Applications of Protein Engineering and Directed Evolution in Plant Research

Engqvist, Martin K. M.; Rabe, Kersten S.

doi:10.1104/pp.18.01534

Cited by 62 publications

(25 citation statements)

References 124 publications

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“…As the pET28 and pET11a plasmids (for expression of Rubisco and chaperonins, respectively) share the pBR322 origin of replication, the long‐term coexistence of these plasmids may result in variation of copy number and thus fluctuations in expression levels . Thus, applications sensitive to fluctuations of Rubisco expression, such as directed evolution screening or metabolic engineering , may require the use of compatible plasmids.…”

Section: Discussionmentioning

confidence: 99%

Improved recombinant expression and purification of functional plant Rubisco

et al. 2019

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Improving the performance of the key photosynthetic enzyme Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C‐terminal hexahistidine‐tag further improved assembly, resulting in a ~ 12‐fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco.

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Section: Discussionmentioning

confidence: 99%

Improved recombinant expression and purification of functional plant Rubisco

et al. 2019

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“…SynBio has enormous potential in enzyme engineering, ranging from improving existing catalytic activities to creating new ones [7][8][9]. Early enzyme engineering in the 1980s focused on rational (re)design by site-specific mutagenesis [10]. However, evolving enzymes through semi-rational mutagenesis depends on knowledge of structure-function relationships that is often unavailable [11] and is time-and labor-intensive, particularly when multiple mutations are needed to achieve the desired improvement.…”

Section: Introductionmentioning

confidence: 99%

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

García‐García

Joshi

Patterson

et al. 2020

Life

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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.

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Section: Introductionmentioning

confidence: 99%

Potential For Applying Continuous Directed Evolution To Plant Enzymes

García‐García

Joshi

Patterson

et al. 2020

Preprint

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SUMMARYPlant 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 faster 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.

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Applications of Protein Engineering and Directed Evolution in Plant Research

Cited by 62 publications

References 124 publications

Improved recombinant expression and purification of functional plant Rubisco

Improved recombinant expression and purification of functional plant Rubisco

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

Potential For Applying Continuous Directed Evolution To Plant Enzymes

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