Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.
Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.
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DOI：
10.1186/s12934-023-02149-4
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发表时间：
2023-08-11
影响因子：
6.4
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中科院分区：
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葡萄糖淀粉酶是食品和生物燃料工业中用于淀粉糖化的重要酶，并且主要由嗜温真菌如曲霉属和根霉属物种产生。与中温菌发酵系统相比，嗜热真菌产酶可以节省发酵能量，降低成本。嗜热真菌Myceliophthora thermophila是工业上部署的真菌，在45 °C的最佳生长期间从生物质中产生酶和生物基化学品。本研究旨在构建M.通过拓宽AsCas 12 a变体的基因组靶向范围、鉴定关键候选基因和菌株工程改造，构建了用于葡糖淀粉酶超生产的嗜热菌平台。在这项研究中，为了增加基因组靶向范围，我们通过工程化两种携带突变S542 R/K607 R和S542 R/K548 V/N552 R的AsCas 12 a变体来升级CRISPR-Cas 12 a介导的技术。使用工程化的AsCas 12 a变体，我们删除了鉴定出的参与葡糖淀粉酶在M.嗜热菌，包括Mtstk-12、Mtap 3 m、Mtdsc-1和Mtsah-2。与野生型菌株MtWT相比，四个靶点的缺失导致分泌水平和葡糖淀粉酶活性分别高出1.87倍和1.85倍以上。在缺失突变体中，主要淀粉分解基因的转录水平显着增加。葡糖淀粉酶高产菌株MtGM 12是从我们先前的菌株MtYM 6通过遗传工程改造这些靶标Mtstk-12、Mtap 3 m、Mtdsc-1和Mtsah-2并过表达Mtamy 1和Mtpga 3而产生的。MtGM 12的总蛋白分泌量和淀粉分解酶活性分别是MtWT的35.6倍和51.9 ~ 55.5倍。转录谱分析表明，淀粉分解基因的表达水平显着上调MtGM 12比MtWT。更有趣的是，MtGM 12在淀粉培养基上培养时，主要表现为短而高度膨胀的菌丝，伴随着粗糙ER的增殖和丰富的线粒体、分泌囊泡和空泡。我们的结果表明，这些AsCas 12 a变体在M.嗜热菌成功构建了糖化酶高产菌株M.通过合理的重新设计和工程改造转录调控和分泌途径，这一针对性工程策略将有助于工业真菌菌种的改良和形态工程的推广。在线版本包含补充材料，可通过10.1186/s12934-023-02149-4获得。
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. The online version contains supplementary material available at 10.1186/s12934-023-02149-4.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
5.2
作者：
Karnaouri A;Topakas E;Antonopoulou I;Christakopoulos P
通讯作者：
Christakopoulos P
DOI：
10.1083/jcb.201310021
发表时间：
2014-01-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bonifacino JS
通讯作者：
Bonifacino JS
影响因子：
3.6
作者：
Al-Sheikh, H;Watson, AJ;Archer, DB
通讯作者：
Archer, DB
影响因子：
--
作者：
Meyer, Vera;Basenko, Evelina Y.;Wosten, Han A. B.
通讯作者：
Wosten, Han A. B.