초록
<P><B>Abstract</B><P>Background<P>Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as <I>Aspergillus</I> and <I>Rhizopus</I> species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus <I>Myceliophthora thermophila</I> is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the <I>M. thermophila</I> platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering.</P></P><P>Results<P>In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in <I>M. thermophila</I>, including <I>Mtstk-12</I>, <I>Mtap3m</I>, <I>Mtdsc-1</I> and <I>Mtsah-2</I>. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic gene<I>s</I> showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets <I>Mtstk-12</I>, <I>Mtap3m</I>, <I>Mtdsc-1</I> and <I>Mtsah-2</I> and overexpressing <I>Mtamy1</I> and <I>Mtpga3</I>. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch.</P></P><P>Conclusions<P>Our results showed that these AsCas12a variants worked well for gene deletions in <I>M. thermophila</I>. We successfully constructed the glucoamylase hyper-production strain of <I>M. thermophila</I> by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.</P></P></P>