Construction and application of episomal plasmid in the basidiomycetous yeast <italic style="font-style: italic">Rhodotorula toruloides</italic>

CHEN Qiongqiong; LYU Liting; ZHAO Zongbao

doi:10.12211/2096-8280.2026-045

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Construction and application of episomal plasmid in the basidiomycetous yeast Rhodotorula toruloides

更新时间：2026-08-18

- Construction and application of episomal plasmid in the basidiomycetous yeast Rhodotorula toruloides
- 担子菌门圆红酵母游离型质粒的构建及应用
- Synthetic Biology Journal Pages: 1-18(2026)
- 作者机构：
  
  1.中国科学院大连化学物理研究所生物技术部，辽宁大连 116023
  2.中国科学院大学，北京 100049
  3.大连理工大学生物工程学院，辽宁大连116024
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2026-045
  CLC： Q782
- Received：19 May 2026，
  
  Revised：2026-08-15，
  
  Online First：18 August 2026，
- 稿件说明：
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陈琼琼，吕力婷，赵宗保. 担子菌门圆红酵母游离型质粒的构建及应用［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-045

CHEN Qiongqiong， LYU Liting， ZHAO Zongbao. Construction and application of episomal plasmid in the basidiomycetous yeast Rhodotorula toruloides［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-045
陈琼琼，吕力婷，赵宗保. 担子菌门圆红酵母游离型质粒的构建及应用［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-045 DOI：

CHEN Qiongqiong， LYU Liting， ZHAO Zongbao. Construction and application of episomal plasmid in the basidiomycetous yeast Rhodotorula toruloides［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-045 DOI：

摘要

含自主复制序列（ARSs）的附加体质粒可脱离染色体独立复制，转化效率高、操作简便，已广泛应用于子囊菌酵母遗传改造。然而担子菌酵母普遍缺乏成熟附加体编辑体系，产油菌种圆红酵母（

Rhodotorula toruloides

）亦缺少相关工具。本研究利用

R. toruloides

内源ARS构建游离型质粒系统，并证实该质粒可在无筛选压力下丢失。从酵母细胞中回收完整质粒，进一步验证了其游离型复制特性，表明该系统适用于无痕迭代遗传改造。将该质粒与CRISPR/Cas9编辑系统结合，本研究不仅实现了

CAR2、CRTI

和

NAT

等靶基因的随机突变，还通过同源重组途径分别完成了内源基因敲除与增强型绿色荧光蛋白编码基因（

eGFP

）表达盒的染色体定点整合。依托质粒可自主丢失特性实现筛选标记无痕回收，大幅简化菌株迭代改造操作。综上，该游离型质粒系统是一套灵活且可循环使用的编辑工具，能够有力支撑

R. toruloides

基因功能解析、多基因表达及代谢通路重构等合成生物学研究。

Abstract

Episomal plasmids containing autonomously replicating sequences (ARSs) can replicate independently of the host chromosome

exhibiting high transformation efficiency

simple operation and excellent flexibility

and have been widely applied for the genetic modification of ascomycetous yeasts. However

mature episomal editing platforms remain scarce in basidiomycetous yeasts. The oleaginous yeast

Rhodotorula toruloides

lacks recyclable genetic tools

which hinders gene functional research and targeted metabolic engineering. In this study

a new episomal plasmid system specifically adapted to

R. toruloides

was constructed using an endogenous ARS element. A series of functional validation experiments with long-term continuous passage culture confirmed that the episomal plasmid could be spontaneously eliminated under non-selective culture conditions. Repeated plasmid recovery assays and sequencing identification of intact plasmids from transformed

R. toruloides

cells further verified its stable episomal replication feature

laying a solid foundation for marker-free iterative genetic engineering. To further improve its editing performance

this system was combined with the classical CRISPR/Cas9 system and established a multifunctional genome editing platform in

R. toruloides

. Genetic amplification and sequencing verification confirmed that random indel mutations were generated at several functional loci

including

CAR2

CRTI

and

NAT

. In addition

endogenous homologous recombination was induced by co-transformation with homologous donor templates achieving accurate target gene knockout and site-specific chromosomal integration of the enhanced green fluorescent protein (

eGFP

) expression cassette. Fluorescence observation and molecular detection further confirmed the s

uccessful integration and exogenous gene expression. Benefiting from the self-eliminating property of this episomal plasmid system

selection markers can be seamlessly recycled after each round of genome editing

which significantly simplifies the cumbersome screening and purification procedures required for iterative strain reconstruction. In conclusion

the flexible and recyclable episomal plasmid system constructed in this work offers a stable and multifunctional editing toolkit for

R. toruloides

. It provides reliable technical support for subsequent synthetic biology research

including gene function verification

multi-gene coordinated expression

and precise reconstruction and optimization of complex metabolic pathways.

关键词

Keywords

references

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Construction and application of episomal plasmid in the basidiomycetous yeast Rhodotorula toruloides

DOI：10.12211/2096-8280.2026-045

摘要

Abstract

关键词

Keywords

references