Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield
Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield
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DOI：
10.1016/j.ymben.2011.09.005
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发表时间：
2011-11-01
影响因子：
8.4
通讯作者：
Stambuk, Boris U.
中科院分区：
文献类型：
--
作者：
Basso, Thiago O.;de Kok, Stefan;Stambuk, Boris U.
蔗糖是酿酒酵母工业生产生物乙醇的主要碳源。在酵母中，发生两种蔗糖代谢模式：（i）通过转化酶的细胞外水解，随后是葡萄糖和果糖的摄取和代谢，以及（ii）通过蔗糖-质子共转运的摄取，随后是细胞内水解和代谢。尽管SUC 2基因中的替代起始密码子能够合成细胞外和细胞内转化酶亚型，但S.酿酒酵母主要发生在细胞外。在厌氧培养中，由于蔗糖-质子共输的能量成本，细胞内水解理论上使乙醇产率比细胞外水解高9%。通过改造SUC 2的启动子和5'编码序列来测试该预测，导致转化酶的主要（94%）胞质定位。在厌氧蔗糖限制恒化器中，该iSUC 2-菌株仅显示出4%的乙醇产率增加，并且高残留蔗糖浓度指示次优蔗糖运输动力学。为了提高蔗糖吸收亲和力，它进行了90代的实验室进化厌氧，蔗糖有限恒化培养，导致残留蔗糖浓度降低20倍，蔗糖运输能力增加10倍。一个单细胞分离株在恒化培养中的蔗糖乙醇产率比同基因SUC 2参考菌株高11%，而转录组分析显示AGT 1（编码一种二硫键胺-质子同向转运体）和其他麦芽糖相关基因的表达升高。在删除重复AGT 1的两个拷贝后，生长特性恢复到未进化的SUC 2和iSUC 2菌株的生长特性。这项研究表明，工程蔗糖代谢的拓扑结构是一个有吸引力的策略，以提高乙醇产量在工业过程中。(C)2011 Elsevier Inc. All rights reserved.
Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. (C) 2011 Elsevier Inc. All rights reserved.