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In JoVE (1)
Other Publications (14)
- Applied and Environmental Microbiology
- Applied Microbiology and Biotechnology
- Biotechnology Journal
- Microbial Cell Factories
- Applied Microbiology and Biotechnology
- Applied Microbiology and Biotechnology
- Applied Microbiology and Biotechnology
- Applied Microbiology and Biotechnology
- Applied Microbiology and Biotechnology
- Microbial Cell Factories
- Applied Microbiology and Biotechnology
- Biotechnology for Biofuels
- Journal of Biotechnology
- Enzyme and Microbial Technology
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Articles by Ryosuke Yamada in JoVE
JoVE Clinical and Translational Medicine
أسلوب الرواية تطوير أدوية لمكافحة السرطان باستخدام Explants ورم العينات الجراحية
1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
هنا أنشأنا وسيلة لاختبار نجاعة الدواء مع العينات الجراحية للأورام الدماغ ، ويسمى "طريقة يزدرع ورم سرطاني". مع هذا الأسلوب ، يمكننا تقييم نجاعة الأدوية دون كسر المكروية من الأورام الصلبة. للتحقق من مصداقية هذه الطريقة ، ونحن تصف بيانات عينة ممثلة مع دبقي لدينا تعامل مع وكيل أول السطر الحالي العلاج الكيميائي ، temozolomide.
Other articles by Ryosuke Yamada on PubMed
Improved Production of Homo-D-lactic Acid Via Xylose Fermentation by Introduction of Xylose Assimilation Genes and Redirection of the Phosphoketolase Pathway to the Pentose Phosphate Pathway in L-Lactate Dehydrogenase Gene-deficient Lactobacillus Plantarum
The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.
Repeated Batch Fermentation from Raw Starch Using a Maltose Transporter and Amylase Expressing Diploid Yeast Strain
We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, alpha-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that alpha-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest alpha-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was alpha-amylase activity at 6.43 U/ml.
Ethanol Production from Cellulosic Materials Using Cellulase-expressing Yeast
We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.
Cocktail Delta-integration: a Novel Method to Construct Cellulolytic Enzyme Expression Ratio-optimized Yeast Strains
The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as beta-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail delta-integration.
Direct Ethanol Production from Cellulosic Materials at High Temperature Using the Thermotolerant Yeast Kluyveromyces Marxianus Displaying Cellulolytic Enzymes
To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 degrees C and 37 degrees C, while the activity of cellulolytic enzymes is highest at around 50 degrees C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus beta-glucosidase on the cell surface, which successfully converts a cellulosic beta-glucan to ethanol directly at 48 degrees C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of beta-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.
Gene Copy Number and Polyploidy on Products Formation in Yeast
Yeast, such as Saccharomyces cerevisiae or Kluyveromyces lactis is appropriate strain for ethanol production or some useful compounds production. Cellulases expressing yeast can ferment ethanol from cellulosic materials; however, the productivity should be increase more and more. To improve and engineer the productivity, the target gene(s) were introduced into yeast genome. Generally, using genetic engineering, increasing integrated gene numbers are increased, the expressed protein ability such as enzymatic activities are also increased. In this mini-review, we focused on the effect of integrated gene copy number and the polyploidy on the productivity such as enzymatic activity and/or product yield.
Construction of a Xylose-metabolizing Yeast by Genome Integration of Xylose Isomerase Gene and Investigation of the Effect of Xylitol on Fermentation
A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.
Novel Strategy for Yeast Construction Using Delta-integration and Cell Fusion to Efficiently Produce Ethanol from Raw Starch
We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining delta-integration and polyploidization through cell fusion. Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase/alpha-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The alpha-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.
Efficient and Direct Glutathione Production from Raw Starch Using Engineered Saccharomyces Cerevisiae
Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. We demonstrated that expression of amylase genes in glutathione-producing S. cerevisiae enables direct use of starch as a carbon source, thus eliminating the Crabtree effect that is caused by excess glucose. Consequently, cell growth and glutathione productivity were significantly improved. This approach is potentially applicable to a variety of fermentative processes for production of value-added chemicals under aerobic conditions.
Metabolic Pathway Engineering Based on Metabolomics Confers Acetic and Formic Acid Tolerance to a Recombinant Xylose-fermenting Strain of Saccharomyces Cerevisiae
The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance.
Direct Ethanol Production from Cassava Pulp Using a Surface-engineered Yeast Strain Co-displaying Two Amylases, Two Cellulases, and β-glucosidase
In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.
Direct Ethanol Production from Cellulosic Materials Using a Diploid Strain of Saccharomyces Cerevisiae with Optimized Cellulase Expression
Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.
Direct Ethanol Production from Hemicellulosic Materials of Rice Straw by Use of an Engineered Yeast Strain Codisplaying Three Types of Hemicellulolytic Enzymes on the Surface of Xylose-utilizing Saccharomyces Cerevisiae Cells
The cost of the lignocellulose-hydrolyzing enzymes used in the saccharification process of ethanol production from biomass accounts for a relatively high proportion of total processing costs. Cell surface engineering technology has facilitated a reduction in these costs by integrating saccharification and fermentation processes into a recombinant microbe strain expressing heterologous enzymes on the cell surface. We constructed a recombinant Saccharomyces cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from Trichoderma reesei, β-xylosidase from Aspergillus oryzae, and β-glucosidase from Aspergillus aculeatus but that also assimilated xylose through the expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. The recombinant strain successfully produced ethanol from rice straw hydrolysate consisting of hemicellulosic material containing xylan, xylooligosaccharides, and cellooligosaccharides without requiring the addition of sugar-hydrolyzing enzymes or detoxication. The ethanol titer of the strain was 8.2g/l after 72h fermentation, which was approximately 2.5-fold higher than that of the control strain. The yield (grams of ethanol per gram of total sugars in rice straw hydrolysate consumed) was 0.41g/g, which corresponded to 82% of the theoretical yield. The cell surface-engineered strain was thus highly effective for consolidating the process of ethanol production from hemicellulosic materials.
Direct and Efficient Ethanol Production from High-yielding Rice Using a Saccharomyces Cerevisiae Strain That Express Amylases
Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.
