Inhibitory effects of galacturonic acid on Saccharomyces cerevisiae: involvement of hexose transporters

Aim: Analyse inhibitory effects of galacturonic acid, an important constituent of plant biomass hydrolysates, on growing and starving cultures of Saccharomyces cerevisiae CEN.PK113-7D. Method & Results: Biomass yields in aerobic and anaerobic glucose-limited chemostat cultures (pH 3.5) were reduced by 25 and 10%, respectively, upon addition of 10 g∙l-1 galacturonic acid. Genes previously reported to show a transcriptional response to other organic acids were overrepresented in a set of galacturonic-acid responsive genes identified by microarray analysis. These results suggested that galacturonic acid causes weak-acid uncoupling of the yeast plasma membrane pH gradient. Consistent with this hypothesis, galacturonate-accelerated loss of viability in starving cell suspensions was strongly pH dependent. Loss of viability was much slower in a strain in which all HXT (hexose transporter) genes were deleted. Moreover, deletion of HXT genes alleviated growth inhibition on ethanol observed at galacturonic acid concentrations of 10 g∙l-1 and above. Conclusions: At low pH, galacturonic acid negatively affects the physiology of S. cerevisiae. Reduced sensitivity of hexose-transporter mutants indicated that one or more HXT transporters are involved in transport of galacturonic acid. Significance and Impact: This study shows that galacturonic acid toxicity should be taken into account in process development for yeast-based fermentative conversion of pectin-rich feedstocks such as sugar beet pulp and citrus peel. Involvement of hexose transporters in galacturonic acid toxicity provides leads for improving tolerance. To investigate the impact of galacturonic acid on S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic, glucose-limited chemostat cultures grown in the presence and absence of 10 g∙l-1 galacturonic acid at pH3.5.

研究目标：分析植物生物质水解液的重要组分之一——半乳糖醛酸(galacturonic acid)对酿酒酵母(Saccharomyces cerevisiae)CEN.PK113-7D的生长及饥饿培养物的抑制作用。方法与结果：在pH 3.5的需氧及厌氧葡萄糖限制恒化培养体系中，添加10 g·L⁻¹半乳糖醛酸后，菌体生物量得率分别下降25%与10%。通过微阵列(microarray)分析鉴定的半乳糖醛酸应答基因集合中，此前被报道可响应其他有机酸的基因占比显著偏高。上述结果表明，半乳糖醛酸会引发酵母质膜pH梯度的弱酸解偶联效应。与该假说一致，饥饿细胞悬浮液中半乳糖醛酸加速的细胞活力丧失现象具有极强的pH依赖性。在所有HXT（己糖转运蛋白，hexose transporter）基因均被敲除的菌株中，细胞活力丧失的速率显著减缓。此外，敲除HXT基因可缓解10 g·L⁻¹及更高浓度半乳糖醛酸存在时所引发的乙醇生长抑制效应。结论：在低pH环境下，半乳糖醛酸会对酿酒酵母的生理状态产生负面影响。己糖转运蛋白突变体的敏感性降低这一现象表明，一种或多种HXT转运蛋白参与了半乳糖醛酸的跨膜转运过程。意义与影响：本研究表明，在以酿酒酵母为菌株的果胶类丰富原料（如甜菜粕、柑橘皮）发酵转化工艺开发中，需考虑半乳糖醛酸的毒性效应。己糖转运蛋白参与半乳糖醛酸毒性这一发现，为提升菌株耐受性提供了研究方向。为探究半乳糖醛酸对酿酒酵母的影响，本研究针对pH 3.5条件下添加与未添加10 g·L⁻¹半乳糖醛酸的需氧葡萄糖限制恒化培养物，开展了基于DNA微阵列(DNA microarray)的转录组分析。