Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose redu...

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Autores principales:	Jung-Hoon Bae, Mi-Jin Kim, Bong Hyun Sung, Yong-Su Jin, Jung-Hoon Sohn
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Xylose Xylose isomerase Secretion Saccharomyces cerevisiae Co-fermentation Fuel TP315-360 Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/70cb32d8ee8841b082d62ca6c2907acd
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spelling	oai:doaj.org-article:70cb32d8ee8841b082d62ca6c2907acd2021-11-28T12:22:24ZDirected evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae10.1186/s13068-021-02073-y1754-6834https://doaj.org/article/70cb32d8ee8841b082d62ca6c2907acd2021-11-01T00:00:00Zhttps://doi.org/10.1186/s13068-021-02073-yhttps://doaj.org/toc/1754-6834Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.Jung-Hoon BaeMi-Jin KimBong Hyun SungYong-Su JinJung-Hoon SohnBMCarticleXyloseXylose isomeraseSecretionSaccharomyces cerevisiaeCo-fermentationFuelTP315-360BiotechnologyTP248.13-248.65ENBiotechnology for Biofuels, Vol 14, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Xylose Xylose isomerase Secretion Saccharomyces cerevisiae Co-fermentation Fuel TP315-360 Biotechnology TP248.13-248.65
spellingShingle	Xylose Xylose isomerase Secretion Saccharomyces cerevisiae Co-fermentation Fuel TP315-360 Biotechnology TP248.13-248.65 Jung-Hoon Bae Mi-Jin Kim Bong Hyun Sung Yong-Su Jin Jung-Hoon Sohn Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
description	Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.
format	article
author	Jung-Hoon Bae Mi-Jin Kim Bong Hyun Sung Yong-Su Jin Jung-Hoon Sohn
author_facet	Jung-Hoon Bae Mi-Jin Kim Bong Hyun Sung Yong-Su Jin Jung-Hoon Sohn
author_sort	Jung-Hoon Bae
title	Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
title_short	Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
title_full	Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
title_fullStr	Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
title_full_unstemmed	Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae
title_sort	directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in saccharomyces cerevisiae
publisher	BMC
publishDate	2021
url	https://doaj.org/article/70cb32d8ee8841b082d62ca6c2907acd
work_keys_str_mv	AT junghoonbae directedevolutionandsecretoryexpressionofxyloseisomeraseforimprovedutilisationofxyloseinsaccharomycescerevisiae AT mijinkim directedevolutionandsecretoryexpressionofxyloseisomeraseforimprovedutilisationofxyloseinsaccharomycescerevisiae AT bonghyunsung directedevolutionandsecretoryexpressionofxyloseisomeraseforimprovedutilisationofxyloseinsaccharomycescerevisiae AT yongsujin directedevolutionandsecretoryexpressionofxyloseisomeraseforimprovedutilisationofxyloseinsaccharomycescerevisiae AT junghoonsohn directedevolutionandsecretoryexpressionofxyloseisomeraseforimprovedutilisationofxyloseinsaccharomycescerevisiae
_version_	1718408022960635904

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

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