Abstract:

In recent years, xylose fermentation to ethanol has become a research hotspot, and genetic engineering is a
promising means to achieve this goal. In this study, multiple splicing PCR method was applied to transform xylose into
xylulose, and relevant genes such as xylose reductase gene (xyl1), xylitol off gene (xyl2), turning aldehyde enzyme gene (tal1)
were spliced and cloned into the expression vector pAUR123 to obtain the fusion expression vector pAUR123-X12A. In
order to overexpress downstream genes in metabolic pathways, xylulosekinase gene (xks1) and ketonethe gene (tkl1), which
are responsible for controlling the conversion of xylulose into ethanol, were spliced and cloned into the expression vector
pAUR123 to achieve the fusion expression vector pAUR123-SK. pAUR123-X12A and pAUR123-SK were transferred into S.
cerevisiae INVSc1 by the lithium acetate method to obtain the recombinants of S. cerevisiae INVSc1-X12A and S. cerevisiae
INVSc1-SK. The recombinants were expressed and then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE)
and enzyme activity to suggest successful expression of the target gene. Preliminary results from this study reveal that two
recombinant strains have been built successfully.

Key words: multi-gene tandem expression, xylose, expression, Saccharomyces cerevisiae