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Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids

  • Junqing Wang
  • Jian Peng
  • Han Fan
  • Xiang Xiu
  • Le Xue
  • Lei Wang
  • Jing Su
  • Xiaohui Yang
  • Ruiming Wang
Metabolic Engineering and Synthetic Biology - Original Paper
  • 68 Downloads

Abstract

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH–cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.

Keywords

Candida tropicalis Markerless genome editing system Suicide plasmid Long-chain dicarboxylic acid Fermentation 

Notes

Acknowledgements

This work was supported by the Shandong Provincial Natural Science Foundation (ZR2016CB04), Major Program of National Natural Science Foundation of Shandong (ZR2017ZB0208), National Science Foundation of China (31801527) and Independent Innovation and Achievement Transformation Project in Shandong Province (201422CX02602). The authors are grateful to Prof. Minchen Wu (Wuxi Medical School, Jiangnan University) for providing technical assistance.

Supplementary material

10295_2018_2074_MOESM1_ESM.doc (2.5 mb)
Supplementary material 1 (DOC 2541 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Junqing Wang
    • 1
    • 2
  • Jian Peng
    • 1
    • 2
  • Han Fan
    • 1
    • 2
  • Xiang Xiu
    • 1
    • 2
  • Le Xue
    • 1
    • 2
  • Lei Wang
    • 1
    • 2
  • Jing Su
    • 1
    • 2
  • Xiaohui Yang
    • 1
    • 2
  • Ruiming Wang
    • 1
    • 2
  1. 1.State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences)JinanPeople’s Republic of China
  2. 2.Key Laboratory of Shandong Microbial Engineering, Qilu University of Technology (Shandong Academy of Sciences)JinanPeople’s Republic of China

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