Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates

156

170

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.

Section: Discussionmentioning

confidence: 67%

“…In contrast, Saccharomyces cerevisiae fermentations can be performed at low pH values. Moreover, S. cerevisiae has further beneficial properties for industrial production processes like high robustness, high resistance to toxic inhibitors and fermentation products, resistance to microbial contamination, and a high level of public acceptance (32,35,50).…”

mentioning

confidence: 99%

Biosynthesis of cis , cis -Muconic Acid and Its Aromatic Precursors, Catechol and Protocatechuic Acid, from Renewable Feedstocks by Saccharomyces cerevisiae

Weber

Brückner

Weinreb

et al. 2012

156

170

“…These include reduction of furfural production by using phosphoric acid rather than sulfuric acid during pretreatment (6,9,10), the isolation of furfural-resistant mutants of yeast (Saccharomyces cerevisiae) (19) and bacteria (26), and directed genetic modifications (19,26,33). Native genes have been identified and overexpressed that catalyze furfural reduction with NADH or NADPH in yeast (7,14,17,18) and bacterial biocatalysts (25,33). Furfural and other aldehydes have many biological effects on eukaryotes and prokaryotes which may contribute to the inhibition of growth (36).…”

mentioning

confidence: 99%

Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression of thyA

Zheng

Wang

Yomano

et al. 2012

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilisYB886,Escherichia coliNC3, andZymomonas mobilisCP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyAgene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novopathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyAwas no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coliand to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coliwould be expected to increase the cellular requirement for dTMP. Increased expression ofthyA(E. coli,B. subtilis, orZ. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

“…These furan aldehydes are known as the most potent inhibitors of microbial cell growth (4,5). The toxic aldehyde groups of furfural and HMF can be reduced to hydroxyl groups by several oxidoreductases, including alcohol dehydrogenases (Adh1, Adh6, and Adh7) (6)(7)(8), aldehyde reductase (Ari1) (9), and methylglyoxal reductases (Gre2 and Gre3) in Saccharomyces cerevisiae cells (8,(10)(11)(12)(13) and aldehyde dehydrogenase (YqhD) and methylglyoxal reductase (DkgA) in Escherichia coli (14). These enzymes consume NADH and NADPH as cofactors during the reduction process.…”

mentioning

confidence: 99%

Roles of the Yap1 Transcription Factor and Antioxidants in Saccharomyces cerevisiae's Tolerance to Furfural and 5-Hydroxymethylfurfural, Which Function as Thiol-Reactive Electrophiles Generating Oxidative Stress

Kim

Hahn

2013

Development of the tolerance of Saccharomyces cerevisiae strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H 2 O 2 -independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in Saccharomyces cerevisiae. However, furfural showed higher reactivity than did HMF toward GSH in vitro and in vivo. In line with such toxic mechanisms, overexpression of YAP1 C620F , a constitutively active mutant of YAP1, and Yap1 target genes encoding catalases (CTA1 and CTT1) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (GSH1 and GLR1) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF.