Potential For Applying Continuous Directed Evolution To Plant Enzymes

Abstract

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.

Continuous directed evolution of a feedback-resistant Arabidopsis arogenate dehydratase in plantized E. coli

“…Function in minimal medium. As eMutaT7 has so far been used only in rich media 4,11 and might be metabolically burdensome enough to prevent growth in minimal media, 5 we first tested its operation in MOPS minimal medium. A ∆ung strain was used as platform; the target was the gene encoding PheS A294G, a tRNA ligase that sensitizes E. coli to p-chlorophenylalanine.…”

“…3,4 Classical directed evolution campaigns can thus only handle relatively few evolutionary cycles and independent evolving populations, and tend to incrementally climb local adaptive peaks rather than explore the fitness landscape more widely because they lack the mutational power needed to cross fitness valleys. [3][4][5] Continuous directed evolution (CDE) systems overcome these limitations of depth and scale by hypermutating the target gene in vivo and, for enzymes, by coupling activity of the target gene to growth of the platform cell. 3,4 CDE systems thus require only serial subculturing because they work by selecting for growth alone, and in principle they can run indefinitely.…”

“…3,4 CDE systems thus require only serial subculturing because they work by selecting for growth alone, and in principle they can run indefinitely. [3][4][5] So far, CDE systems 2 are largely confined to Escherichia coli and yeast, 4 and while progress in CDE in mammalian cells 6 and plants 7 continues, long generation times and small library sizes are enduring obstacles.…”

“…9 This strategy is still at the concept stage. Further, for the E. coli systems, the differences between conditions in plant and prokaryote cells (e.g., metabolic pathway architecture, metabolite levels, redox poise, protein-folding and degradation systems 8 ) and the uncertain durability of the hypermutation machinery in long evolution campaigns 4,5,8 are potential roadblocks. Nor is it clear that E. coli CDE systems work in minimal media, which is critical because many selection schemes require auxotrophs.…”

“…Nor is it clear that E. coli CDE systems work in minimal media, which is critical because many selection schemes require auxotrophs. 5 We therefore undertook a proof-of-concept study in E. coli to evolve a plant arogenate dehydratase (ADT) (EC 4.2.1.91) for resistance to feedback inhibition by phenylalanine. We used eMutaT7, 11 one of several CDE systems whose mutagenesis machinery is a T7RNA polymerase (T7RNAP)-nucleobase deaminase fusion that is directed to the target gene by a T7 promoter.…”