Over-expression, purification and characterization of Adh5p from Saccharomyces cerevisiae

[摘要] English: In the past few decades much research has been conducted on yeast alcoholdehydrogenases with emphasis on their role in S. cerevisiae. There are sevenknown Adh proteins that were identified in S. cerevisiae. Six of them areintensively studied on gene and translational level. After sequencing of S.cerevisiae chromosome II (Feldman et al. 1994), ADH5 was identified which is76% and 77% identical to ADH1 and ADH2 respectively. Later studies onglobal localization on genes showed that ADH5 is localized in the cytoplasm(Hue et al. 2003). No further research has been performed on ADH5.In this study Adh5p was over-expressed, purified and characterized towards itsprimary substrate, ethanol, and preferred co-factor NAD+. Furthermore, indepthkinetic studies were performed using various alcohols, increasing inchain length and branching. Adh5p is capable of oxidizing alcohols from twocarbons to ten carbons. However, the catalytic efficiency decreases withincreasing chain length. Results showed that Adh5p functions in vitro the sameas Adh1p and Adh2p, sharing a primary substrate (ethanol). Adh1p and Adh2pare capable of converting more substrate per unit enzyme per second Adh5p.The second part of this study was to determine if Adh5p could substitute thecatalytic function of Adh1p in vivo. For this purpose, ADH5 expression neededto be similar to Adh1p levels in the cell. Thus an expression vector was usedcontaining ADH5 gene flanked with the promoter and terminator regions ofADH1. S. cerevisiae TΔ123 was constructed with ADH4 and ADH5 still intact.S. cerevisiae TΔ123 was transformed with a pRS413 and pRS423 constructcontaining the ADH1 promoter and terminator fused to the ADH5 ORF. Growthwas monitored in chemically defined media containing 7 g l-1 ethanol or 8 g l-1glucose. Growth parameters were also compared to the S. cerevisiae W303-1Aand the adh quadruple deletion strain (Q1) containing only ADH1. The S.cerevisiae TΔ123::ADH5_S and S. cerevisiae TΔ123::ADH5_M constructs werecapable of limited growth on both glucose and ethanol as carbon source When comparing the biomass yield of S. cerevisiae TΔ123::ADH5_S and S.cerevisiae TΔ123::ADH5_M to the biomass yield for both S. cerevisiae W303-1A and S. cerevisiae Q1 the constructs delivered a much lower biomass yield.To verify the expression of Adh5p, a Micro BCA�?protein assay kit supplied byPierce (Smith et al. 1985) was used. Protein concentrations were determined atvarious time intervals. Cell homogenization was standardized, S. cerevisiae Q1cells were diluted to fit the OD600 obtained for both the S. cerevisiaeTΔ123::ADH5_S and S. cerevisiae TΔ123::ADH5_M strains.To conclude, Adh5p is capable of oxidizing various alcohols, but ethanol is itsprimary substrate. Furthermore Adh5p is not capable of replacing Adh1p incellular metabolic function. The low turnover number illustrated by Adh5p andthe lack thereof to reduce acetaldehyde is the most prominent cause of cellulardeath. Unlike Adh1p, Adh5p is not capable of reducing acetaldehyde toethanol and thus not capable of NAD+ - NADH regeneration.

[发布机构] University of the Free State