Guo Zhongpeng, Duquesne Sophie, Bozonnet Sophie, Cioci Gianluca, Nicaud Jean-Marc, Marty Alain, O'Donohue Michael Joseph
LISBP-Biocatalysis Group, INSA/INRA UMR 792, Université de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse, France ; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400 Toulouse, France ; CNRS, UMR5504, 31400 Toulouse, France.
INRA, UMR1319 Micalis, 78352 Jouy-en-Josas, France ; AgroParisTech, UMR Micalis, 78352 Jouy-en-Josas, France.
Biotechnol Biofuels. 2015 Aug 4;8:109. doi: 10.1186/s13068-015-0289-9. eCollection 2015.
Yarrowia lipolytica, one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellobiose. Engineering cellobiose-degrading ability into this yeast is a vital step towards the development of cellulolytic biocatalysts suitable for consolidated bioprocessing.
In the present work, we identified six genes encoding putative β-glucosidases in the Y. lipolytica genome. To study these, homologous expression was attempted in Y. lipolytica JMY1212 Zeta. Two strains overexpressing BGL1 (YALI0F16027g) and BGL2 (YALI0B14289g) produced β-glucosidase activity and were able to degrade cellobiose, while the other four did not display any detectable activity. The two active β-glucosidases, one of which was mainly cell-associated while the other was present in the extracellular medium, were purified and characterized. The two Bgls were most active at 40-45°C and pH 4.0-4.5, and exhibited hydrolytic activity on various β-glycoside substrates. Specifically, Bgl1 displayed 12.5-fold higher catalytic efficiency on cellobiose than Bgl2. Significantly, in experiments where cellobiose or cellulose (performed in the presence of a β-glucosidase-deficient commercial cellulase cocktail produced by Trichoderma reseei) was used as carbon source for aerobic cultivation, Y. lipolytica ∆pox co-expressing BGL1 and BGL2 grew better than the Y. lipolytica strains expressing single BGLs. The specific growth rate and biomass yield of Y. lipolytica JMY1212 co-expressing BGL1 and BGL2 were 0.15 h(-1) and 0.50 g-DCW/g-cellobiose, respectively, similar to that of the control grown on glucose.
We conclude that the bi-functional Y. lipolytica developed in the current study represents a vital step towards the creation of a cellulolytic yeast strain that can be used for lipid production from lignocellulosic biomass. When used in combination with commercial cellulolytic cocktails, this strain will no doubt reduce enzyme requirements and thus costs.
解脂耶氏酵母是研究最为广泛的“非常规”产油酵母之一，无法利用纤维二糖生长。赋予该酵母纤维二糖降解能力是开发适用于同步糖化发酵的纤维素分解生物催化剂的关键一步。
在本研究中，我们在解脂耶氏酵母基因组中鉴定出6个编码假定β-葡萄糖苷酶的基因。为了研究这些基因，我们尝试在解脂耶氏酵母JMY1212 Zeta中进行同源表达。两个过表达BGL1（YALI0F16027g）和BGL2（YALI0B14289g）的菌株产生了β-葡萄糖苷酶活性，并能够降解纤维二糖，而其他四个菌株未表现出任何可检测到的活性。我们对两种具有活性的β-葡萄糖苷酶进行了纯化和表征，其中一种主要与细胞相关，另一种存在于细胞外培养基中。这两种Bgl在40 - 45°C和pH 4.0 - 4.5时活性最高，并对各种β-糖苷底物表现出水解活性。具体而言，Bgl1对纤维二糖的催化效率比Bgl2高12.5倍。值得注意的是，在以纤维二糖或纤维素（在里氏木霉生产的缺乏β-葡萄糖苷酶的商业纤维素酶混合物存在下进行）作为需氧培养碳源的实验中，共表达BGL1和BGL2的解脂耶氏酵母Δpox比表达单个BGL的解脂耶氏酵母菌株生长得更好。共表达BGL1和BGL2的解脂耶氏酵母JMY1212的比生长速率和生物量产量分别为0.15 h⁻¹和0.50 g-DCW/g-纤维二糖，与在葡萄糖上生长的对照相似。
我们得出结论，本研究中构建的双功能解脂耶氏酵母是朝着创建一种可用于从木质纤维素生物质生产脂质的纤维素分解酵母菌株迈出的重要一步。当与商业纤维素分解混合物联合使用时，该菌株无疑将降低酶的需求，从而降低成本。
Zotero 插件
