Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

Abstract

Transformation of Saccharomyces cerevislae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 103-fold less active.

GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae

“…While XIs from anaerobic fungus Piromyces sp. (Kuyper et al, , ), and some bacteria, such as Bacteroides stercoris (Ha et al, ), Clostridium phytofermentans (Brat et al, ), and Thermus thermophilus (Walfridsson et al, ), have shown to be functional in S. cerevisiae putatively due to specific protein folding properties, most attempts to express bacterial XIs, including E. coli XI, have failed (Amore et al, ; Moes et al, ; Sarthy et al, ; Walfridsson et al, ). Similarly, E. coli arabinose isomerase (AI) could not be functionally expressed in S. cerevisiae neither (Sedlak and Ho, ; Wisselink et al, ).…”

Metabolomic and 13C‐metabolic flux analysis of a xylose‐consuming Saccharomyces cerevisiae strain expressing xylose isomerase

“…While many bacteria use a xylose isomerase (XI) enzyme to catalyze this conversion directly without the use of pyridine nucleotide cofactors, xylose‐consuming eukaryotes generally effect the isomerization through a two‐step redox pathway in which xylose reductase (XR) first catalyzes the reduction of xylose to xylitol, which is then oxidized via xylitol dehydrogenase (XDH) to form xylulose. Initial attempts to express heterologous xylA (encoding XI) genes in S. cerevisiae were unsuccessful; in several cases putative xylA transcripts were detected in Northern blots but putative XI protein products were insoluble and inactive (Amore et al, ; Gárdonyi and Hahn‐Hägerdal, ; Sarthy et al, ). Consequently, the majority of xylose‐consuming strains have been constructed using the XR‐XDH pathway.…”

Fermentations with New Recombinant Organisms

“…Advances in the genetics of engineered yeasts for pentose fermentation, particularly S. cerevisiae, are most attractive to the corn processing industry because of their familiarity and experience with yeast fermentations and the potential robustness of the organisms. Stevis and Ho (1985), Gong et al (1981), Sarthy et al (1987), Amore et al (1989), Moes et al (1996), and Walfridsson et al, (1996) have introduced bacterial xylose isomerase genes into S. cerevisiae, which does not normally metabolize xylose. This approach for producing a Saccharomyces capable of converting xylose to ethanol has met with limited success because of the following possibilities (Dumsday et al, 1997a): differences in internal pH between bacteria and yeasts; incorrect folding of the enzyme; and unsuitable post-translational modifications.…”