Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>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.</p> <p>Results</p> <p>The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.</p> <p>Conclusions</p> <p>We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.</p

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>T. reesei </it>effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-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 <it>Saccharomyces cerevisiae</it>, 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 δ-integration.</p> <p>Results</p> <p>In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.</p

A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution.The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations

Vertical distribution patterns of pelagic cnidarians in the oceanic zone off Adélie Land, East Antarctica

第3回極域科学シンポジウム/第34回極域生物シンポジウム 11月26日（月） 国立極地研究所 ３階ラウン

Changes in Selected Food Groups Consumption and Quality of Meals in Japanese School Children during the COVID-19 Pandemic

In 2020, a state of emergency was declared to control the devastating impact of coronavirus, leading to temporary school closures in Japan, meaning that school lunches were not provided to the majority of schoolchildren. Using questionnaires completed by participants’ guardians, we examined the relationship between household income and the quality of meals in Japanese schoolchildren before, during, and after the state of emergency. Participants (1111 children, 10–14 years old) were chosen to form a nationally representative sample of the Japanese population. “Well-balanced dietary intake” was defined as the intake of (i) meat, fish, or eggs and (ii) vegetables. The desired prevalence was defined as equal to or more than twice a day. Household income was divided into quartiles. “Well-balanced dietary intake” was lower in all households during the state of emergency compared with before. The proportion of those with a “well-balanced dietary intake” at least twice a day was notably low in both Q3 and Q4 during the state of emergency compared with before the declared state of emergency; relative risk increase (95% CI) were Q1: −19.0% (−19.6% to −18.4%), p &lt; 0.001, Q2: −21.3% (−22.1% to −20.6%) p &lt; 0.001, Q3: −25.4% (−26.1% to −24.7%), p &lt; 0.001, and Q4: −34.8% (−35.6% to −34.0%), p &lt; 0.001. The interaction p (vs. Q1) of Q2, Q3, and Q4 were all &lt;0.001. Guardians from low-income households had significantly higher rates of having less: time, psychological room, and financial position to prepare meals during the state of emergency. Our results suggest that schoolchildren’s quality of meals worsened during the state of emergency, especially in low-income households, because school lunches were not provided

Convenience Food Options and Adequacy of Nutrient Intake among School Children during the COVID-19 Pandemic

The COVID-19 pandemic has caused changes in the family food environment, resulting in more families relying on convenience food options. This study aimed to investigate diet quality by convenience food options (namely instant, frozen, and take-out foods) among Japanese school children during the COVID-19 pandemic. We examined the relationship between the frequency of consumption of convenience food options and nutritional status of the school children. The participants (671 children, 10–14 years old) were chosen to form a nationally representative sample of the Japanese population. Using questionnaires completed by the participants’ guardians, information was collected on the frequency of instant, frozen, and take-out food consumption. Habitual food and nutrient intake were collected using a validated food frequency questionnaire, completed by the children with help from their guardian(s). “Frequent” consumption was defined as consumption of instant, frozen, and/or take-out foods on more than 5 days per week. Using 19 nutrients and their respective dietary reference intake (DRI) values, an index was created to label each child’s nutrient intake as “Adequate”, “Inadequate”, “Excess”, or “Deficient.” Compared to children with non-frequent consumption, school children with frequent instant food consumption had significantly higher rates of inadequate nutrient intake (risk ratio (RR) = 3.0 [95% CI: 1.6–5.6]) and excess nutrient intake (RR = 2.3 [95% CI: 1.3–4.2]), while school children with frequent take-out food consumption had significantly higher rates of inadequate nutrient intake (RR = 2.1 [95% CI: 1.3–3.3]). There were no significant differences for children with frequent frozen-food intake. These associations did not change when adjusting for sociodemographic factors. Our results suggest that the frequent consumption of instant or take-out foods among school children results in non-adequate nutritional intake