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For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulosedegrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on ␣-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His 6 ) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-␣-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley ␤-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and ␤-glucosidase 1 from Aspergillus aculeatus No.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15 degrees C, EG II-CD at 20 degrees C and EG III at 37 degrees C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3-1,4-beta-D-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3-1,4-beta-D-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3-1,4-beta-D-glucan, xyloglucan, xylan, and mannan.

L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, L-sorbose was found to regulate coordinately six cellulase genes (including eg13, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when L-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45-60% and 20-30% of the cbh1 transcript, whereas small amounts of mRNA, 1-2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and L-sorbose, respectively.

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ABCG8, contribute to worsening of the FH phenotype and we defined such a situation as "oligogenic FH". 7 According to The Exome Aggregation Consortium exome browser, 1 in ≈220 individuals has loss-of-function mutations in ABCG5 or ABCG8, indicating that a substantial number of individuals have deleterious mutation(s) in those genes. 8 Based on this information, we aimed to evaluate the prevalence and clinical effect of mutations in ABCG5/ABCG8 on LDL cholesterol (LDL-C) and CAD among individuals with a clinical diagnosis of FH. Methods Study Population We retrospectively investigated 543 subjects who met the Japanese clinical diagnostic criteria of FH at Kanazawa University Hospital from April 2014 to March 2017. We excluded 54 subjects for lack of lipid profile and/or genetic analysis. We also excluded 2 patients with homozygous or compound heterozygous FH. Thus, a total of 487 subjects

The stability and specific activity of endo-beta-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4-8.8) and thermotolerance (entirely active at 55 degrees C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4-5.2; thermostability, inactive at 55 degrees C for 30 min). k (cat) of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K (m) became twice of that of the wild type.