Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coliand biochemical characterization

Abstract

BackgroundKluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown.ResultsThe ethanol fermentation capability of K. marxianus GX-UN120 at 40°С was found to be the same as that of Saccharomyces cerevisiae at 34°С. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable. The genes encoding the four alcohol dehydrogenases (ADHs) were cloned from strain GX-UN120. Each KmADH was expressed in Escherichia coli and each recombinant protein was digested with enterokinase to remove the fusion protein. The optimum pH of the purified recombinant KmAdh1 was 8.0 and that of KmAdh2, KmAdh3 and KmAdh4 was 7.0. The optimum temperatures of KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were 50, 45, 55 and 45°C, respectively. The Km values of the recombinant KmAdh1 and KmAdh2 were 4.0 and 1.2 mM for acetaldehyde and 39.7 and 49.5 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh1 and KmAdh2 were 114.9 and 21.6 μmol min-1 mg-1 for acetaldehyde and 57.5 and 1.8 μmol min-1 mg-1 for ethanol, respectively. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol, and the K m values of the recombinant KmAdh3 and KmAdh4 were 26.0 and 17.0 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh3 and KmAdh4 were 12.8 and 56.2 μmol min-1 mg-1 for ethanol, respectively.ConclusionThese data in this study collectively indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The relatively high optimum temperature of KmAdh1 may partially explain the ability of K. marxianus to produce ethanol at high temperature. Understanding the biochemical characteristics of KmAdhs will enhance our fundamental knowledge of the metabolism of ethanol fermentation in K. marxianus.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

“…The gene encoding alcohol dehydrogenase has been cloned and characterized from many different yeast strains, such as P. stipites , 84 S. cerevisiae 85 and K. marxianus . 86 , 87 There are two (i.e., Psadh1 and Psadh2 ), seven (i.e., adh1 to adh7 ) and four genes (i.e., adh1 to adh4 ) encoding alcohol dehydrogenases in P. stipitis , S. cerevisiae and K. marxianus , respectively. The transcriptional activation of these genes varies depending on the yeast strains, growth phases and environmental conditions.…”

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

“…However, there are still impeded in their large-scale application due to the limited commercially robust biocatalysts, the narrow substrate specificity and the dependency of expensive cofactor as well as the water-insoluble substrates [ 20 ]. With the development of genomics, proteomics, and bioinformatics, genome hunting and data mining have become powerful tools for exploiting novel enzymes [ 21 , 22 ]. In the present study, on the basis of screening results of microorganisms, and the combination of the BLASTp searching using the reported carbonyl reductase (GenBank accession no.…”

Alcohol dehydrogenases from Proteus mirabilis contribute to alcoholic flavor

“…Based on sequence analysis and protein domain conservation analysis by using Basic Local Alignment Search Tool program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and NCBI Conserved Domain Search program (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), ADH‐4 and ADH‐5 belong to zinc‐containing type I, ADH‐1, ADH‐2 and ADH‐3 belong to iron‐activated type III. Additionally, the optimal pH value of ADHs was 7.0 or 8.0, and the optimal temperature was 40 or 45 °C, as previously reported for ADHs …”

“…Additionally, the optimal pH value of ADHs was 7.0 or 8.0, and the optimal temperature was 40 or 45 ∘ C, as previously reported for ADHs. 17,18 During cheese ripening, temperature and pH may affect cheese flavor and texture.…”