Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Biotechnol Biofuels. 2011 Apr 15;4:8. doi: 10.1186/1754-6834-4-8.
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.
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.
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.
纤维素的水解需要纤维素酶内切葡聚糖酶、纤维二糖水解酶和β-葡萄糖苷酶的作用。这些酶的表达比例和协同效应显著影响纤维素的降解程度和比速率。在本研究中，我们使用先前开发的在酵母中优化纤维素酶表达水平的方法，构建了优化表达纤维素酶的二倍体酿酒酵母菌株，并试图提高纤维素的降解活性，使水稻秸秆（最丰富的木质纤维素生物质来源之一）能够直接用于生产乙醇。
该工程化的二倍体菌株，其基因组中整合了三个纤维素酶基因的多个拷贝，在糖蜜培养基（381.4 mU/g 湿细胞）中预培养，与亲本单倍体菌株（63.5 mU/g 湿细胞）相比，其磷酸膨胀纤维素（PASC）降解活性提高了约六倍。当用于发酵 PASC 时，二倍体菌株在 72 小时内产生了 7.6 g/L 的乙醇，乙醇得率达到理论值的 75%，并在 72 小时内从预处理的水稻秸秆中产生了 7.5 g/L 的乙醇。
我们已经开发出了优化表达纤维素酶的二倍体酵母菌株，能够直接从纤维素材料发酵。尽管这是一项概念验证研究，但据我们所知，这是首次使用表达纤维素酶的酵母从农业废弃物生物质中生产乙醇，而无需添加外源性酶。我们的结果表明，在酵母中结合多基因表达优化和二倍体化是提高各种木质纤维素生物质乙醇生产的一种很有前途的方法。
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