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A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola

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Abstract

The traditional homologous recombination (HR) gene-editing method faces problems such as low editing efficiency and absence of marker genes. CRISPR–Cas9-editing efficiency is high and has been widely used in bacteria and yeast. In comparison with CRISPR–Cas9, CRISPR–Cas12a has many outstanding advantages. Here, we report an Acidaminococcus sp. BV3L6 (As) Cas12a-based genome-editing method used for Starmerella bombicola. To demonstrate the high efficiency of the CCMGE system, we verified a counter-selectable marker in S. bombicola, orotidine 5’-phosphate decarboxylase (100% for URA3). We also tested the common gene UDP-glucosyltransferase (100% for UGTA) using a 300 bp donor containing hygromycin expression cassette. This toolkit was further extended to simultaneously edit two genes (18% for UGTA and leu) and three genes (13.8% for UGTA, leu and URA3). The system greatly reduces the screening time for such multi-site editing. Based on the CCMGE system, the PHA (polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase (PHAC). The PHA content and DCW reached 11.8% and 30.1 g/L, respectively. The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.

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Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The work was supported by the 111 Project (No. 111-2-06), National Natural Science Foundation of China (32001064), Key Research and Development Program of China (2021YFC2100102-03), China Postdoctoral Science Foundation (2020M671331), and Postgraduate Research and Practice Innovation Program of Jiangsu Province (No. KYCX20-1807).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China

    Min Zhang, Yibo Shi, Lihua Zhang, Shiying Zhu, Haiquan Yang, Wei Shen, Yuanyuan Xia & Xianzhong Chen

  2. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Min Zhang, Yibo Shi, Lihua Zhang, Shiying Zhu, Haiquan Yang, Wei Shen, Yuanyuan Xia & Xianzhong Chen

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  1. Min Zhang
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  2. Yibo Shi
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  3. Lihua Zhang
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Correspondence to Xianzhong Chen.

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Zhang, M., Shi, Y., Zhang, L. et al. A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola. Syst Microbiol and Biomanuf 2, 665–675 (2022). https://doi.org/10.1007/s43393-022-00093-9

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  • DOI: https://doi.org/10.1007/s43393-022-00093-9

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