Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 1552 University Ave, Madison, WI, USA.
Lehrstuhl für Chemie Biogener Rohstoffe, Technische Universität München, Schulgasse 16, 94315, Straubing, Germany.
Microb Cell Fact. 2018 Jan 12;17(1):5. doi: 10.1186/s12934-017-0848-9.
Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast.
Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain.
We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.
γ-戊内酯（GVL）处理木质纤维素生物质是一种有前途的生物燃料生产技术，可降解生物质；然而，GVL 对发酵微生物有毒。我们使用化学基因组学与酵母（酿酒酵母）缺失文库相结合的方法来鉴定敏感和抗性突变体，并使用化学蛋白质组学来监测 GVL 存在下的蛋白质丰度，旨在了解 GVL 的毒性和抗性机制，以期构建耐受 GVL、能发酵木糖的酵母。
GVL 的化学基因组学分析预测，这种化学物质会影响膜和膜结合过程。我们表明，GVL 会导致快速、剂量依赖性的细胞通透性，并与乙醇协同作用。GVL 的化学基因组学分析显示，功能相关酶 Pad1p 和 Fdc1p 的缺失会增加酵母对 GVL 的耐受性，这两种酶共同作用将肉桂酸及其衍生物脱羧基转化为乙烯基形式。此外，过表达 Pad1p 会使细胞对 GVL 毒性敏感。为了提高 GVL 的耐受性，我们在木糖发酵酵母菌株中删除了 PAD1 和 FDC1。修饰后的菌株在含有 1.5%GVL 的合成水解物中表现出更高的厌氧生长、糖利用率和乙醇产量，在其他条件下也是如此。工程菌株的化学蛋白质组学分析表明，与对照菌株相比，GVL 存在时与麦角固醇生物合成相关的酶更丰富。工程 GVL 菌株的麦角固醇含量高于对照菌株。
我们发现 GVL 通过破坏细胞膜对酵母产生毒性，并且这种毒性与乙醇协同作用。PAD1 和 FDC1 的缺失通过增加有氧生长细胞中麦角固醇的积累赋予了木糖发酵酵母菌株对 GVL 的抗性。在对照菌株受到抑制的水平下，耐 GVL 菌株可发酵糖。该菌株代表了专门针对 GVL 产生的水解物进行了改造的木糖发酵酵母。
