A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum
ABSTRACT Clostridium pasteurianum is a microorganism for production of 1,3‐propanediol (1,3‐PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modificatio...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley-VCH
2025-01-01
|
| Series: | Engineering in Life Sciences |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/elsc.202400026 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832581973660401664 |
|---|---|
| author | Tom Nguyen Luca W. G. Meleski Minu P. Belavatta Sivasubramanian Gurumoorthi Chijian Zhang Anna‐Lena Heins An‐Ping Zeng |
| author_facet | Tom Nguyen Luca W. G. Meleski Minu P. Belavatta Sivasubramanian Gurumoorthi Chijian Zhang Anna‐Lena Heins An‐Ping Zeng |
| author_sort | Tom Nguyen |
| collection | DOAJ |
| description | ABSTRACT Clostridium pasteurianum is a microorganism for production of 1,3‐propanediol (1,3‐PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR‐Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3‐PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3‐butanediol (2,3‐BDO) production of up to 0.48 g L−1, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain. |
| format | Article |
| id | doaj-art-990a9c4d80d742f091cc7d3d3517c277 |
| institution | Kabale University |
| issn | 1618-0240 1618-2863 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley-VCH |
| record_format | Article |
| series | Engineering in Life Sciences |
| spelling | doaj-art-990a9c4d80d742f091cc7d3d3517c2772025-01-30T06:40:30ZengWiley-VCHEngineering in Life Sciences1618-02401618-28632025-01-01251n/an/a10.1002/elsc.202400026A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium PasteurianumTom Nguyen0Luca W. G. Meleski1Minu P. Belavatta2Sivasubramanian Gurumoorthi3Chijian Zhang4Anna‐Lena Heins5An‐Ping Zeng6Institute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyHamburgGermanyABSTRACT Clostridium pasteurianum is a microorganism for production of 1,3‐propanediol (1,3‐PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR‐Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3‐PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3‐butanediol (2,3‐BDO) production of up to 0.48 g L−1, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.https://doi.org/10.1002/elsc.202400026Clostridium pasteurianumconsecutive genome engineeringCRISPR‐Casmetabolic engineeringsubstrate utilization |
| spellingShingle | Tom Nguyen Luca W. G. Meleski Minu P. Belavatta Sivasubramanian Gurumoorthi Chijian Zhang Anna‐Lena Heins An‐Ping Zeng A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum Engineering in Life Sciences Clostridium pasteurianum consecutive genome engineering CRISPR‐Cas metabolic engineering substrate utilization |
| title | A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum |
| title_full | A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum |
| title_fullStr | A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum |
| title_full_unstemmed | A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum |
| title_short | A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum |
| title_sort | consecutive genome engineering method reveals a new phenotype and regulation of glucose and glycerol utilization in clostridium pasteurianum |
| topic | Clostridium pasteurianum consecutive genome engineering CRISPR‐Cas metabolic engineering substrate utilization |
| url | https://doi.org/10.1002/elsc.202400026 |
| work_keys_str_mv | AT tomnguyen aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT lucawgmeleski aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT minupbelavatta aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT sivasubramaniangurumoorthi aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT chijianzhang aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT annalenaheins aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT anpingzeng aconsecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT tomnguyen consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT lucawgmeleski consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT minupbelavatta consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT sivasubramaniangurumoorthi consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT chijianzhang consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT annalenaheins consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum AT anpingzeng consecutivegenomeengineeringmethodrevealsanewphenotypeandregulationofglucoseandglycerolutilizationinclostridiumpasteurianum |