Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis

Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

胞质乙酰辅酶A是酿酒酵母（Saccharomyces cerevisiae）合成诸多生物技术相关化合物的前体物质。在该酵母中，胞质乙酰辅酶A的合成与生长过程严格依赖于乙酰辅酶A合成酶（acetyl-CoA synthetase, ACS）的Acs1或Acs2同工酶的表达。由于ACS催化反应中ATP水解为AMP与焦磷酸的步骤限制了乙酰辅酶A衍生产物的最大得率，本研究探索了采用两种不依赖ATP的乙酰辅酶A合成途径替代ACS的可行性。在评估了编码乙酰化乙醛脱氢酶（acetylating acetaldehyde dehydrogenase, A-ALD）与丙酮酸甲酸裂解酶（pyruvate-formate lyase, PFL）的不同细菌基因的表达情况后，研究人员构建了acs1Δ acs2Δ酿酒酵母菌株，其中A-ALD或PFL成功替代了ACS的功能。在依赖A-ALD的菌株中，观测到的有氧生长速率可达0.27 h⁻¹；而依赖PFL的酿酒酵母在厌氧条件下的生长速率（0.21 h⁻¹）与甲酸生成量呈化学计量耦合关系。在葡萄糖限制恒化培养体系中，胞内代谢物分析未发现依赖A-ALD的菌株与参考菌株间存在显著差异。然而，依赖A-ALD与PFL的菌株在葡萄糖上的生物量得率均低于参考菌株。转录组分析结果表明，生物量得率降低分别由依赖A-ALD菌株中的乙醛与依赖PFL菌株中的甲酸所引发。转录谱分析还显示，此前提出的Acs2在组蛋白乙酰化中的作用，可能与胞质乙酰辅酶A水平相关，而非Acs2直接参与组蛋白乙酰化过程。本研究首次证实酿酒酵母的ACS可被替代反应完全取代，同时表明仍需开展进一步改造，以实现作为产物前体的乙酰辅酶A在体内供应的最优效率。为探究酿酒酵母胞质乙酰辅酶A生成的天然途径引入改变后的影响，本研究针对有氧或厌氧、葡萄糖限制的恒化培养样本开展了基于DNA微阵列（DNA microarray）的转录组分析。