Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli
Abstract Background Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natura...
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| Format: | Article |
| Language: | English |
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BMC
2019-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-019-1080-6 |
| _version_ | 1819151872576978944 |
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| author | Elke R. Duell Paul M. D’Agostino Nicole Shapiro Tanja Woyke Thilo M. Fuchs Tobias A. M. Gulder |
| author_facet | Elke R. Duell Paul M. D’Agostino Nicole Shapiro Tanja Woyke Thilo M. Fuchs Tobias A. M. Gulder |
| author_sort | Elke R. Duell |
| collection | DOAJ |
| description | Abstract Background Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. Results Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA–D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible Ptet O promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. Conclusions Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways. |
| first_indexed | 2024-12-22T14:40:18Z |
| format | Article |
| id | doaj.art-0dda3d42b2724358acf156204c67637d |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-22T14:40:18Z |
| publishDate | 2019-02-01 |
| publisher | BMC |
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| series | Microbial Cell Factories |
| spelling | doaj.art-0dda3d42b2724358acf156204c67637d2022-12-21T18:22:34ZengBMCMicrobial Cell Factories1475-28592019-02-0118111110.1186/s12934-019-1080-6Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coliElke R. Duell0Paul M. D’Agostino1Nicole Shapiro2Tanja Woyke3Thilo M. Fuchs4Tobias A. M. Gulder5Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technical University of MunichBiosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technical University of MunichDepartment of Energy, Joint Genome InstituteDepartment of Energy, Joint Genome InstituteZIEL Institute for Food & Health, Lehrstuhl für Mikrobielle Ökologie, Department biowissenschaftliche Grundlagen, Technical University of MunichBiosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technical University of MunichAbstract Background Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. Results Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA–D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible Ptet O promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. Conclusions Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.http://link.springer.com/article/10.1186/s12934-019-1080-6SodorifenSerratia plymuthicaTerpenesGenome miningDiPaCHeterologous expression |
| spellingShingle | Elke R. Duell Paul M. D’Agostino Nicole Shapiro Tanja Woyke Thilo M. Fuchs Tobias A. M. Gulder Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli Microbial Cell Factories Sodorifen Serratia plymuthica Terpenes Genome mining DiPaC Heterologous expression |
| title | Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli |
| title_full | Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli |
| title_fullStr | Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli |
| title_full_unstemmed | Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli |
| title_short | Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli |
| title_sort | direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in e coli |
| topic | Sodorifen Serratia plymuthica Terpenes Genome mining DiPaC Heterologous expression |
| url | http://link.springer.com/article/10.1186/s12934-019-1080-6 |
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