Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield. Saccharomyces cerevisiae

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-H+ 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 relocation of invertase to the cytosol. 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 95 generations of 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 and batch 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. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. Overall design: The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

蔗糖是酿酒酵母（Saccharomyces cerevisiae）工业生物乙醇生产的主要碳源。在酵母中存在两种蔗糖代谢模式：(i) 经蔗糖酶（invertase）胞外水解后，摄取并代谢葡萄糖与果糖；(ii) 通过蔗糖-质子同向转运体（sucrose-H+ symport）摄取蔗糖，随后在胞内完成水解与代谢。尽管SUC2基因中的可变起始密码子可合成胞外与胞内两种蔗糖酶同工型，但酿酒酵母中的蔗糖水解主要以胞外方式进行。在厌氧培养体系中，由于蔗糖-质子同向转运存在能量消耗，理论上胞内水解路径的乙醇产率比胞外水解路径高9%。为验证该预测，研究人员对SUC2基因的启动子及5'编码序列进行工程改造，使蔗糖酶被重新定位至细胞质基质。在厌氧蔗糖限制恒化（chemostat）培养中，该iSUC2工程菌株的乙醇产率仅提升4%，且较高的残留蔗糖浓度提示其蔗糖转运动力学性能未达最优。为提升蔗糖摄取亲和力，研究人员对该菌株进行了95代厌氧蔗糖限制恒化培养驯化，最终使残留蔗糖浓度降低至原水平的1/20，蔗糖转运能力提升10倍。单细胞分离得到的菌株，在恒化与分批培养中，其蔗糖基乙醇产率较同基因背景的SUC2参考菌株提升11%。转录组分析（transcriptome analysis）显示，该驯化菌株中编码二糖-质子同向转运体的AGT1基因及其他麦芽糖相关基因的表达水平显著上调。实验室驯化过程中发生复制的AGT1基因被敲除后，恢复了未驯化iSUC2菌株的生长特性。本研究证实，改造蔗糖代谢的亚细胞定位策略，可用于提升工业生产中的乙醇产率。总体实验设计：本研究旨在探究将蔗糖水解重新定位至细胞质基质，是否可提升蔗糖基乙醇产率，以及仅表达胞内蔗糖酶的菌株需通过哪些额外步骤优化蔗糖利用效率。为此，研究人员对SUC2基因进行改造，使其仅在胞内定位。将该工程菌株的生长与产物生成情况，与亲本菌株在厌氧蔗糖限制恒化培养中的表现进行对比。随后通过进化工程优化菌株的蔗糖转运动力学，并对驯化得到的菌株在恒化培养中的生长与产物生成特性进行表征。此外，通过转录组分析与基因敲除实验，鉴定驯化菌株中有助于提升蔗糖转运动力学的遗传变异。