Evolving artificial metalloenzymes via random mutagenesis

Abstract

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Figure 1. Model reaction and ArM structure

a , Model cyclopropanation reaction used for ArM evolution. The dirhodium cofactor catalyzes diazo decomposition to generate a rhodium carbenoid intermediate that can insert into the olefin π bond or (because the reactions are carried out in aqueous solution) the water O-H bond. The protein scaffold provides chemoselectivity and enantioselectivity. Reactions conducted using 22 mM styrene, 4.4 mM diazo, and 1 mol% ArM in 10% v/v THF/50 mM PIPES (pH 7.4) containing 1.75 M NaBr at 4 °C. b, ArM formation via SPAAC between bicyclononyne-substituted metal complexes and a genetically encoded 4-l-azidophenylalnine residue, allowing covalent attachment of the cofactor, even in cellular lysate. c, Structure of dirhodium cofactor 1.

Figure 2. Overview of ArM evolution protocol

From Top-Left: The gene encoding a POP scaffold (e.g. 0-ZA₄) in pET-28 (the POP β-domain is shown in grey-blue in both plasmid and the ArM structure model) is used to generate a library of β-domain variants with random mutations via error prone PCR. The remaining backbone is amplified separately, and the gene library is ligated via Gibson assembly. The gene library is co-transformed into E. coli with pEVOL-pAzF, a plasmid containing an orthogonal tRNA and aaRS for Amber stop codon suppression. A colony picker robot is used to array colonies into 96 well plates, where the POP genes are expressed, and cells are lysed, heated, and centrifuged. Next, a liquid handling robot is used to transfer the lysate to a fresh 96 well plate, cofactor is added to generate POP ArMs, and an azide-substituted resin is added to scavenge unreacted cofactor. The ARM library is screened by HPLC or SFC for increased enantioselectivity of olefin cyclopropanation. Putative hits are isolated and cultured for verification on a larger scale without the presence of cellular lysate debris. If the putative hit is validated, it is used as parent for an additional round of mutagenesis until the desired activity/selectivity is observed.

Figure 3. Overview of the directed evolution lineages generated and time-course comparison of several catalysts

a , Enantioselectivities and yields (inset) for cyclopropanation of styrene catalyzed by evolved ArM variants to give 3 (Fig. 1A). Although enantiomeric excess was the screening criteria, yields of the desired product also increased across the lineages. Each variant contains the mutations indicated plus those from the previous round(s) of evolution. The residues identified from deconvolution experiments were cloned into a minimal mutant (1-GSH), which was able to provide equivalent enantioselectivity as the final mutant (3-VRVH) but significantly lower yield. This highlights the complexity of biomolecule scaffolds and the importance of random mutagenesis for ARM engineering. Reactions conducted as shown in Figure 1A using 22 mM styrene, 4.4 mM diazo, and 1 mol% catalyst in 10% v/v THF/50 mM PIPES (pH 7.4) containing 1.75 M NaBr or NaCl at 4 °C for 4h. Enantiomeric excess and yield of 3 were determined by analysis of HPLC chromatograms for crude reaction mixtures relative to internal standards. b, time course experiments for reactions catalyzed by different dirhodium catalysts. The yield and qualitative rate for the evolved variant 3-VRVH exceeds parent 0-ZA₄ as well as a previously reported ArM produced via rational design. The reaction in aqueous buffer of the free cofactor is also shown. All data points shown are an average of 2 reactions.

Figure 4. Location of mutations in evolved ArMs

a , side and b, top views of POP ribbon model with spheres showing the location of mutations in variants 0-ZA₄ (gray), 1-NAGS (red), 2-NSIA (orange), and 3-VRVH (blue). Mutations with greatest impact on enantioselectivity are labeled. Residues modified via carbene insertion (vide infra) are also shown as spheres (purple: W175; green: W142).

Figure 5. Combinatorial codon mutagenesis sites and protocol

a , Ribbon model of POP variant 0-ZA₄ showing the location of residue 477 (green sphere), residue 413 (blue sphere), and residues selected for combinatorial codon mutagenesis (remaining spheres). Mutations in 1-RFY (Q98R, G99F, and P239Y) are shown as red, orange, and yellow spheres, respectively. b, Scaffold immobilization and ArM formation on Ni-NTA resin in 96-well plates using libraries of scaffold variants generated via error prone PCR or CCM as shown in Figure 2.

Figure 6. Time course experiments of ArM catalyzed cyclopropanations of styrene with (4-methoxyphenyl) methyldiazoacetate

(see Supplementary Table 2 for full data). a, Plot of cyclopropane yield versus time, showing increases for each generation of mutants. b, Plot of cyclopropane enantiomeric excess (e.e.) versus time. A decrease in %e.e. over the course of reaction was observed, which decreases along the lineage. The cause of this was investigated but not identified (see Main Text). Reactions were performed in triplicate with standard deviation shown. *2-NSIA reactions were performed in duplicate, prohibiting standard deviation calculation.