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首页> 外文期刊>Microbial Cell Factories >Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues
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Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues

机译：通过遗传利用里氏木霉内源纤维素酶水解玉米芯残基的潜力来生产高效纤维素酶混合物

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Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass. In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8?U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2–BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48?h enzymatic saccharification. These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.
机译：里氏木霉是用于纤维素酶生产的最重要的真菌之一。然而，已经证明其纤维素酶系统以次优的比例存在，以解构木质纤维素底物。尽管先前的酶促优化研究已经获得了用于有效木质纤维素水解的不同类型的体外合成混合物，但是通过工业菌株生产体内优化的纤维素酶混合物仍然是降低木质纤维素生物质生产生物燃料中酶成本的障碍之一。在这项研究中，我们使用了基于pyrG标记的系统遗传策略来过度表达超纤维素分解里氏木霉菌株中的主要纤维素酶成分，并产生用于糖化玉米芯残基的高效纤维素酶混合物。我们发现，CBH2的过表达在转录水平上增加了32倍，蛋白质水平与里氏木霉中最丰富的分泌蛋白CBH1相近，但对纤维素分解能力的贡献不大。但是，当EG2过表达，转录水平增加46倍，蛋白质水平与CBH2相当时，工程菌株QPE36的总纤维素酶活性提高了1.5倍（最高5.8？U / mL FPA），而大大提高了糖化效率对不同预处理的玉米芯残留物的影响。为了辅助以下遗传操作，基于对5-FOA的抗性，通过同源重组成功切除了标记pyrG。此外，BGL1在EG2过表达菌株QE51中被过表达（pyrG切除），BGL活性提高了11.6倍。 EG2-BGL1双过表达菌株QEB4对预处理的玉米芯残基表现出明显的纤维素分解能力增强。尤其是，经过48h的酶促糖化作用后，去木质素的玉米芯残留物的纤维素转化率几乎达到了94.2％。这些结果表明，遗传上利用里氏木霉内源纤维素酶生产高效纤维素酶混合物的潜力是提高糖化效率的有力策略，这最终将有助于降低木质纤维素基生物燃料的成本。

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