Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401 USA.
Biotechnol Biofuels. 2014 Oct 16;7(1):148. doi: 10.1186/s13068-014-0148-0. eCollection 2014.
Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia.
Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates.
Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to directly convert lignocellulosic substrates to biofuels.
解脂耶氏酵母是一种能够将葡萄糖代谢为脂类并在细胞内积累的油脂酵母。然而，它缺乏分解生物质纤维素所需的整套纤维素酶，因此不能直接利用生物质作为碳源。为了开发一种直接利用微生物从纤维素生物质生产碳氢燃料的转化平台，我们首先对其内源糖苷水解酶进行基因组搜索和功能测试，以研究解脂耶氏酵母作为综合生物加工菌株的潜力。
最初，我们试图在解脂耶氏酵母中表达里氏木霉内切葡聚糖酶 II（EGII）和纤维二糖水解酶 II（CBHII），结果成功分泌了活性酶。然而，一种关键的纤维素酶，里氏木霉 CBHI，虽然在解脂耶氏酵母中成功表达和分泌，但酶活性低于预期，表明其在解脂耶氏酵母中的表达存在不兼容（可能在翻译后水平）。这一结果促使我们评估替代或改良的 CBHI 酶。随后，我们表达了一种里氏木霉-塔宾曲霉（Tr-Te）嵌合 CBHI、嗜热毁丝霉 CBHI 和灰色犁头霉 CBHI，它们表现出显著提高的酶活性。具体而言，纯化的嵌合 Tr-Te CBHI 在微晶纤维素上的比活性与天然里氏木霉 CBHI 相当。此外，该嵌合 Tr-Te CBHI 还在降解纤维素底物时与 EGII 和 CBHII 表现出显著的协同作用，无论是使用混合上清液还是相应的解脂耶氏酵母转化子的共培养物。共培养系统方法还允许根据有效降解纤维素底物所需的纤维素酶的最佳比例，合理地混合转化子培养物的体积。
总之，这项工作首次证明了在解脂耶氏酵母中成功表达具有基本完整天然活性的嵌合 CBHI，支持了通过异源表达真菌纤维素酶和其他酶来遗传工程改造解脂耶氏酵母菌株的观点，以直接将木质纤维素底物转化为生物燃料。
