Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230027, China.
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Microb Cell Fact. 2023 Aug 11;22(1):150. doi: 10.1186/s12934-023-02149-4.
Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering.
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 M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. 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 genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. 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.
Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila 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.
葡糖淀粉酶是食品和生物燃料工业中淀粉糖化的重要酶，主要由嗜温真菌如曲霉属和根霉属产生。与使用嗜温菌的发酵系统相比，来自嗜热真菌的酶可以节省发酵能源和降低成本。嗜热真菌嗜热毁丝霉是一种工业上用于在 45°C 最佳生长时从生物质中生产酶和生物基化学品的真菌。本研究旨在通过拓宽 AsCas12a 变体的基因组靶向范围、鉴定关键候选基因和菌株工程来构建产葡糖淀粉酶的嗜热毁丝霉平台。
在这项研究中，为了增加基因组靶向范围，我们通过工程改造携带 S542R/K607R 和 S542R/K548V/N552R 突变的两种 AsCas12a 变体来升级 CRISPR-Cas12a 介导的技术。使用工程化的 AsCas12a 变体，我们删除了鉴定出的参与嗜热毁丝霉葡糖淀粉酶表达和分泌的关键因素，包括 Mtstk-12、Mtap3m、Mtdsc-1 和 Mtsah-2。与野生型菌株 MtWT 相比，四个靶标的缺失导致分泌和葡糖淀粉酶活性分别提高了 1.87 倍和 1.85 倍以上。转录水平的主要淀粉分解基因在缺失突变体中显著增加。通过遗传工程这些靶标 Mtstk-12、Mtap3m、Mtdsc-1 和 Mtsah-2 以及过表达 Mtamy1 和 Mtpga3，从我们之前的菌株 MtYM6 中生成了产葡糖淀粉酶的 MtGM12 菌株。MtGM12 中的总分泌蛋白和淀粉分解酶活性分别比 MtWT 高约 35.6 倍和 51.9-55.5 倍。转录谱分析显示，MtGM12 中的淀粉分解基因表达水平明显高于 MtWT。更有趣的是，当在淀粉上培养时，MtGM12 表现出主要的短而突出的菌丝，粗糙内质网增殖，大量线粒体、分泌小泡和液泡。
我们的结果表明，这些 AsCas12a 变体在嗜热毁丝霉中很好地进行了基因缺失。我们通过合理的重新设计和转录调控及分泌途径的工程改造，成功构建了产葡糖淀粉酶的嗜热毁丝霉高产菌株。这种靶向工程策略将非常有助于提高工业真菌菌株，并促进形态工程以提高酶产量。
