Design and engineering of multi-step (bio)catalytic systems

Nature has been a perpetual source of inspiration for biochemists. It is not only the vast diversity of compounds that living beings can create, but also the extraordinary strategies of synthesis deployed. Evidently, the catalysts used by living beings -enzymes- are key to nature’s synthesis strategies. Biocatalysis is undoubtfully one of the most invaluable gifts given by nature to flourish the development of green chemical and pharmaceutical industries. With the development of protein and metabolic engineering tools and strategies, more and more enzymes have been used in the industries to improve the chemical processing; and microbes such as Escherichia coli and Saccharomyces cerevisiae have been engineered to produce a wide variety of value-added and bulk chemicals to replace traditional chemical synthesis. However, researchers have just explored the tip of the iceberg in the biocatalysis area. Proteins with new catalytic functionality should be discovered or engineered to broaden current biotransformation boundaries. New in vitro enzymatic or chemoenzymatic cascade reactions need to be designed and optimized to realize stronger synthetic power and more stable systems. Metabolic networks of traditional or new microorganisms should be largely rewired to meet the manufacturing standards. In this work, I aimed at designing and engineering multi-step (bio) catalytic systems for selective synthesis of value-added chemicals. Microorganisms synthesize complex molecules from simple substrates by a series of enzymes working cooperatively. Inspired by how aromatic polyketides are synthesized by the teamwork between enzymes, I sought to couple biocatalysis with organometallic catalysis, two distinct catalytic disciplines, in one pot to realize synthetic power that cannot be achieved by either of them. I first developed a modular, one-pot, sequential chemoenzymatic system for the formal enantioselective construction of C-C bond in 2-aryl 1,4-dicarbonyl compounds. This sequence comprises a rhodium-catalyzed diazocoupling that provides >9:1 selectivity for heterocoupling of two diazoesters and a reduction mediated by an ene-reductase (ER), which occurs in up to 99% enantiomeric excess (ee). The high yield and enantioselectivity of this system were resulted from the preferential generation of an (E)-alkene from the diazo coupling reaction and selective reduction of the (E)-alkene in a mixture of (E) and (Z) isomers by the ER. This work demonstrates the benefit of combining organometallic and enzymatic catalysis to create unusual overall transformations that do not require the isolation and purification of intermediates. To make the system works better on a broader range of substrates, I later developed a new class of cooperative chemoenzymatic reactions that combine photocatalysts that isomerize alkenes with ene-reductases that reduce carbon-carbon double bonds to generate valuable enantioenriched products. I demonstrated that this method enables the stereoconvergent reduction of E/Z mixtures of alkenes or reduction of the unreactive stereoisomer of an alkene in yields and ee’s that match those obtained from the reduction of the pure, more reactive isomer. This new cooperative system overcomes the limitations of both individual catalysts and affords a range of synthetically valuable and biologically active enantioenriched compounds. More generally, these results illustrate the value of driving a chemical reaction with light to ensure compatibility between the chemical and enzymatic catalysts. In vitro biocatalytic reaction normally has poor tolerance to harsh conditions such as low pH or high substrate concentrations. Cells membrane is natural compartmentalization and protects the enzymes from extracellular inhibitors. In addition, cell factories-based production provides an attractive alternative to chemical synthesis of value-added chemicals. I also worked on engineering a S. cerevisiae strain as a whole-cell catalyst for L-lactic acid overproduction in industrially preferred low pH environment (pH 3) by metabolic engineering and genome-wide engineering methods. In addition, to establish an automated cellular engineering platform, I developed a growth-based L-lactic acid biosensor and an automated quantification assay by BioProfile Analyzer