The Modification Of Cre/Loxp Recombination System And The Application In Scheffersomyces Stipitis

Scheffersomyces stipitis has the capacity to ferment xylose to ethanol. It is one of themost industrial prospect pentose yeasts. There are abundant plant fiber resources in nature,andsuch Agricultural and forestry waste can be fully and reasonably used to produce humanuseful substances through biotransformation, which has practical and long-term significance.The growth and bioconversion of the wild type has not yet reached the requirements ofindustrial strains. What’s more, xylose wasn’t effectively converted into ethanol throughMetabolic Engineering of Saccharomyces cerevisiae for xylose fermentation. Therefore, geneengineering was applied to improve the conversion of xylose in S. stipitis and a new geneticengineering S. stipitis was constructed,which has become a research hotspot. However, S.stipitis uses CUG to code for serin rather than leucine, as is the case for the universal geneticcode thereby limiting the expression of exogenous gene rich in CTG in S. stipitis. In this study,we constructed the selection marker for S. stipitis and modified the Cre/Loxp recombinationsystem through site-directed mutagenesis. Aldehyde dehydrogenase gene (ALD5) wasknocked out with no trace, which provided the basic tools for the study on genetictransformation of S. stipitis, and paved the way for the development of exogenous geneexpression system on this basis.The chromosome DNA ploidy of S. stipitis was researched firstly in this paper.According to the differences of characteristics between the content of haploid and diploidchromosome in S. cerevisiae, DNA content was assayed and analysed. We initially confirmedS. stipitis was the haploid cell, and homologous recombination technology could be applieddirectly.The uracil auxotrophy and drug resistance markers were constructed for molecularbiology operation of S. stipitis. Homologous recombinant fragments of URA3gene wasconstructed by overlap PCR. URA3gene deleted by double homologous recombination, and aS. stipitis (ura3-)strain was constructed. G418and Hygromycin B are common drug resistancemarkers in yeast. Experimental results showed that S. stipitis was sensitive to Hygromycin B,but had strong resistance to G418. Hygromycin B was chosen as the new drug resistancemarker.Cre/Loxp system contained Cre recombinase gene and Hygromycin B gene, and thesetwo exogenous genes could code for active proteins in S. stipitis after CTG codons weremutagenized to TTG. Homologous recombinant fragments of ALD5gene was constructed byoverlap PCR. URA3gene was inserted into homologous recombinant fragments as a selectionmarker. The ALD5gene was deleted and a S. stipitis (ald5-) was obtained. Galactose inducedthe expression of Cre recombinase gene and URA3gene was removed, which led S.stipitis(ald5-ura3-)to be obtained.In the research, the recombinant rate reached about50%. SoGAL promoter could be applied in S. stipitis.