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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2023)

In the last decade, biocatalysis has experienced a surge in both industrial and academic researchefforts, development, and acknowledgement in the form of the Nobel Prize for directed evolution. This attention and popularity is due to the exquisite activity and selectivity of enzymes in addition to their promiscuity, which can be leveraged by the synthetic chemistry through directed evolution. Despite the advancements in directed evolution for the unnatural activity of enzymes, iterative mutagenesis can only augment a native enzyme scaffold to a point. This limitation has been addressed in part by combination of enzymes with artificial transition metal catalysts to produce artificial metalloenzymes (ArMs). The development of ArMs has also experienced a surge in recent years due to advancements in high throughput experimentation. A key limitation to ArMs, however, is that most synthetic metal cofactors are not cell-permeable. The lack of cell permeability limits experimentation with ArMs to manipulations in vitro, which severely hampers efforts to implement ArMs in directed evolution campaigns as well as employing them as part of biosynthetic pathways. In addition, few publications exist that seek to fully investigate the influence that a protein scaffold has on a metal center during the mechanism of a reaction catalyzed by ArMs. Both the lack of compatibility in vivo and mechanistic understanding of ArMs has limited the rate at which novel generations of ArM variants can be developed and studied. The work in this thesis addresses both the in vivo compatibility and mechanistic understanding of an ArM formed by the replacement of the iron-porphyrin in cytochromes P450 with an iridium porphyrin, namely the enzyme Ir(Me)-CYP119 developed by our laboratory. First, a system to assemble CYP119 within E. coli cells was developed. Next, the mechanism of cyclopropanation catalyzed by Ir(Me)-CYP119 was studied to reveal pertinent protein-cofactor dynamics and their influence on the mechanism of carbene transfer. In the final chapters, the in vivo platform was leveraged to evolve Ir(Me)-CYP119 to catalyze reactions with selectivities difficult or even impossible to achieve with small molecule catalysts. These studies have laid the foundations for and have created opportunities for nearly limitless application of hemoprotein-based ArMs to novel reactions and biosynthetic pathways in different organisms.

    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2022)
    Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non-natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high-throughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α-diazopropanoate esters (Me-EDA) into the N-H bonds of N-alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDA with N-methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo.
      Cover page: Directed Evolution of Artificial Metalloenzymes in Whole Cells
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2021)
      Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.
        Cover page: Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme
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