0.14% at days 1 and 3, respectively, upon incubation with different pH levels and 1% NaCl. Growth and ethanol production at pH 4 and 5 was close to that at pH 3, with a slight increase in production by Metschnikowia cibodasensis Y34 at pH 4 up to day 3.

Characterization of lignocellulose particles during lignocellulose solubilization by Clostridium thermocellum

Clostridium thermocellum is a candidate bacterium for lignocellulose utilization due to its efficient lignocellulose solubilization ability. It has been reported that C. thermocellum efficiently degrades purified cellulose substrates, but cannot completely degrade milled lignocellulose powders. Evaluation of cellulose and hemicellulose contents in a lignocellulose residue after the cultivation of C. thermocellum indicated that C. thermocellum degraded cellulose and hemicellulose equally. Microscopic observations demonstrated that C. thermocellum significantly degraded small-sized lignocellulose particles, but it only partially degraded the larger sized particles. The lignin content of the large-sized particles was higher than that of the small particles. The remained large-sized particles included vascular tissues. These results suggest that the lignified structures such as vascular tissues in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization. Large-sized lignified structures in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization.

Changes in nutrient composition and in vitro ruminal fermentation of total mixed ration silage stored at different temperatures and periods

BACKGROUNDnTotal mixed ration (TMR) is widely used for dairy cattle and needs to be prepared daily because it deteriorates rapidly. Ensiling TMR allows preservation and saves labour at the farm; however, silage fermentation may influence various nutritional components. The objectives of this study were to evaluate nutritional changes and in vitro rumen fermentation of TMR silage that was stored at different temperatures and durations on a laboratory scale in comparison with those of typical TMR before ensiling.nnnRESULTSnNo distinct changes in crude protein (CP), neutral detergent fibre and non-fibrous carbohydrate contents were observed during silage fermentation. However, clear changes were observed in the soluble CP and soluble sugar fractions; solubilisation of the CP fraction in TMR silage was enhanced by prolonged storage and higher storage temperatures, and most soluble sugars were lost during ensiling. Short-chain fatty acid concentrations in the in vitro rumen from TMRs before and after ensiling were not significantly different; however, throughout incubation, NH3 -N concentrations from TMR silages were significantly higher than those from TMR before ensiling.nnnCONCLUSIONnA higher ruminal NH3 -N concentration from TMR silage may be a result of a shortage of fermentable sugars and enhanced deamination of CP. Feeding TMR ensiled under a high temperature must be investigated to balance proteins and carbohydrates for rumen fermentation.