Use of the Saccharomyces cerevisiae endopolygalacturonase promoter to direct expression in Escherichia coli

Abstract

In Saccharomyces cerevisiae, an endopolygalacturonase encoded by the PGL1 gene catalyzes the random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid. To study the regulation of the PGL1 gene, we constructed a reporter vector containing the lacZ gene under the control of PGL1 promoter. Surprisingly, when Escherichia coli DH5α was transformed by this vector, cells harboring the constructed plasmid produced blue colonies. Sequence analysis of this promoter revealed that E. coli consensus sequences required to express an in-frame lacZ alpha product were present. We next decided to investigate how the PGL1 promoter is regulated in E. coli compared to yeast. In this study, we examined the modulation of the PGL1 promoter in E. coli, and the results indicated that its activity is greatly induced by saturated digalacturonic acid and is indirectly regulated by the transcriptional regulators the 2-keto-3-deoxygluconate repressor. Moreover, PGL1 expression is enhanced under aerobic conditions. We found that β-galactosidase activity in E. coli could reach 180 units, which is 40-fold greater than the activity produced in S. cerevisiae, and greater than recombinant protein expression previously reported by other researchers. We thus demonstrate that this vector can be considered as a dual expression plasmid for both E. coli and S. cerevisiae hosts. So far, no modulation of endoPG promoters expressed in E. coli has been reported.

Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning‐free workflows

“…As previously reported, many gene products, either noncoding RNA or proteins, can be toxic in E. coli (Kimelman et al, ). Although in this study ZEP genes were cloned under the control of yeast PGK1 promoter, some eukaryotic promoters can still drive gene expression in E. coli (Antonucci, Wen, & Rutter, ; Gognies, Bahkali, Moslem, & Belarbi, ). Thus, ZEP genes may have been expressed in the transformed cells causing toxicity.…”