Cargando…

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae

BACKGROUND: To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below en...

Descripción completa

Detalles Bibliográficos
Autores principales: Klimacek, Mario, Kirl, Elisabeth, Krahulec, Stefan, Longus, Karin, Novy, Vera, Nidetzky, Bernd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007572/
https://www.ncbi.nlm.nih.gov/pubmed/24606998
http://dx.doi.org/10.1186/1475-2859-13-37
_version_ 1782314352454402048
author Klimacek, Mario
Kirl, Elisabeth
Krahulec, Stefan
Longus, Karin
Novy, Vera
Nidetzky, Bernd
author_facet Klimacek, Mario
Kirl, Elisabeth
Krahulec, Stefan
Longus, Karin
Novy, Vera
Nidetzky, Bernd
author_sort Klimacek, Mario
collection PubMed
description BACKGROUND: To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (m(ATP)). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Y(ethanol) = 0.35 g/g) but slow (q(ethanol) = 0.05 ± 0.01 g/g(BM)/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway. RESULTS: BP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h(-1) but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h(-1); q(ethanol) = 0.28 ± 0.04 g/g(BM)/h), efficient (Y(ethanol) = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO(2)/HCO(3)(-), was accompanied by a 2.3-fold increase of m(ATP) while Y(ATP) (= 87 ± 7 mmol(ATP)/g(BM)) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical. CONCLUSIONS: In just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that m(ATP) was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific.
format Online
Article
Text
id pubmed-4007572
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-40075722014-05-19 Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae Klimacek, Mario Kirl, Elisabeth Krahulec, Stefan Longus, Karin Novy, Vera Nidetzky, Bernd Microb Cell Fact Research BACKGROUND: To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (m(ATP)). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Y(ethanol) = 0.35 g/g) but slow (q(ethanol) = 0.05 ± 0.01 g/g(BM)/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway. RESULTS: BP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h(-1) but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h(-1); q(ethanol) = 0.28 ± 0.04 g/g(BM)/h), efficient (Y(ethanol) = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO(2)/HCO(3)(-), was accompanied by a 2.3-fold increase of m(ATP) while Y(ATP) (= 87 ± 7 mmol(ATP)/g(BM)) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical. CONCLUSIONS: In just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that m(ATP) was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific. BioMed Central 2014-03-08 /pmc/articles/PMC4007572/ /pubmed/24606998 http://dx.doi.org/10.1186/1475-2859-13-37 Text en Copyright © 2014 Klimacek et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.
spellingShingle Research
Klimacek, Mario
Kirl, Elisabeth
Krahulec, Stefan
Longus, Karin
Novy, Vera
Nidetzky, Bernd
Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title_full Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title_fullStr Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title_full_unstemmed Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title_short Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
title_sort stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant saccharomyces cerevisiae
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007572/
https://www.ncbi.nlm.nih.gov/pubmed/24606998
http://dx.doi.org/10.1186/1475-2859-13-37
work_keys_str_mv AT klimacekmario stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae
AT kirlelisabeth stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae
AT krahulecstefan stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae
AT longuskarin stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae
AT novyvera stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae
AT nidetzkybernd stepwisemetabolicadaptionfrompuremetabolizationtobalancedanaerobicgrowthonxyloseexploredforrecombinantsaccharomycescerevisiae