Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

中文翻译：

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工程毕赤酵母与表面展示微纤维素体用于羧甲基纤维素水解和乙醇生产。

通过纤维素小体的表面组装，工程酵母作为一种整合的生物加工 (CBP) 微生物已被积极用于纤维素乙醇的生产。然而，之前的大多数研究都集中在酿酒酵母上，实现了磷酸溶胀纤维素（PASC）或微晶纤维素（Avicel）而不是羧甲基纤维素（CMC）向乙醇的有效转化，平均滴度低于 2 g/L。利用超高亲和力 IM7/CL7 蛋白对，我们在此描述了一种通过体外组装三种重组纤维素酶（包括内切葡聚糖酶 (EG)、外切葡聚糖酶 (CBH) 和 β-葡萄糖苷酶）用微纤维素体改造毕赤酵母的方法。 BGL)，以及细胞表面上的碳水化合物结合模块 (CBM)。首次，工程酵母能够将 CMC 高效直接地转化为生物乙醇，乙醇滴度高达 5.1 g/L。该研究促进了巴斯德毕赤酵母作为CBP细胞工厂在纤维素乙醇生产中的应用，并为筛选不同物种的纤维素酶构建表面组装纤维素体提供了一个有前景的平台。