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Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.
Microbial Cell Factories ( IF 4.3 ) Pub Date : 2018-01-14 , DOI: 10.1186/s12934-017-0848-9
Scott Bottoms 1, 2 , Quinn Dickinson 1, 3 , Mick McGee 1 , Li Hinchman 1 , Alan Higbee 4 , Alex Hebert 5 , Jose Serate 1 , Dan Xie 1 , Yaoping Zhang 1 , Joshua J Coon 5, 6, 7, 8 , Chad L Myers 9 , Robert Landick 1 , Jeff S Piotrowski 1, 10
Affiliation  

BACKGROUND 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. RESULTS 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. CONCLUSIONS 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.

中文翻译:

化学基因组学指导的耐γ-戊内酯酵母的工程设计。

背景技术木质纤维素纤维团的γ-戊内酯(GVL)处理是用于生物质降解以生产生物燃料的有前途的技术。但是,GVL对发酵微生物有毒。使用化学基因组学与酵母(Saccharomyces cerevisiae)缺失集合的组合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,我们试图了解GVL的机制毒性和抗性,目的是工程化耐GVL的木糖发酵酵母。结果GVL的化学基因组分析预测该化学物质会影响膜和膜结合过程。我们表明,GVL引起快速的,剂量依赖性的细胞通透性,并且与乙醇协同作用。GVL的化学基因组分析表明,功能相关的酶Pad1p和Fdc1p的缺失共同起作用,以使肉桂酸及其衍生物脱羧成乙烯基,从而增加了酵母对GVL的耐受性。此外,Pad1p的过表达使细胞对GVL毒性敏感。为了提高GVL耐受性,我们删除了木糖发酵酵母菌株中的PAD1和FDC1。修饰的菌株在含1.5%GVL的合成水解物中和其他条件下表现出增加的厌氧生长,糖利用和乙醇生产。工程菌株的化学蛋白质组学分析表明,与背景菌株相比,在存在GVL的情况下,参与麦角固醇生物合成的酶更加丰富。与背景菌株相比,工程改造的GVL菌株所含麦角甾醇的含量更高。结论我们发现GVL通过破坏细胞膜对酵母产生毒性,并且这种毒性与乙醇具有协同作用。通过增加需氧生长细胞中麦角固醇的积累,删除PAD1和FDC1赋予GVL对木糖发酵酵母菌株的抗性。在存在对未修饰菌株有抑制作用的GVL水平下,耐GVL菌株发酵糖。该菌株代表了专门针对GVL生产的水解产物的木糖发酵酵母。在存在对未修饰菌株有抑制作用的GVL水平下,耐GVL菌株发酵糖。该菌株代表了专门针对GVL生产的水解产物的木糖发酵酵母。在存在对未修饰菌株有抑制作用的GVL水平下,耐GVL菌株发酵糖。该菌株代表了专门针对GVL生产的水解产物的木糖发酵酵母。
更新日期:2018-01-12
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