Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae

Abstract

A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37-48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.

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

“…E. coli JM109, used for the transfection of the sequencing vector, phage M13mp19, was maintained on M9 plates containing thiamine (15 /zg m1-1) and grown in L broth. The plasmid constructions for yeast expression were made using pAAH5 [4] as the source of the ADH1 promoter, pALK304 [1] as the source of gamP gene and pALK318, a derivative of pALK307 [1], where the 44 bp EcoRV-MIuI fragment is deleted. The construction of pALK305 containing the gamP cDNA under the control of the long ADH1 promoter has been described previously [1].…”

“…The plasmid constructions for yeast expression were made using pAAH5 [4] as the source of the ADH1 promoter, pALK304 [1] as the source of gamP gene and pALK318, a derivative of pALK307 [1], where the 44 bp EcoRV-MIuI fragment is deleted. The construction of pALK305 containing the gamP cDNA under the control of the long ADH1 promoter has been described previously [1]. The S. cerevisiae strains used in this study were YF135 (a, leu2-3, leu2-112, his3-11, his3-15) and ALKO2681, the latter a mutant of ALKO807 (a, leu2-3, /eu2-112, ura3-5OA, canl-lO1) selected for increased heterologous protein production at our laboratory [9].…”

“…Glucoamylase activity was determined as described previously [1,16], using soluble starch as substrate. One unit was defined as the amount of enzyme releasing 1 /zmol glucose per min at 30°C.…”

“…We have previously constructed yeast strains expressing H. resinae gamP under the control of the ADH1 promoter [1]. In these strains, glu-coamylase is produced mainly by logarithmically growing cells.…”

“…The promoter of the yeast alcohol dehydrogenase 1 gene, ADH1, is widely used for the expression of heterologous genes in Saccharomyces cerevisiae. We have constructed yeast strains expressing the Hormoconis resinae glucoamylase P gene, gamP, under the control of the ADH1 promoter [1]. To enhance expression, it was decided to attempt to modify the promoter since it * Corresponding author.…”