Genomic and phenotypic characterization of a refactored xylose-utilizing  Saccharomyces cerevisiae  strain for lignocellulosic biofuel production

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Abstract

Background

Engineered strains of Saccharomyces cerevisiae have significantly improved the prospects of biorefinery by improving the bioconversion yields in lignocellulosic bioethanol production and expanding the product profiles to include advanced biofuels and chemicals. However, the lignocellulosic biorefinery concept has not been fully applied using engineered strains in which either xylose utilization or advanced biofuel/chemical production pathways have been upgraded separately. Specifically, high-performance xylose-fermenting strains have rarely been employed as advanced biofuel and chemical production platforms and require further engineering to expand their product profiles.

Results

In this study, we refactored a high-performance xylose-fermenting S. cerevisiae that could potentially serve as a platform strain for advanced biofuels and biochemical production. Through combinatorial CRISPR–Cas9-mediated rational and evolutionary engineering, we obtained a newly refactored isomerase-based xylose-fermenting strain, XUSE, that demonstrated efficient conversion of xylose into ethanol with a high yield of 0.43 g/g. In addition, XUSE exhibited the simultaneous fermentation of glucose and xylose with negligible glucose inhibition, indicating the potential of this isomerase-based xylose-utilizing strain for lignocellulosic biorefinery. The genomic and transcriptomic analysis of XUSE revealed beneficial mutations and changes in gene expression that are responsible for the enhanced xylose fermentation performance of XUSE.

Conclusions

In this study, we developed a high-performance xylose-fermenting S. cerevisiae strain, XUSE, with high ethanol yield and negligible glucose inhibition. Understanding the genomic and transcriptomic characteristics of XUSE revealed isomerase-based engineering strategies for improved xylose fermentation in S. cerevisiae. With high xylose fermentation performance and room for further engineering, XUSE could serve as a promising platform strain for lignocellulosic biorefinery.

Electronic supplementary material

The online version of this article (10.1186/s13068-018-1269-7) contains supplementary material, which is available to authorized users.

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Most cited references 56

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Stress-activated genomic expression changes serve a preparative role for impending stress in yeast.

David Berry, Audrey P. Gasch (2008)

Yeast cells respond to stress by mediating condition-specific gene expression changes and by mounting a common response to many stresses, called the environmental stress response (ESR). Giaever et al. previously revealed poor correlation between genes whose expression changes in response to acute stress and genes required to survive that stress, raising question about the role of stress-activated gene expression. Here we show that gene expression changes triggered by a single dose of stress are not required to survive that stimulus but rather serve a protective role against future stress. We characterized the increased resistance to severe stress in yeast preexposed to mild stress. This acquired stress resistance is dependent on protein synthesis during mild-stress treatment and requires the "general-stress" transcription factors Msn2p and/or Msn4p that regulate induction of many ESR genes. However, neither protein synthesis nor Msn2/4p is required for basal tolerance of a single dose of stress, despite the substantial expression changes triggered by each condition. Using microarrays, we show that Msn2p and Msn4p play nonredundant and condition-specific roles in gene-expression regulation, arguing against a generic general-stress function. This work highlights the importance of condition-specific responses in acquired stress resistance and provides new insights into the role of the ESR.

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Hypusine-containing Protein eIF5A Promotes Translation Elongation

Preeti Saini, Daniel E. Eyler, Rachel Green … (2009)

Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors EF-Tu/eEF1A and EF-G/eEF2 that deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively 1 . The factor eIF5A, the sole protein in eukaryotes and archaea containing the unusual amino acid hypusine [N ε-(4-amino-2-hydroxybutyl)lysine] 2 , was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation 3,4 . Hypusine is required for eIF5A to associate with ribosomes 5,6 , and to stimulate methionyl-puromycin synthesis 7 . As eIF5A did not stimulate earlier steps of translation initiation 8 , and depletion of eIF5A in yeast only modestly impaired protein synthesis 9 , it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular mRNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay (NMD) pathway 10,11 , and to nucleocytoplasmic transport 12,13 . Here we show using molecular genetic and biochemical studies that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in yeast resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. As eIF5A is a structural homolog of the bacterial protein EF-P 14,15 , we propose that eIF5A/EF-P is a universally conserved translation elongation factor.

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Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

Maurice Toirkens, M J H Almering, M Hartog … (2005)

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

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Author and article information

Contributors

Sun-Mi Lee:

ORCID: http://orcid.org/0000-0002-5303-5464

+82-2-958-5836 , smlee@kist.re.kr

Journal

Journal ID (nlm-ta): Biotechnol Biofuels

Journal ID (iso-abbrev): Biotechnol Biofuels

Title: Biotechnology for Biofuels

Publisher: BioMed Central (London )

ISSN (Electronic): 1754-6834

Publication date (Electronic): 29 September 2018

Publication date PMC-release: 29 September 2018

Publication date Collection: 2018

Volume: 11

Electronic Location Identifier: 268

Affiliations

[1 ]ISNI 0000000121053345, GRID grid.35541.36, Clean Energy Research Center, , Korea Institute of Science and Technology (KIST), ; Seoul, 02792 Republic of Korea

[2 ]ISNI 0000 0004 1791 8264, GRID grid.412786.e, Division of Energy and Environment Technology, , University of Science and Technology (UST), ; Daejeon, 34113 Republic of Korea

[3 ]ISNI 0000 0001 0840 2678, GRID grid.222754.4, Green School, , Korea University, ; Seoul, 02841 Republic of Korea

Author information

Sun-Mi Lee http://orcid.org/0000-0002-5303-5464

Article

Publisher ID: 1269

DOI: 10.1186/s13068-018-1269-7

PMC ID: 6162923

PubMed ID: 30288173

SO-VID: 6c02d6a6-78dd-40a8-8c1f-72b4ce170baa

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 2 July 2018

Date accepted : 21 September 2018

Funding

Funded by: National Research Council of Science and Technology (KR)

Award ID: CAP-11-04-KIST

Funded by: FundRef http://dx.doi.org/10.13039/501100003725, National Research Foundation of Korea;

Award ID: 2017R1A6A3A04009462

Award Recipient : Ja Kyong Ko

Funded by: FundRef http://dx.doi.org/10.13039/501100007053, Korea Institute of Energy Technology Evaluation and Planning;

Award ID: 20153030091360

Award Recipient : Sun-Mi Lee

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: xylose fermentation,cofermentation,crispr–cas9,evolutionary engineering,gene expression landscape

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ScienceOpen disciplines: Biotechnology

Keywords: xylose fermentation, cofermentation, crispr–cas9, evolutionary engineering, gene expression landscape

Genomic and phenotypic characterization of a refactored xylose-utilizing Saccharomyces cerevisiae strain for lignocellulosic biofuel production

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Genome Engineering using CRISPR

Most cited references 56

Stress-activated genomic expression changes serve a preparative role for impending stress in yeast.

Hypusine-containing Protein eIF5A Promotes Translation Elongation

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

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