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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

  • Metabolic Engineering and Synthetic Biology - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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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.

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Author notes

  1. Junqing Wang and Jian Peng, the two first authors, contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250353, Shandong, People’s Republic of China

    Junqing Wang, Jian Peng, Han Fan, Xiang Xiu, Le Xue, Lei Wang, Jing Su, Xiaohui Yang & Ruiming Wang

  2. Key Laboratory of Shandong Microbial Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250353, Shandong, People’s Republic of China

    Junqing Wang, Jian Peng, Han Fan, Xiang Xiu, Le Xue, Lei Wang, Jing Su, Xiaohui Yang & Ruiming Wang

Authors
  1. Junqing Wang
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  2. Jian Peng
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  3. Han Fan
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  4. Xiang Xiu
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  5. Le Xue
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  6. Lei Wang
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  7. Jing Su
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  8. Xiaohui Yang
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  9. Ruiming Wang
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Correspondence to Junqing Wang.

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Wang, J., Peng, J., Fan, H. et al. Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids. J Ind Microbiol Biotechnol 45, 971–981 (2018). https://doi.org/10.1007/s10295-018-2074-9

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  • DOI: https://doi.org/10.1007/s10295-018-2074-9

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