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Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system / 生物工程学报
Chinese Journal of Biotechnology ; (12): 472-481, 2019.
Artigo em Chinês | WPRIM | ID: wpr-771360
ABSTRACT
Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Engenharia Genética / Recombinação Homóloga / Sistemas CRISPR-Cas / Edição de Genes Idioma: Chinês Revista: Chinese Journal of Biotechnology Ano de publicação: 2019 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Engenharia Genética / Recombinação Homóloga / Sistemas CRISPR-Cas / Edição de Genes Idioma: Chinês Revista: Chinese Journal of Biotechnology Ano de publicação: 2019 Tipo de documento: Artigo