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Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System

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Abstract

Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.

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Abbreviations

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats/Cas9

sgRNAs:

Single-guide RNAs

RGR :

Ribozyme-gRNA-Ribozyme

HH:

Hammerhead

HDV:

Hepatitis delta virus

PKS:

Polyketide synthase

CGMCC:

China General Microbiological Culture Collection Center

HR:

Highly reducing

NR:

Non-reducing

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Acknowledgements

We acknowledge our colleague Dr. Liming Ouyang at ECUST China for her constructive discussion on this work.

Funding

This work was financially subsidized by NOW-MoST Joint Program (2013DFG32630) and partially supported by National Basic Research Program (973 program 2012CB721006).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China

    Guozhi Chen & Ju Chu

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  1. Guozhi Chen
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  2. Ju Chu
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Correspondence to Ju Chu.

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We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with submission to Applied Biochemistry and Biotechnology.

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The authors declare that they have no competing interests.

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Chen, G., Chu, J. Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System. Appl Biochem Biotechnol 188, 1134–1144 (2019). https://doi.org/10.1007/s12010-019-02960-z

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