Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae

Table 4 Cloning strategy for the construction of yeast integrating plasmids YCt XRWt/Gm XDH/XKS1 and YCt XRDm/Gm XDH/XKS1. The shown primer sets were used to amplify the target sequences from the template plasmids. The amplification products were cloned into the corresponding restriction sites of the target plasmid.

	Target sequence	Template plasmid	Primers (restriction sites underlined)	Target plasmid	Restriction sites	Resulting plasmid
1	Ct XRWt gene	pET11-Ct XRWt	Fwd: 5'-GGTGGTGGATCC ATGAG CGCAAGTATCCGAGAC-3'	pRS416GPD	BamH I Sal I	pRS416GPD-Ct XRWt
	Ct XRDm gene	pET11-Ct XRDm	Rev: 5'CTAGTGGGTCGAC TTAAAC GAAGATTGGAATGTTGTC-3'	pRS416GPD	BamH I Sal I	pRS416GPD-Ct XRDm
	Gm XDH gene	pBTac1	Fwd: 5'-GGTGGTGGATCC ATGTCT ACTCCTGAAAACTTATCT-3'	pRS416GPD	BamH I Sal I	pRS416GPD-Gm XDH
			Rev: 5'-CTAGTGGGTCGAC TTAC TCAGGGCCGTTAATGATG-3'
	XKS1 gene	Genomic S. cerevisiae DNA	Fwd: 5'-GGTGGTGGATCC ATGTTG TGTTCAGTAATTCAGAGA-3'	pRS416GPD	BamH I Sal I	pRS416GPD-XKS1
			Rev: 5'-GGTGGTGTCGAC TTAGAT GAGAGTCTTTTCCAGTTC-3'
2	Gene cassette^a XKS1	pRS416GPD-XKS1	Fwd: 5'-CATGGTGACGTC AGTTTATC ATTATCAATACTCGCCATTTC-3'	YiP5	Aat II	YXKS1
			Rev: 5'-GGTGGTGACGTC GGCCGCA AATTAAAGCCTTCG-3'
3	Gene cassette Gm XDH	pRS416GPD-Gm XDH	Fwd: 5'-CATGGTATCGAT AGTTTATC ATTATCAATACTCGCCATTTC-3'	YXKS1	Cla I	YGm XDH/XKS1
			Rev: 5'-GGTGGTATCGAT GGCCGCA AATTAAAGCCTTCG-3'
4	Gene cassette Ct XRWt	pRS416GPD-Ct XRWt	Fwd: 5'-GGTGGTGAATTC AGTTTATC ATTATCAATACTCGCCATTTC-3'	YGm XDH/XKS1	EcoR I	YCt XRWt/Gm XHD/XKS1
	Gene cassette Ct XRDm	pRS416GPD-Ct XRDm	Rev: 5'-GGTGGTGAATTC GGCCGCA AATTAAAGCCTTCG-3'	YGm XDH/XKS1	EcoR I	YCt XRDm/Gm XHD/XKS1

^a Gene cassettes contain S. cerevisiae TDH3 promoter (labeled GPD in the Table) – target gene – S. cerevisiae CYC1 terminator

ISSN: 1475-2859