title: Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.
abstract: BACKGROUND
  Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described.
  
  
  RESULTS
  Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their contribution to strain growth and vanillin reduction were balancing the redox state of strain when vanillin was presented.
  
  
  CONCLUSIONS
  Beside the reported Adh6p, the enzymes encoded by YNL134C and YJR096W were proved to have vanillin reduction activity in present study. While ALD6 and ZWF1 did not directly reduce vanillin to vanillyl alcohol, their contribution to vanillin resistance primarily depended on the enhancement of the reducing equivalent supply.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China.
authors.forename: Xinning
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Hou
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China. bxm@sdu.edu.cn.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Bao
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China. shenyu@sdu.edu.cn.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shen
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D001547:Benzaldehydes / Q000494:pharmacology*; D005285:Fermentation; D009249:NADP / Q000032:analysis / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D001547:Benzaldehydes; D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; C100058:vanillin; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase
keywords: ADH6; Budding yeast; Vanillin tolerance; YJR096W; YNL134C