Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

Hasunuma Tomohisa, Sanda Tomoya, Yamada Ryosuke, Yoshimura Kazuya, Ishii Jun, Kondo, Kondo Akihiko

Description

The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae . Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 9
• Langue: English

Extrait

Hasunuma et al . Microbial Cell Factories 2011, 10 :2 http://www.microbialcellfactories.com/content/10/1/2

R E S E A R C H Open Access Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae Tomohisa Hasunuma 1 , Tomoya Sanda 2 , Ryosuke Yamada 2 , Kazuya Yoshimura 2 , Jun Ishii 1 , Akihiko Kondo 2*

Abstract Background: The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results: In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae . Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions: Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

Background bacteria [3]. However, a major drawback is that S. cere- Numerous environmental and social benefits could visiae cannot utilize xylose, the most common pentose result from the replacement of petroleum-based trans- sugar in the hemicellulose that makes up a sizable frac- port fuels with bio-ethanol converted from lignocellulo- tion of lignocellulosic hydrolysates. Thus, most efforts in sic materials such as agricultural residues and industrial the engineering of S. cerevisiae for xylose fermentation waste [1,2]. The commonly used yeast Saccharomyces have focused on manipulation of the initial xylose meta- cerevisiae has many advantages as an ethanol producer, bolic pathway [4]. The reconstruction of an efficient such as fast sugar consumption, high ethanol yield from  xylose assimilation pathway in S. cerevisiae has been glucose, and higher resistance to ethanol and other approached via heterologous expression of genes for compounds present in lignocellulosic hydrolysates than xylose reductase (XR) and xylitol dehydrogenase (XDH) derived from Pichia stipitis along with overexpression of * Correspondence: [e-mail] S. cerev e x l E 2 n D g e i p n a e rt er m in e g n , t K o o f b C e h U e n m i i v c e a r l si S ty c , ie 1 n - c 1 e R a o n kk d o E d n ai g , in N e a e d ri a n , g K , o G b r e ad 65 u 7 at -8 e 5 S 0 c 1 h , o Ja ol pa o n f xylose fe is r i m a enta y ti u o l n ok [ i 5 n -7 a ] s . e X ( y X lo K s ) e t is o fi p r r s o t d re u d ce uc e e t d h t a o no x l yl i i- n Full list of author information is available at the end of the article tol by XR, and then xylitol is oxidized to xylulose by © 2011 Hasunuma et al; licensee BioMed Central Ltd. 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 cited.