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Title: Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

Abstract

Abstract Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae . We then increased the flux through these pathways by making gene deletions of BAT1 , ALD6 , and PHO13 , to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared tomore » glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.« less

Authors:
; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1618761
Alternate Identifier(s):
OSTI ID: 1571260
Grant/Contract Number:  
SC0019363; SC0018420
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 12 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; isobutanol; xylose; 2-methyl-1-butanol; branched-chain higher alcohols; Saccharomyces cerevisiae; mitochondrial engineering

Citation Formats

Zhang, Yanfei, Lane, Stephan, Chen, Jhong-Min, Hammer, Sarah K., Luttinger, Jake, Yang, Lifeng, Jin, Yong-Su, and Avalos‬, José L. Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae. Netherlands: N. p., 2019. Web. https://doi.org/10.1186/s13068-019-1560-2.
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Zhang, Yanfei, Lane, Stephan, Chen, Jhong-Min, Hammer, Sarah K., Luttinger, Jake, Yang, Lifeng, Jin, Yong-Su, & Avalos‬, José L. Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae. Netherlands. https://doi.org/10.1186/s13068-019-1560-2
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Zhang, Yanfei, Lane, Stephan, Chen, Jhong-Min, Hammer, Sarah K., Luttinger, Jake, Yang, Lifeng, Jin, Yong-Su, and Avalos‬, José L. Fri . "Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae". Netherlands. https://doi.org/10.1186/s13068-019-1560-2.
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@article{osti_1618761,
title = {Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae},
author = {Zhang, Yanfei and Lane, Stephan and Chen, Jhong-Min and Hammer, Sarah K. and Luttinger, Jake and Yang, Lifeng and Jin, Yong-Su and Avalos‬, José L.},
abstractNote = {Abstract Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae . We then increased the flux through these pathways by making gene deletions of BAT1 , ALD6 , and PHO13 , to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.},
doi = {10.1186/s13068-019-1560-2},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 12,
place = {Netherlands},
year = {2019},
month = {9}
}
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Journal Article:
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https://doi.org/10.1186/s13068-019-1560-2
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Figures / Tables:

Figure 1 Figure 1: Engineering the mitochondrial isobutanol biosynthetic pathway in a xylose-utilizing strain of S. cerevisiae. Two different heterologous xylose utilization pathways have been used in yeast to convert xylose to xylulose: the isomerase pathway (used in this study), which uses xylose isomerase (XI); and the oxidoreductase pathway, consisting of xylosemore » reductase (XR) and xylitol dehydrogenase (XHD). In both pathways, xylulose is subsequently phosphorylated to xylulose-5- phophate (X5P) by xylulokinase (XK), and then channeled to glycolysis through the non-oxidative pentose phosphate pathway (PPP). (a) Mitochondrial isobutanol biosynthesis involves an upstream pathway that consists of the $ILV$ genes including acetolactate synthase ($ILV2$), ketol-acid reductoisomerase ($ILV5$), and dihydroxyacid dehydratase ($ILV3$); as well as a downstream pathway that consists of mitochondrially targeted $α$-ketoacid decarboxylases (KDC) and alcohol dehydrogenases (ADH). (b) There is considerable overlap between the upstream pathways for isobutanol and 2-methyl-1-butanol production, except the isoleucine precursor α-keto-β- methylvalerate ($α$-KMV) is synthesized by Ilv2p from one pyruvate and one α-ketobutyrate produced from threonine deamination catalyzed by threonine deaminase ($ILV1$); from there, the downstream Ehrlich degradation pathway for both branched chain alcohols is identical. Genes overexpressed in our strains are shown in blue, while genes deleted are shown in red. $ALD6$: cytosolic aldehyde dehydrogenase, $BAT1$: mitochondrial branched-chain amino acid aminotransferase, $BAT2$: cytosolic branched-chain amino acid aminotransferase, PDCs: pyruvate decarboxylases, $PHO13$: alkaline phosphatase, $α$-KIV: $α$-ketoisovalerate, IBAL: isobutyraldehyde, IBU: Isobutyrate, $α$-KMV: α-keto-β-methylvalerate, 2MBAL: 2-methyl-1- butyraldehyde, 2MBU: 2-methyl-1-butyrate.« less
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    Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae
    journal, November 2019

    • Lane, Stephan; Zhang, Yanfei; Yun, Eun Ju
    • Biotechnology and Bioengineering, Vol. 117, Issue 2
    • DOI: 10.1002/bit.27202
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