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.
Highlights
Enzyme evolution is naturally driven by beneficial mutations that are selected over many generations, but this inherently slow and random process does not necessarily produce optimally adapted enzymes, especially for new environments or applications [1,2]
Similar to certain other prokaryotic THI4s previously characterized by our group [39], Thermovibrio ammonificans THI4 (TaTHI4) has only low complementing activity in an E. coli thiazole (∆thiG) auxotrophic strain and prefers anaerobic, high-sulfide conditions, making it ill-suited for function in plant cells
Yeast THI4 was chosen for OrthoRep because, as a suicide enzyme, it requires a high level of expression to complement a thiazole auxotroph and represents a severe test of the expression system
Summary
Enzyme evolution is naturally driven by beneficial mutations that are selected over many generations, but this inherently slow and random process does not necessarily produce optimally adapted enzymes, especially for new environments or applications [1,2]. SynBio lies at the intersection between discovery-driven biology and goal-oriented engineering and brings a fresh perspective to plant improvement by adding to the breeding toolbox [6,7]. SynBio has enormous potential in enzyme engineering, ranging from improving existing catalytic activities to creating new ones [7,8,9]. Enzyme engineering in the 1980s focused on rational (re)design by site-specific mutagenesis [10]. A classic example of the challenges of enzyme improvement by site-specific mutation is Rubisco, ribulose-1, 5-bisphosphate carboxylase/oxygenase [12]. It is close to impossible to predict an optimal set of mutations to maximize catalytic efficiency
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