skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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.
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
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.
@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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1186/s13068-019-1560-2

Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science

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

Save / Share:

Works referenced in this record:

Value-added biotransformation of cellulosic sugars by engineered Saccharomyces cerevisiae
journal, July 2018


Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae
journal, March 2016


Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective
journal, May 2017


Metabolic engineering of carbon overflow metabolism of Bacillus subtilis for improved N-acetyl-glucosamine production
journal, February 2018


Multifunctional yeast high-copy-number shuttle vectors
journal, January 1992


Rearrangement of Coenzyme A-Acylated Carbon Chain Enables Synthesis of Isobutanol via a Novel Pathway in Ralstonia eutropha
journal, February 2018


Energetics of the effect of acetic acid on growth of Saccharomyces cerevisiae
journal, March 2000


Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation
journal, January 2018

  • Osiro, Karen O.; Brink, Daniel P.; Borgström, Celina
  • FEMS Yeast Research, Vol. 18, Issue 1
  • DOI: 10.1093/femsyr/fox096

Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases
journal, December 2017


Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha
journal, August 2012

  • Lu, Jingnan; Brigham, Christopher J.; Gai, Claudia S.
  • Applied Microbiology and Biotechnology, Vol. 96, Issue 1
  • DOI: 10.1007/s00253-012-4320-9

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis
journal, November 2017


Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae : Role of yeast ACDHs
journal, October 2001


Engineering Corynebacterium glutamicum for isobutanol production
journal, April 2010

  • Smith, Kevin Michael; Cho, Kwang-Myung; Liao, James C.
  • Applied Microbiology and Biotechnology, Vol. 87, Issue 3
  • DOI: 10.1007/s00253-010-2522-6

Deconstruction of Lignocellulosic Biomass to Fuels and Chemicals
journal, July 2011


Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response
journal, November 2004


Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling
journal, October 2016

  • Brink, Daniel P.; Borgström, Celina; Tueros, Felipe G.
  • Microbial Cell Factories, Vol. 15, Issue 1
  • DOI: 10.1186/s12934-016-0580-x

Harnessing yeast organelles for metabolic engineering
journal, July 2017

  • Hammer, Sarah K.; Avalos, José L.
  • Nature Chemical Biology, Vol. 13, Issue 8
  • DOI: 10.1038/nchembio.2429

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation
journal, July 1992

  • Verduyn, Cornelis; Postma, Erik; Scheffers, W. Alexander
  • Yeast, Vol. 8, Issue 7
  • DOI: 10.1002/yea.320080703

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels
journal, January 2008

  • Atsumi, Shota; Hanai, Taizo; Liao, James C.
  • Nature, Vol. 451, Issue 7174, p. 86-89
  • DOI: 10.1038/nature06450

Conversion of various types of lignocellulosic biomass to fermentable sugars using kraft pulping and enzymatic hydrolysis
journal, May 2017

  • Przybysz Buzała, Kamila; Kalinowska, Halina; Przybysz, Piotr
  • Wood Science and Technology, Vol. 51, Issue 4
  • DOI: 10.1007/s00226-017-0916-7

Xylose and some non-sugar carbon sources cause catabolite repression in Saccharomyces cerevisiae
journal, October 2003


Corynebacterium glutamicum Tailored for Efficient Isobutanol Production
journal, March 2011

  • Blombach, Bastian; Riester, Tanja; Wieschalka, Stefan
  • Applied and Environmental Microbiology, Vol. 77, Issue 10
  • DOI: 10.1128/AEM.02972-10

Excessive by-product formation: A key contributor to low isobutanol yields of engineered Saccharomyces cerevisiae strains
journal, December 2016


Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose
journal, March 1996


Optogenetic regulation of engineered cellular metabolism for microbial chemical production
journal, March 2018

  • Zhao, Evan M.; Zhang, Yanfei; Mehl, Justin
  • Nature, Vol. 555, Issue 7698
  • DOI: 10.1038/nature26141

A new efficient gene disruption cassette for repeated use in budding yeast
journal, July 1996


The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression
journal, September 2017

  • Nouaille, Sébastien; Mondeil, Sophie; Finoux, Anne-Laure
  • Nucleic Acids Research, Vol. 45, Issue 20
  • DOI: 10.1093/nar/gkx781

Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction
journal, November 2007

  • Berthold, Catrine L.; Gocke, Dörte; Wood, Martin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 63, Issue 12, p. 1217-1224
  • DOI: 10.1107/S0907444907050433

Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae
journal, April 2003


Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains
journal, January 2018

  • Kong, In Iok; Turner, Timothy Lee; Kim, Heejin
  • FEMS Yeast Research, Vol. 18, Issue 1
  • DOI: 10.1093/femsyr/foy001

pH control in biological systems using calcium carbonate: pH control in biological systems using calcium
journal, January 2015

  • Salek, S. S.; van Turnhout, A. G.; Kleerebezem, R.
  • Biotechnology and Bioengineering, Vol. 112, Issue 5
  • DOI: 10.1002/bit.25506

Genetically Engineered SaccharomycesYeast Capable of Effective Cofermentation of Glucose and Xylose
journal, May 1998


Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae
journal, May 2017

  • Generoso, Wesley Cardoso; Brinek, Martin; Dietz, Heiko
  • FEMS Yeast Research, Vol. 17, Issue 3
  • DOI: 10.1093/femsyr/fox029

Homeostasis of protein and mRNA concentrations in growing cells
journal, October 2018


Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome
journal, April 1995


On the Dependency of Cellular Protein Levels on mRNA Abundance
journal, April 2016


Features of promising technologies for pretreatment of lignocellulosic biomass
journal, April 2005


Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol
journal, October 2014

  • Park, Seong-Hee; Kim, Sujin; Hahn, Ji-Sook
  • Applied Microbiology and Biotechnology, Vol. 98, Issue 21
  • DOI: 10.1007/s00253-014-6081-0

Exploring the xylose paradox in Saccharomyces cerevisiae through in vivo sugar signalomics of targeted deletants
journal, May 2019

  • Osiro, Karen O.; Borgström, Celina; Brink, Daniel P.
  • Microbial Cell Factories, Vol. 18, Issue 1
  • DOI: 10.1186/s12934-019-1141-x

Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae
journal, January 2012

  • Brat, Dawid; Weber, Christian; Lorenzen, Wolfram
  • Biotechnology for Biofuels, Vol. 5, Issue 1
  • DOI: 10.1186/1754-6834-5-65

Metabolic Engineering of Microorganisms for the Production of Higher Alcohols
journal, September 2014


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

The Ehrlich Pathway for Fusel Alcohol Production: a Century of Research on Saccharomyces cerevisiae Metabolism
journal, February 2008

  • Hazelwood, L. A.; Daran, J. -M.; van Maris, A. J. A.
  • Applied and Environmental Microbiology, Vol. 74, Issue 8
  • DOI: 10.1128/AEM.02625-07

Biofuels 2020: Biorefineries based on lignocellulosic materials
journal, July 2016

  • Valdivia, Miguel; Galan, Jose Luis; Laffarga, Joaquina
  • Microbial Biotechnology, Vol. 9, Issue 5
  • DOI: 10.1111/1751-7915.12387

Isobutanol production from d -xylose by recombinant Saccharomyces cerevisiae
journal, January 2013


Transposon Mutagenesis To Improve the Growth of Recombinant Saccharomyces cerevisiae on D-Xylose
journal, February 2007

  • Ni, H.; Laplaza, J. M.; Jeffries, T. W.
  • Applied and Environmental Microbiology, Vol. 73, Issue 7
  • DOI: 10.1128/AEM.02564-06

Branched-Chain Keto Acid Decarboxylase from Lactococcus lactis (KdcA), a Valuable Thiamine Diphosphate-Dependent Enzyme for Asymmetric C-C Bond Formation
journal, June 2007

  • Gocke, Dörte; Nguyen, Cong Luan; Pohl, Martina
  • Advanced Synthesis & Catalysis, Vol. 349, Issue 8-9, p. 1425-1435
  • DOI: 10.1002/adsc.200700057

Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains
journal, July 2018

  • Verhoeven, Maarten D.; de Valk, Sophie C.; Daran, Jean-Marc G.
  • FEMS Yeast Research, Vol. 18, Issue 8
  • DOI: 10.1093/femsyr/foy075

Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase
journal, October 2010

  • Runquist, D.; Hahn-Hagerdal, B.; Bettiga, M.
  • Applied and Environmental Microbiology, Vol. 76, Issue 23
  • DOI: 10.1128/AEM.01505-10

Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum
journal, September 2015


Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols
journal, February 2013

  • Avalos, José L.; Fink, Gerald R.; Stephanopoulos, Gregory
  • Nature Biotechnology, Vol. 31, Issue 4
  • DOI: 10.1038/nbt.2509

Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects
journal, August 2012


Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production
journal, November 2012

  • Varman, Arul M.; Xiao, Yi; Pakrasi, Himadri B.
  • Applied and Environmental Microbiology, Vol. 79, Issue 3
  • DOI: 10.1128/AEM.02827-12

Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA
journal, May 2011

  • Takahashi, Yuki; Nishikawa, Makiya; Takiguchi, Naomi
  • Biotechnology and Bioengineering, Vol. 108, Issue 10
  • DOI: 10.1002/bit.23179

    Works referencing / citing this record:

    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

      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.