Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products,abundant thermophilic enzymes,and promising fermentation capacities.However,efficient and versatile genome editing ...Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products,abundant thermophilic enzymes,and promising fermentation capacities.However,efficient and versatile genome editing tools are not available for this species.In this study,we developed an efficient genome editing tool for T.thermophilus HB27 based on its endogenous type IB,I-C,and III-A/B CRISPR-Cas systems.First,we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in T.thermophilus HB27.We found that genomic manipulations such as gene deletion,mutation,and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery.We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths.Additionally,we developed a reporter gene system for T.thermophilus based on a heat-stableβ-galactosidase gene TTP0042,and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.展开更多
基金the National Natural Science Foundation of China(32170096)the Fundamental Research Funds for the Central Universities(2662022S KPY001)Cooperation Fund of Huazhong Agricultural University-Agricultural Genomics Institute at Shenzhen(CAAS)(SZYJY2021002).
文摘Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products,abundant thermophilic enzymes,and promising fermentation capacities.However,efficient and versatile genome editing tools are not available for this species.In this study,we developed an efficient genome editing tool for T.thermophilus HB27 based on its endogenous type IB,I-C,and III-A/B CRISPR-Cas systems.First,we systematically characterized the DNA interference capabilities of the different types of the native CRISPR-Cas systems in T.thermophilus HB27.We found that genomic manipulations such as gene deletion,mutation,and in situ tagging could be easily implemented by a series of genome-editing plasmids carrying an artificial self-targeting mini-CRISPR and a donor DNA responsible for the recombinant recovery.We also compared the genome editing efficiency of different CRISPR-Cas systems and the editing plasmids with donor DNAs of different lengths.Additionally,we developed a reporter gene system for T.thermophilus based on a heat-stableβ-galactosidase gene TTP0042,and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.