Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants
Abstract Background 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 com...
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| Format: | Article |
| Language: | English |
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BMC
2023-08-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-023-02149-4 |
| _version_ | 1797450963400785920 |
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| author | Zhijian Zhu Manyu Zhang Dandan Liu Defei Liu Tao Sun Yujing Yang Jiacheng Dong Huanhuan Zhai Wenliang Sun Qian Liu Chaoguang Tian |
| author_facet | Zhijian Zhu Manyu Zhang Dandan Liu Defei Liu Tao Sun Yujing Yang Jiacheng Dong Huanhuan Zhai Wenliang Sun Qian Liu Chaoguang Tian |
| author_sort | Zhijian Zhu |
| collection | DOAJ |
| description | Abstract Background 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. Results 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. Conclusions 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. |
| first_indexed | 2024-03-09T14:48:05Z |
| format | Article |
| id | doaj.art-272c6c228c7c491cbe4d1526bf053a9b |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-09T14:48:05Z |
| publishDate | 2023-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-272c6c228c7c491cbe4d1526bf053a9b2023-11-26T14:38:09ZengBMCMicrobial Cell Factories1475-28592023-08-0122111710.1186/s12934-023-02149-4Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variantsZhijian Zhu0Manyu Zhang1Dandan Liu2Defei Liu3Tao Sun4Yujing Yang5Jiacheng Dong6Huanhuan Zhai7Wenliang Sun8Qian Liu9Chaoguang Tian10Division of Life Sciences and Medicine, University of Science and Technology of ChinaKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesNational Technology Innovation Center of Synthetic BiologyKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract Background 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. Results 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. Conclusions 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.https://doi.org/10.1186/s12934-023-02149-4Myceliophthora thermophilaGlucoamylaseHyper-productionGenetic engineeringSecretionCRISPR-AsCas12a |
| spellingShingle | Zhijian Zhu Manyu Zhang Dandan Liu Defei Liu Tao Sun Yujing Yang Jiacheng Dong Huanhuan Zhai Wenliang Sun Qian Liu Chaoguang Tian Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants Microbial Cell Factories Myceliophthora thermophila Glucoamylase Hyper-production Genetic engineering Secretion CRISPR-AsCas12a |
| title | Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants |
| title_full | Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants |
| title_fullStr | Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants |
| title_full_unstemmed | Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants |
| title_short | Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants |
| title_sort | development of the thermophilic fungus myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved ascas12a variants |
| topic | Myceliophthora thermophila Glucoamylase Hyper-production Genetic engineering Secretion CRISPR-AsCas12a |
| url | https://doi.org/10.1186/s12934-023-02149-4 |
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