Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

García-García, Jorge D.; Joshi, Jaya; Patterson, Jenelle A.; Trujillo-Rodriguez, Lidimarie; Reisch, Christopher R.; Javanpour, Alex A.; Liu, Chang C.; Hanson, Andrew D.

doi:10.3390/life10090179

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Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Jorge D. García-García

^1,*,†

Jaya Joshi

^1,*,†,

Jenelle A. Patterson

^1,*,

Lidimarie Trujillo-Rodriguez

Christopher R. Reisch

²,

Alex A. Javanpour

³,

Chang C. Liu

^3,4,5 and

Andrew D. Hanson

Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32603, USA

Department of Biomedical Engineering, University of California, Irvine, CA 92617, USA

⁴

Department of Chemistry, University of California, Irvine, CA 92617, USA

⁵

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Life 2020, 10(9), 179; https://doi.org/10.3390/life10090179

Received: 17 August 2020 / Revised: 31 August 2020 / Accepted: 1 September 2020 / Published: 5 September 2020

(This article belongs to the Special Issue Plant Synthetic Biology)

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Abstract

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 Saccharomyces cerevisiae and EvolvR in Escherichia coli, 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. View Full-Text

Keywords: protein engineering; synthetic biology; linear plasmids; error-prone polymerases; CRISPR/Cas9; directed evolution

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García-García, J.D.; Joshi, J.; Patterson, J.A.; Trujillo-Rodriguez, L.; Reisch, C.R.; Javanpour, A.A.; Liu, C.C.; Hanson, A.D. Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study. Life 2020, 10, 179. https://doi.org/10.3390/life10090179

AMA Style

García-García JD, Joshi J, Patterson JA, Trujillo-Rodriguez L, Reisch CR, Javanpour AA, Liu CC, Hanson AD. Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study. Life. 2020; 10(9):179. https://doi.org/10.3390/life10090179

Chicago/Turabian Style

García-García, Jorge D., Jaya Joshi, Jenelle A. Patterson, Lidimarie Trujillo-Rodriguez, Christopher R. Reisch, Alex A. Javanpour, Chang C. Liu, and Andrew D. Hanson. 2020. "Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study" Life 10, no. 9: 179. https://doi.org/10.3390/life10090179

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