Cloning and improving the expression ofPichia stipitis xylose reductase gene inSaccharomyces cerevisiae

Abstract

The intactPichia stipitis xylose reductase gene (XR) has been cloned and expressed inSaccharomyces cerevisiae. The possible further improvement of the expression of thePichia gene in the new host was studied. To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5′-noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes. It was found that thePichia genetic signal for transcription of XR is more effective than the yeast TRP5 promoter, but is about half as effective as the yeast strong promoter of the alcohol dehydrogenase gene (ADC1). However, the nucleotide sequence immediately adjacent to the initiation codon of XR, which controls the translation of the gene product, seemed to be five times less effective than the corresponding sequence of the ADC1 gene. By totally replacing its 5′-noncoding sequence with that of the yeast ADC1 gene, the expression of XR in yeast was found to be nearly ten times higher. Furthermore, the clonedPichia XR described in this article contains very little of its 3′-noncoding sequence. In order to study whether the 3′-noncoding sequence is important to its expression inS. cerevisiae, the intact 3′-noncoding sequences of the yeast xylulokinase gene was spliced to the 3′ end of theP _ADC1-XR structural gene. This latter modification has resulted in a twofold further increase in the expression of thePichia XR in yeast.