Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (8): 198-205.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1482

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Editing pyrG Gene of Monascus by CRISPR/Cas 9 and Its Effects on Secondary Metabolism

TANG Guang-fu(), GUI Yan-ling, MAN Hai-qiao, ZHAO Jie-hong()   

  1. Key Lab of Pharmacognostics of Guizhou Province,Guizhou University of Traditional Chinese Medicine,Guiyang 550025
  • Received:2021-11-29 Online:2022-08-26 Published:2022-09-14
  • Contact: ZHAO Jie-hong E-mail:1838921871@qq.com;105798711@qq.com

Abstract:

In order to study the effects of pyrG gene of monascus on its secondary metabolism,the monascus chassis strain with Cas9 was obtained by agrobacterium-mediated genetic transformation. Then the directed-editing sgRNA of pyrG gene were designed and synthesized in vitro. After genetic transformation of protoplast of chassis strain,4 mutant strains were screened out,including 2 strains of single-base deleted mutation and 2 strains of double-base deleted mutation,analyzed by resistant on 5-FOA,PCR amplification,T7EI digestion and sequencing. Comparative analysis showed that the content of lovastatin of the wild-type strain significantly increased,and the content of citrinin significantly decreased when cultured in the medium supplemented with uracil. The contents of lovastatin and citrinin of pyrG mutants increased. The results showed that uracil nucleoside and pyrG gene had regulatory effects on secondary metabolism of monascus. It provides reference for functional gene research and secondary metabolism regulation of monascus.

Key words: CRISPR/Cas 9, Monascus purpureus, PyrG mutant strain, protoplast, chassis strain