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Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

ABSTRACT: 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.

SUBMITTER: Bottoms S

PROVIDER: S-EPMC5767017 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

Bottoms Scott S Dickinson Quinn Q McGee Mick M Hinchman Li L Higbee Alan A Hebert Alex A Serate Jose J Xie Dan D Zhang Yaoping Y Coon Joshua J JJ Myers Chad L CL Landick Robert R Piotrowski Jeff S JS

Microbial cell factories 20180112 1

<h4>Background</h4>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 ...[more]

PMID: 29329531

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Project description:Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids. Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ± 5 mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ± 17.7 mg/L in shake flasks, and 1232 ± 64 mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.

Dataset Information

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

Publications

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

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