Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study
Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic p...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-09-01
|
| Series: | Life |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2075-1729/10/9/179 |
| _version_ | 1797554521086361600 |
|---|---|
| author | Jorge D. García-García Jaya Joshi Jenelle A. Patterson Lidimarie Trujillo-Rodriguez Christopher R. Reisch Alex A. Javanpour Chang C. Liu Andrew D. Hanson |
| author_facet | Jorge D. García-García Jaya Joshi Jenelle A. Patterson Lidimarie Trujillo-Rodriguez Christopher R. Reisch Alex A. Javanpour Chang C. Liu Andrew D. Hanson |
| author_sort | Jorge D. García-García |
| collection | DOAJ |
| description | Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in <i>Saccharomyces cerevisiae</i> and EvolvR in <i>Escherichia coli</i>, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering. |
| first_indexed | 2024-03-10T16:33:20Z |
| format | Article |
| id | doaj.art-e0a2d6cc0928450eac6aaffeed498ee0 |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-10T16:33:20Z |
| publishDate | 2020-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Life |
| spelling | doaj.art-e0a2d6cc0928450eac6aaffeed498ee02023-11-20T12:42:01ZengMDPI AGLife2075-17292020-09-0110917910.3390/life10090179Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory StudyJorge D. García-García0Jaya Joshi1Jenelle A. Patterson2Lidimarie Trujillo-Rodriguez3Christopher R. Reisch4Alex A. Javanpour5Chang C. Liu6Andrew D. Hanson7Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USAHorticultural Sciences Department, University of Florida, Gainesville, FL 32611, USAHorticultural Sciences Department, University of Florida, Gainesville, FL 32611, USADepartment of Microbiology and Cell Science, University of Florida, Gainesville, FL 32603, USADepartment of Microbiology and Cell Science, University of Florida, Gainesville, FL 32603, USADepartment of Biomedical Engineering, University of California, Irvine, CA 92617, USADepartment of Biomedical Engineering, University of California, Irvine, CA 92617, USAHorticultural Sciences Department, University of Florida, Gainesville, FL 32611, USAPlant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in <i>Saccharomyces cerevisiae</i> and EvolvR in <i>Escherichia coli</i>, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.https://www.mdpi.com/2075-1729/10/9/179protein engineeringsynthetic biologylinear plasmidserror-prone polymerasesCRISPR/Cas9directed evolution |
| spellingShingle | Jorge D. García-García Jaya Joshi Jenelle A. Patterson Lidimarie Trujillo-Rodriguez Christopher R. Reisch Alex A. Javanpour Chang C. Liu Andrew D. Hanson Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study Life protein engineering synthetic biology linear plasmids error-prone polymerases CRISPR/Cas9 directed evolution |
| title | Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study |
| title_full | Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study |
| title_fullStr | Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study |
| title_full_unstemmed | Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study |
| title_short | Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study |
| title_sort | potential for applying continuous directed evolution to plant enzymes an exploratory study |
| topic | protein engineering synthetic biology linear plasmids error-prone polymerases CRISPR/Cas9 directed evolution |
| url | https://www.mdpi.com/2075-1729/10/9/179 |
| work_keys_str_mv | AT jorgedgarciagarcia potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT jayajoshi potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT jenelleapatterson potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT lidimarietrujillorodriguez potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT christopherrreisch potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT alexajavanpour potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT changcliu potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy AT andrewdhanson potentialforapplyingcontinuousdirectedevolutiontoplantenzymesanexploratorystudy |