Overproduction of pentose phosphate pathway enzymes using a new CRE–loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Abstract

Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxPzeocin-loxP cassette using pCRE3. The 2m-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counterselection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.

Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant

“…Integration was con¢rmed using PCR by ampli-¢cation of the PGK promoter together with the XYL1 gene as described earlier [10]. The cleavage product was used for transformation of TMB3001 and TMB3255, generating TMB3260 and TMB3261 after selection for zeocin resistance.…”

High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae

“…First, protein engineering approaches have been undertaken to change cofactor specificities of NAD(P)H-specific XR and NAD-specific XDH for balancing their cofactor requirements (Bengtsson et al, 2009;Krahulec et al, 2010;Petschacher and Nidetzky, 2008). Second, selected or entire genes coding for enzymes in the pentose phosphate pathway have been perturbed as knockout or overexpression targets to improve the rate of xylose fermentation by engineered S. cerevisiae (Jin et al, 2005;Johansson and Hahn-Hägerdal, 2002;Karhumaa et al, 2005;Lu and Jeffries, 2007;Sonderegger et al, 2004;Walfridsson et al, 1995). Third, studies have been carried out on characterizing xylose transport by engineered S. cerevisiae (Hamacher et al, 2002) and introducing heterologous xylose-specific transporters such as SUT1/2 from Sch.…”

A New Method for Repeated “Self-Cloning” Promoter Replacement in Saccharomyces cerevisiae

“…This problem has been overcome by the use of promoter replacement cassettes with markers eliminated by the Cre-loxP recombination system of bacteriophage P1 [5,6]. In spite of its high efficiency for research applications, this method cannot be applied for strain construction in the food industry because the resulting strains contain heterologous loxP scar DNA sequences after marker excision.…”