文献类型： 外文期刊

第一作者： Lan, Qing

作者： Lan, Qing;Duan, Yitong;Wu, Pingping;Li, Xueyin;Yu, Yao;Zhou, Jungang;Lu, Hong;Lan, Qing;Duan, Yitong;Wu, Pingping;Li, Xueyin;Yu, Yao;Zhou, Jungang;Lu, Hong;Lu, Hong;Shi, Bo

作者机构：

关键词： Kluyveromycesmarxianus; Hemicellulases; Ribosomes skipping; Enzymatic hydrolysis; Ethanol

期刊名称：BIOTECHNOLOGY FOR BIOFUELS （ 影响因子：6.04； 五年影响因子：6.485 ）

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年卷期： 2021 年 14 卷 1 期

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收录情况： SCI

摘要： Background Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results Two polycistronic genes IMPX and IMP alpha X were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a beta-mannanase gene M330 (without the stop codon), a P2A sequence and a beta-xylanase gene Xyn-CDBFV in turn. In the IMP alpha X gene, there was an additional alpha-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular beta-mannanase activities of the IMPX and IMP alpha X strains were 21.34 and 15.50 U/mL, respectively, but the extracellular beta-xylanase activity of IMP alpha X strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXP alpha R and IMP alpha XP alpha R, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and beta-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of beta-xylanase and beta-xylosidase in the IXP alpha R strain were 1664.2 and 0.90 U/mL. Similarly, the IMP alpha XP alpha R strain secreted the three enzymes, beta-mannanase, beta-xylanase, and beta-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMP alpha XP alpha R strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMP alpha XP alpha R strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

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