圆红冬孢酵母自主复制序列分离和利用游离型质粒进行基因敲除

Isolation of autonomously replicating sequence and gene knockout using an episomal plasmid in Rhodosporidium toruloides

【目的】与整合型表达载体相比,游离型表达载体通常具有更高的拷贝数以实现目标基因的高强度表达,并且对于DNA操作应用更加方便和灵活。然而,目前的研究尚未确定适用于圆红冬孢酵母的游离型质粒,该酵母外源基因的表达或者基于CRISPR/Cas9的基因组编辑都需要通过整合方式来完成,这也是对其遗传改造进展缓慢的一个重要原因。本研究目的是构建圆红冬孢酵母的游离型质粒,使得其外源基因的表达和基因组编辑更方便省时。【方法】首先对圆红冬孢酵母苯丙氨酸氨裂解酶基因(phenylalanine ammonia-lyase gene,PAL)中可能存在的自主复制序列(autonomously replicating sequences,ARSs)进行挖掘和表征,将该基因及其上下游序列进行分段扩增,构建到带有β-异丙基苹果酸脱氢酶基因(β-isopropyl malate dehydrogenase gene,LEU2)的质粒中,通过电转化的方法导入LEU2基因缺陷的圆红冬孢酵母中,根据转化效率高低鉴定了该酵母的一个ARS。其次,以编码香叶基香叶基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase,GGPPS)的BTS1基因为敲除靶点,将其gRNA构建到基于ARS的游离型质粒中,通过转化子直观的颜色变化来验证该游离型质粒是否成功应用于圆红冬孢酵母的CRISPR/Cas9体系。【结果】本工作鉴定了圆红冬孢酵母的ARS,构建了基于ARS元件的游离型质粒,并将该质粒应用于圆红冬孢酵母CRISPR/Cas9体系,成功实现了基于游离型质粒的基因敲除。【结论】本研究丰富了圆红冬孢酵母现有的工具库,为圆红冬孢酵母的合成生物学应用提供了良好的研究基础和技术支持。

[Objective]Episomal expression vectors typically have higher copy number to achieve strong gene expression than chromosomal expression vectors.Moreover,they are more convenient and flexible for DNA manipulation.However,the episomal plasmids suitable for the application in Rhodosporidium toruloides remain to be determined,and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be achieved by integration,which is a key reason for the slow progress in its genetic modification.Thus,this work aims to construct an episomal plasmid of R.toruloides,which facilitates the expression of heterologous genes and promotes the gene editing in a time-saving manner.[Methods]First,the possible autonomously replicating sequences(ARSs)in the phenylalanine ammonia-lyase gene(PAL)of R.toruloides were mined.Specifically,PAL and its upstream and downstream sequences were amplified in segments and constructed into a plasmid containing theβ-isopropyl malate dehydrogenase gene(LEU2).The recombinant plasmids were then introduced into LEU2-deficient R.toruloides by the electroporation method.An ARS was then identified according to transformation efficiency.Then,the BTS1 gene encoding geranylgeranyl pyrophosphate synthase was selected as the knockout target,and its gRNA was constructed into the episomal plasmid based on the identified ARS.The color change of the transformant was observed to verify whether the episomal plasmid was successfully applied to the CRISPR/Cas9 system of R.toruloides.[Results]In this work,an ARS was identified,based on which an episomal plasmid was constructed and applied to CRISPR/Cas9 editing in R.toruloides.Finally,the episomal plasmid-based gene knockout of R.toruloides was successfully achieved.[Conclusion]This work enriched the existing tool library and provided a research basis and technical support for the application of R.toruloides in synthetic biology.