从S.stipitis BCC47637基因组DNA中扩增质粒pRS416Tef1 SsXKS:XKS。将SsXKS扩增子连接到p416Tef1-Ura3的BamHI/EcoRI位点，得到pRS416Tef1 SsXKS。使用两组引物从酿酒酵母BY4742基因组DNA中扩增出两个片段的质粒pRS416Tef1-HXT7-F79S:HXT7-F79ES。一组引物含有氨基酸置换所需的点突变。这两个片段被放置在p416Tef1-Ura3中的PTEF1启动子后面，通过酵母中的同源重组形成pRS416Tef1-HXT7-F79S。从热带假丝酵母BCC59435基因组DNA中扩增质粒pRS416Tef1-CtXYL1:CtXYL1。将CtXYL1扩增子连接到p416Tef1-Ura3的BamHI/EcoRI位点，得到pRS416Tef1-CtXYL1。质粒pRS416Tef1-CsXYL1:CsXYL1连接到p416Sef1-Ura3的BamHI/EcoRI位点上，得到pRS4216Tef1-Cs XYL1。从S.stipitis BCC47637基因组DNA中扩增质粒pRS416Ef1-SsXYL1:SsXYL1，将SsXYL1扩增子连接到p416Tef1-Ura3的BamHI/SmaI位点，得到pRS416Tef1-SsXYL1。质粒pRS421Tef1-SpXYL1.1:SpXYL1.1连接到p416Sef1-Ura3的BamHI/EcoRI位点，得到pRS416Tef1-SpXYL1。热带假丝酵母BCC59435基因组DNA中扩增出质粒pRS416Tef1-CtXYL2:CtXYL2。将CtXYL2扩增子连接到p416Tef1-Ura3的SacII/EcoRI位点，得到pRS416Tef1-CtXYL2。使从S.stipitis BCC47637基因组DNA中扩增出质粒pRS416Tef1-SsXYL2:SsXYL2。SsXYL2扩增子连接到p416Tef1-Ura3的SacII/EcoRI位点，得到pRS416Tef1-SsXYL2。

参考文献：Promdonkoy P, Siripong W, Downes JJ, Tanapongpipat S, Runguphan W. Systematic improvement of isobutanol production from D-xylose in engineered Saccharomyces cerevisiae. AMB Express. 2019 Oct 10;9(1):160.