CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus
ABSTRACT: Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to rel...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-08-01
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| Series: | Journal of Dairy Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S002203022200337X |
| _version_ | 1828784992652623872 |
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| author | Linghui Kong Zhiqiang Xiong Xin Song Yongjun Xia Lianzhong Ai |
| author_facet | Linghui Kong Zhiqiang Xiong Xin Song Yongjun Xia Lianzhong Ai |
| author_sort | Linghui Kong |
| collection | DOAJ |
| description | ABSTRACT: Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus. |
| first_indexed | 2024-12-11T23:41:36Z |
| format | Article |
| id | doaj.art-3beb7364784b4397b7ed817a96b71e4c |
| institution | Directory Open Access Journal |
| issn | 0022-0302 |
| language | English |
| last_indexed | 2024-12-11T23:41:36Z |
| publishDate | 2022-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Dairy Science |
| spelling | doaj.art-3beb7364784b4397b7ed817a96b71e4c2022-12-22T00:45:43ZengElsevierJournal of Dairy Science0022-03022022-08-01105864996512CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilusLinghui Kong0Zhiqiang Xiong1Xin Song2Yongjun Xia3Lianzhong Ai4Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China; School of Pharmacy (School of Enology), Binzhou Medical University, Yantai, 264003, Shandong Province, ChinaShanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaShanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaShanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, ChinaShanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China; Corresponding authorABSTRACT: Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.http://www.sciencedirect.com/science/article/pii/S002203022200337XCRISPR interferencemultiplex gene repressionexopolysaccharide biosynthesisuridine diphosphate glucose sugar metabolismStreptococcus thermophilus |
| spellingShingle | Linghui Kong Zhiqiang Xiong Xin Song Yongjun Xia Lianzhong Ai CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus Journal of Dairy Science CRISPR interference multiplex gene repression exopolysaccharide biosynthesis uridine diphosphate glucose sugar metabolism Streptococcus thermophilus |
| title | CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus |
| title_full | CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus |
| title_fullStr | CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus |
| title_full_unstemmed | CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus |
| title_short | CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus |
| title_sort | crispr dcas9 based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in streptococcus thermophilus |
| topic | CRISPR interference multiplex gene repression exopolysaccharide biosynthesis uridine diphosphate glucose sugar metabolism Streptococcus thermophilus |
| url | http://www.sciencedirect.com/science/article/pii/S002203022200337X |
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