Protein and pathway engineering of microbial aromatic amino acid metabolism for diverse applications
Creators: name: Reed, Kevin Bennett
Publisher: name: The University of Texas at Austin
Year: 2022
Subjects: Directed evolution
Metabolic engineering
Protein engineering
Aromatic amino acids
Herbicide resistance
Halogenated products
Biocatalysis

description: Microbes are extraordinary chassis for the evolution of proteins with tailored functions and for the sustainable bio-production of societally relevant chemicals. Presented herein are two vignettes exemplifying the directed evolution and metabolic engineering of aromatic amino acid pathways towards two disparate outcomes. In the first study, a new approach to select for dual-trait EPSP synthase enzymes in S. cerevisiae is described. Herein we present a directed evolution strategy to select for both glyphosate resistance and improved catalytic properties (i.e. k [subscript cat] /K [subscript m]) simultaneously in S. cerevisiae. We first created a growth-based selection by knocking out yeast’s native EPSP synthase function and requiring flux through the shikimate pathway to proceed via a heterologously expressed wild-type Z. mays EPSPS (ZmEPSPS). Through further characterization, we observe a grow deficit when a common EPSPS mutant T102I, P106S (TIPS) is expressed, showcasing that this plant property can be recapitulated in yeast. We then sought to evolve a more optimal EPSPS mutant through an oscillating selection protocol, which alternated between high and low glyphosate concentration stages, selecting for glyphosate tolerance and improved catalytic properties, respectively. Through multiple growth stages, we discover a novel EPSPS mutant that grows better in glyphosate and shows improved enzyme kinetics compared to the starting point, EPSPS TIPS. We envision this system being used to further explore the trade-off between glyphosate resistance and enzymatic efficiency through more complex directed evolution approaches. Altogether, this study highlights the potential for dynamic selection criteria to tune proteins towards a specific phenotype of interest. In the second study, the bioproduction of a diverse suite of tryptophan-derived halogenated products is realized. Using a variety of engineering approaches, we have created a platform to produce a large diversity of halogenated tryptophan derivatives in Escherichia coli. First, through metabolic engineering, we have engineered Escherichia coli to produce high quantities near gram per liter scale (between 0.3 and 0.7 g/L) of six halogenated tryptophan variants de novo from glucose in flask fermentations. Next, we optimize a transcription factor-based biosensor screen that can be used to both evolve halogenases towards improved function and act to facilitate rapid bioprospecting of currently undiscovered tryptophan halogenases. We subsequently introduce multiple downstream enzymes, encompassing a variety of biologically available “reaction centers” of tryptophan that are accessible through enzymatic means to generate a greater diversity of halogenated products, culminating in the de novo production of 26 distinct halogenated molecules via one-pot co-culture fermentations. Lastly, we develop a high-throughput biosensor-directed evolution scheme to screen for downstream enzyme variants that are capable of selectively converting halogenated tryptophan, thereby opening the door to new-to-nature tryptophan derivatives. This work as a whole takes a significant step towards a future where high-value halogenated chemicals can be produced sustainably using microbial factories.