دورية أكاديمية
Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.
| العنوان: | Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production. |
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| المؤلفون: | Sun X; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Gao F; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Fan C; Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Jinan, China., Yang S; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Zhao T; Institute of Microbiology, Chinese Academy of Sciences, Beijing, China., Tu T; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Luo H; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Yao B; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Huang H; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China., Su X; State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. |
| المصدر: | Applied and environmental microbiology [Appl Environ Microbiol] 2024 Jul 24; Vol. 90 (7), pp. e0208223. Date of Electronic Publication: 2024 Jun 20. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7605801 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-5336 (Electronic) Linking ISSN: 00992240 NLM ISO Abbreviation: Appl Environ Microbiol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, American Society for Microbiology. |
| مواضيع طبية MeSH: | Cellulase*/genetics , Cellulase*/metabolism , RNA Interference* , Hypocreales*/genetics , Hypocreales*/metabolism, Fungal Proteins/genetics ; Fungal Proteins/metabolism ; Gene Expression Regulation, Fungal ; Genetic Engineering/methods |
| مستخلص: | Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes ( tre49304 , tre120391 , and tre123541 ) were identified to affect cellulase expression in T. reesei . Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain.IMPORTANCEGenetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli . Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens , a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites. Competing Interests: The authors declare no conflict of interest. |
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| معلومات مُعتمدة: | 2021YFC2100204 MOST | National Key Research and Development Program of China (NKPs); BX20220347, 2022M723448 China Postdoctoral Science Foundation (China Postdoctoral Foundation Project); 32302785 MOST | National Natural Science Foundation of China (NSFC); CAAS-ZDRW202305 CAAS | Agricultural Science and Technology Innovation Program (ASTIP); CARS-41 China Agricultural Research System (CARS) of MOF and MARA |
| فهرسة مساهمة: | Keywords: Humicola insolens; RNA interference; Trichoderma reesei; genome engineering; suppression subtractive hybridization |
| المشرفين على المادة: | EC 3.2.1.4 (Cellulase) 0 (Fungal Proteins) |
| SCR Organism: | Trichoderma reesei |
| تواريخ الأحداث: | Date Created: 20240620 Date Completed: 20240724 Latest Revision: 20240726 |
| رمز التحديث: | 20240726 |
| مُعرف محوري في PubMed: | PMC11267940 |
| DOI: | 10.1128/aem.02082-23 |
| PMID: | 38899886 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1098-5336 |
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| DOI: | 10.1128/aem.02082-23 |