Effect of upstream sequences of the ADH1 promoter on the expression of Hormoconis resinae glucoamylase P by Saccharomyces cerevisiae

Abstract

The effect of the upstream sequences of the yeast ADH1 promoter on the expression of Hormoconis resinae glucoamylase P by Saccharomyces cerevisiae was studied. Sequence analysis of the 5'-terminal region of the promoter revealed sequence patterns resembling a transcription start point and the binding site for the regulatory protein ADR1. A short promoter was constructed by deleting all the promoter sequences upstream of nucleotide -409, including the upstream activating sequence UASRPG. A medium-length promoter was constructed by deleting a fragment of 558 bp containing the putative upstream transcription start point but not the UAS. The short promoter increased the glucoamylase expression level 1.6-fold compared with the long promoter, but the beginning of secretion was delayed by about 10 h probably because of the absence of the UAS. The medium-length promoter directed expression of the glucoamylase without an initial delay, with the enzyme activity lying between the activities produced under the long and short promoters. Northern blot analysis confirmed the secretion patterns of the strains with different promoters but failed to reveal any transcripts starting at the putative upstream start point.

Production of β‐galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

“…For the joint glucose and galactose fermentation, the lag phase for sugar metabolisation was not observed as the secreted enzyme was not needed for glucose metabolisation. However, in respect of the β‐galactosidase secretion the 10 h lag phase was again observed, confirming the results obtained for lactose fermentation on the promotor characteristics described by Vainio 11. Also, the amounts of β‐galactosidase produced were considerably less than those obtained with lactose, despite the final biomass concentration being similar to that obtained with 50 g dm −3 lactose.…”

“…This effect will be more marked with greater β‐galactosidase activity in the inoculum supernatant. The promotor used may also explain the observed 10 h lag phase11 as well as the increase in β‐galactosidase activity during the ethanol‐consumption phase 12, 13…”

“…However, the increase in β‐galactosidase activity was not proportional to the increase in biomass concentration. As the promotor used was the short ADHI , the ethanol‐consumption phase also had a stimulatory effect on the promotor 11, 12. In conclusion, the increase in the yeast extract concentration gave higher extracellular β‐galactosidase production.…”