Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis
Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis
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DOI：
10.1128/aem.00230-10
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发表时间：
2010-10-01
影响因子：
4.4
通讯作者：
Darzins, Al
中科院分区：
文献类型：
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作者：
Linger, Jeffrey G.;Adney, William S.;Darzins, Al
被称为联合生物处理（CBP）的策略的开发涉及使用单一微生物通过同时产生糖解酶和发酵释放的单糖将预处理的木质纤维素生物质转化为乙醇。在这份报告中，朝着实现这一目标的发酵宿主运动发酵单胞菌的初步步骤进行了研究，通过表达异源纤维素酶，并随后检查这些纤维素酶胞外分泌的潜力。许多菌株的Z. mobilis具有对羧甲基纤维素的内源性胞外活性，这表明该微生物可能具有用于产生另外的纤维素分解酶的有利环境。研究了两种纤维素分解酶E1和GH12的异源表达。这两种蛋白在Z.但在不同的层次上。虽然E1酶表达量较低，但GH12酶占总细胞蛋白的4.6%。此外，融合预测的分泌信号天然Z。发现将mobilis连接到E1和GH12的N末端可指导显著水平的活性E1和GH12酶的细胞外分泌。纤维素酶的细胞内池的亚细胞定位显示，E1和GH12分泌结构的一个显着的部分驻留在周质空间。我们的研究结果强烈表明Z. mobilis能够支持与生物燃料生产相关的高水平纤维素酶的表达和分泌，从而作为开发Z. mobilis转化为CBP平台生物。
Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.