We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Marker-disruptive gene integration and URA3 recycling for multiple gene manipulation in Saccharomyces cerevisiae.
- Authors
Kaneko, Shohei; Tanaka, Tsutomu; Noda, Hideo; Fukuda, Hideki; Akada, Rinji; Kondo, Akihiko
- Abstract
The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying β-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL–URA3 fragment integration were achieved via a PCR fragment consisting of the BGL–URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3:: URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.
- Subjects
SACCHAROMYCES cerevisiae; GENETIC regulation; YEAST fungi genetics; GENETIC markers; FERMENTATION; INDUSTRIAL applications
- Publication
Applied Microbiology & Biotechnology, 2009, Vol 83, Issue 4, p783
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-009-2038-0