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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1482

Abstract

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

TANG Guang-fu, GUI Yan-ling, MAN Hai-qiao, ZHAO Jie-hong. Editing pyrG Gene of Monascus by CRISPR/Cas 9 and Its Effects on Secondary Metabolism[J]. Biotechnology Bulletin, 2022, 38(8): 198-205.

Figures/Tables 8

Table 1 Primers and sgRNA sequences for PCR detection

引物名称 Primer name	引物或sgRNA序列 Primer or sgRNA sequence（5'-3'）	退火温度 Annea-ling temperature/℃	靶标序列长度 Target sequence length/bp
Cas9-F	GTTTAAGGTCCTGGGCAACA	55	312
Cas9-R	ATCGTGGGGTACTTCTCGTG	55	312
PyrGgr-F	GTTGAAACGAACCCCAGCAC	57	380
pyrGgr-R	CCACATCGACATCCTCTCCG	57	380
sgRNA	TTAATACGACTCACTATAGGGggcttgaagttcctgcgttgGTTTTAGAGCTAGAAATA	/	/

Fig. 1 Target sequence of sgRNA for gene pyrG The arrow indicates the position of the pyrGgr primer pair

Fig. 2 Standard curve of citrinin

Fig. 3 Transformation of Cas 9 into monascus mediated by A. tumefaciens A：PCR detection of A. tumefaciens. B：Co-culture of A. tumefaciens and monascus. C：PCR detection of the Cas 9 of monascus chassis strain. 1-8：Different strains. M：DL1000 DNA marker

Fig. 4 Transformation of monascus protoplasts by sgRNA in vitro A：Inhibition of 5-FOA on monascus. B：Electrophoresis of the sgRNA. C：Prepared protoplasts. D：Screening resistant monascus

Fig. 5 Variation analysis of pyrG gene of monascus mutant A：Part nested peak sequence. B：PCR detection of pyrG gene in some strains. C：PCR product cutted by T7EI. D：Mutation loci of pyrG gene in some strains. MU：Mutant. WT：Wild-type. “+”：With DNA. “-”：Without DNA. Black box：Refers to the PAM site. Grey background：Refers to the sgRNA target sequence

Fig. 6 Colony morphology and microscopic characteristics of partial mutant strains Magnification of micrographs 400×

Fig. 7 Effect of uracil riboside on the chemical composition content of different strains in medium A：Lovastatin absorbance. B：Monascus pigment content. C：Citrinin content. “-”：Not added. “+”：Added. WT：Wild type strain. MU：Mutant strain

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