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Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2020)

This dissertation presents the development of artificial metalloenzymes (ArMs) for unnatural reactions, the in vivo engineering and catalysis of ArMs, and the integration of ArMs with a natural biosynthetic pathway to produce products previously inaccessible by nature in a microbial host.Chapter 1 provides an overview of ArMs that are generated from incorporation of abiotic metal complexes into protein scaffolds. Assembly strategies and non-natural reactivity of this class of biohybrid catalysts are presented in detail. While a majority of work in this field has focused on the design and application of ArMs in vitro, the overview discusses the emerging trend towards integrating ArMs into whole-cell systems and metabolic networks and the challenges associated with the development of ArMs that function in vivo. Chapter 2 describes the directed evolution of an artificial iridium-containing P450 that catalyzed the cyclopropanation of monocyclic terpenes and their derivatives with diastereoselectivities that are complementary to those attained with chiral transition metal catalysts. The combination of the broad reactivity of iridium porphyrin and the exquisite selectivity provided by proteins generates catalysts with activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone. Chapter 3 describes the application of a nonpathogenic Escherichia coli (E. coli) strain, Nissle 1917, for recombinant expression of P450s containing artificial cofactors. The outer-membrane receptor in Nissle 1917 transports iridium porphyrin into cells for binding to the apo-CYP119 expressed in the cytoplasm. We show that the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds with remarkable enantioselectivity and site-selectivity. The application of Nissle 1917 as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Chapter 4 describes the integration of artificial metalloenzymes that catalyze unnatural reactions into metabolic networks that could broaden the cache of molecules produced biosynthetically by microorganisms. We report the assembly of an artificial P450 containing an iridium-porphyrin complex in the cytoplasm of an E. coli that produces a terpene by heterologous expression and insertion of this ArM into the biosynthetic pathway to produce an unnatural terpenoid. This work shows that synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce molecules previously inaccessible by natural metabolic pathways.

    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2019)
    The selective functionalization of one C-H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site-selective functionalizations of C-H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium-porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C-H bonds of a range of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site-selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C-H bonds with site selectivity that is difficult to achieve with small-molecule catalysts.
      Cover page: Site‐Selective Functionalization of (sp3)C−H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium‐Porphyrin Cofactor
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2019)
      Abstract: The selective functionalization of one C−H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site‐selective functionalizations of C−H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium‐porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C−H bonds of a range of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site‐selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C−H bonds with site selectivity that is difficult to achieve with small‐molecule catalysts.
        Cover page: Site‐Selective Functionalization of (sp3)C−H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium‐Porphyrin Cofactor
        • 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 (2022)
          The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.
            Cover page: Assembly and Evolution of Artificial Metalloenzymes within E. coli Nissle 1917 for Enantioselective and Site-Selective Functionalization of CH and CC BondsCreative Commons 'BY-NC' version 4.0 license
            • 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
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2017)
              Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C-H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.
                Cover page: Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes
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