Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

Development and verification of an FLP/FRT system for gene deletion in Candida amazonensis

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。

[Objective] To develop an FLP/FRT system for gene disruption in Candida amazonensis that can repeatedly use a single selectable marker, and to verify the effectiveness of this system by deleting the PDC gene encoding pyruvate decarboxylase. [Methods] Four promoters（SpXYLp, SpMAL6 p, SpMAL1p and SpGAL1p） from Spathaspora passalidarum and ScGAL1p promoter from Saccharomyces cerevisiae were amplified and fused to the reporter gene of green fluorescent protein（gfpm） to study the regulation under corresponding inducible conditions. A strictly inducible promoter was selected to control the expression of the C. amazonensis-adapted FLP gene（caFLP）, encoding the site-specific recombinase FLP. The promoter-ca FLP fusion fragment was used to ligated with the hphm marker gene that conferred resistance to Hygromycin B, and the ligation product was flanked by direct repeats of the FLP recognition target（FRT）. Then with the addition of the homologous arms, we constructed the PDC deletion cassette（PRFg HRP）. The cassette was transformed into C. amazonensis CBS 12363 and transformants with hphm were derived. When the transformants were incubated into inducible medium, FLP-mediated recombination resulted in the deletion of DNA located between the repeats. [Results] SpMAL1p（induced by maltose） and SpGAL1p（induced by galactose） were identified to be strictly inducible promoters. SpGAL1p was used to regulate the expression of the FLP, and the PDC deletion cassette（PRFgHRP） was constructed and transformed into C. amazonensis successfully. After selection of Hyg-resistant transformant（designated as C. amazonensis PDC01） in which the deletion cassette was inserted into the PDC target gene, FLP expression was induced by growth of the transformant in galactose-containing medium, and Hyg-sensitive transformant in which hphm and ca FLP flippers were excised from the genome was obtained, designated as C. amazonensis PDC02. [Conclusion] It is the first time to construct an FLP/FRT system for gene disruption in C. amazonensis, and we obtained a PDC mutant without resistant marker gene successfully through this system. These research results lay a good foundation for further metabolic engineering of C. amazonensis.