Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
Metab Eng. 2011 Nov;13(6):694-703. doi: 10.1016/j.ymben.2011.09.005. Epub 2011 Sep 22.
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.
蔗糖是酿酒酵母工业生物乙醇生产的主要碳源。在酵母中，蔗糖代谢有两种方式：（i）通过转化酶进行细胞外水解，然后吸收和代谢葡萄糖和果糖，（ii）通过蔗糖-质子协同转运吸收，然后进行细胞内水解和代谢。尽管 SUC2 基因中的替代起始密码子可以合成细胞外和细胞内的转化酶同工酶，但在酿酒酵母中，蔗糖水解主要发生在细胞外。在厌氧培养物中，由于蔗糖-质子协同转运的能量成本，细胞内水解理论上可以使乙醇产量比细胞外水解高 9%。通过工程改造 SUC2 的启动子和 5'编码序列来测试这一预测，导致转化酶主要（94%）定位于细胞质中。在厌氧蔗糖限制恒化器中，该 iSUC2 菌株的乙醇产量仅增加了 4%，并且高残留的蔗糖浓度表明蔗糖转运动力学不佳。为了提高蔗糖吸收亲和力，它在厌氧、蔗糖限制恒化器培养中经历了 90 代的实验室进化，导致残留蔗糖浓度降低了 20 倍，蔗糖转运能力增加了 10 倍。单细胞分离株在恒化器培养物中对蔗糖的乙醇产量比同基因 SUC2 参考菌株高 11%，而转录组分析显示，编码二糖-质子协同转运体的 AGT1 和其他麦芽糖相关基因的表达水平升高。在删除两个重复的 AGT1 拷贝后，生长特性恢复到未进化的 SUC2 和 iSUC2 菌株的特性。本研究表明，工程改造蔗糖代谢的拓扑结构是提高工业过程中乙醇产量的一种有吸引力的策略。
