Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Abstract

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.

HS-SPME/GC-MS and chemometrics for volatile composition of Chinese traditional aromatic vinegar in the Zhenjiang region

“…The compounds 3-methyl-1-butanol and 2,3-butanediol (3-3100 mg/L and 3-1700 mg/L, respectively) were detected in all thirteen Zhenjiang aromatic vinegars. The compound 3-methyl-1-butanol has a fruity and alcohol flavour note (32) or caramel odour (33), and is produced during the catabolism of branched chain amino acids leucine, isoleucine and valine, or synthesized during the biosynthesis of the branched chain amino acids described previously (34). The compound 2,3-butanediol can be produced via a mixed acid pathway and catalyzed to acetoin (3-hydroxy-2-butanone) by acetoin reductase (35,36), and has been detected in traditional (37,38).…”

Impact of yeast systems biology on industrial biotechnology

“…To a great extent, research in the area of systems biology is also dependent on the market demand. For example, in the present times, when the demand for the production of alternate energy sources is high, the capability of S. cerevisiae to produce higher alcohols such as butanol and methyl butanol is investigated (Schoondermark-Stolk et al, 2006;Steen et al, 2008).…”

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

“…For example, the genes involved in the synthesis of isopropanol [ 16,17] and butanol [ 18] from -) ! Clostriduim were recently expressed in E. coli.…”