Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing
Acarbose, a pseudo-tetrasaccharide produced by <i>Actinoplanes</i> sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-12-01
|
| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/13/1/1 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832587908743168000 |
|---|---|
| author | Saskia Dymek Lucas Jacob Alfred Pühler Jörn Kalinowski |
| author_facet | Saskia Dymek Lucas Jacob Alfred Pühler Jörn Kalinowski |
| author_sort | Saskia Dymek |
| collection | DOAJ |
| description | Acarbose, a pseudo-tetrasaccharide produced by <i>Actinoplanes</i> sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in <i>Actinoplanes</i> sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into <i>Actinoplanes</i> sp. These vectors were validated by silencing the known acarbose biosynthesis genes <i>acbB</i> and <i>acbV</i>, as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein–DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production. |
| format | Article |
| id | doaj-art-260bf14fd30c4c89b25f03bab0716fe6 |
| institution | Kabale University |
| issn | 2076-2607 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj-art-260bf14fd30c4c89b25f03bab0716fe62025-01-24T13:42:14ZengMDPI AGMicroorganisms2076-26072024-12-01131110.3390/microorganisms13010001Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi SilencingSaskia Dymek0Lucas Jacob1Alfred Pühler2Jörn Kalinowski3Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, GermanyMicrobial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, GermanySenior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, GermanyMicrobial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, GermanyAcarbose, a pseudo-tetrasaccharide produced by <i>Actinoplanes</i> sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in <i>Actinoplanes</i> sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into <i>Actinoplanes</i> sp. These vectors were validated by silencing the known acarbose biosynthesis genes <i>acbB</i> and <i>acbV</i>, as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein–DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production.https://www.mdpi.com/2076-2607/13/1/1<i>Actinoplanes</i>acarboseCRISPR interferenceCRISPRilibrarytranscriptional regulators |
| spellingShingle | Saskia Dymek Lucas Jacob Alfred Pühler Jörn Kalinowski Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing Microorganisms <i>Actinoplanes</i> acarbose CRISPR interference CRISPRi library transcriptional regulators |
| title | Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing |
| title_full | Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing |
| title_fullStr | Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing |
| title_full_unstemmed | Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing |
| title_short | Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in <i>Actinoplanes</i> sp. SE50/110 Using CRISPRi Silencing |
| title_sort | targeting transcriptional regulators affecting acarbose biosynthesis in i actinoplanes i sp se50 110 using crispri silencing |
| topic | <i>Actinoplanes</i> acarbose CRISPR interference CRISPRi library transcriptional regulators |
| url | https://www.mdpi.com/2076-2607/13/1/1 |
| work_keys_str_mv | AT saskiadymek targetingtranscriptionalregulatorsaffectingacarbosebiosynthesisiniactinoplanesispse50110usingcrisprisilencing AT lucasjacob targetingtranscriptionalregulatorsaffectingacarbosebiosynthesisiniactinoplanesispse50110usingcrisprisilencing AT alfredpuhler targetingtranscriptionalregulatorsaffectingacarbosebiosynthesisiniactinoplanesispse50110usingcrisprisilencing AT jornkalinowski targetingtranscriptionalregulatorsaffectingacarbosebiosynthesisiniactinoplanesispse50110usingcrisprisilencing |