Assembly and Evolution of Artificial Metalloenzymes within E. coli Nissle 1917 for Enantioselective and Site-Selective Functionalization of C─H and C═C Bonds

Abstract

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.

Figure 1.

Methods to introduce artificial metalloporphyrins at the level of protein expression. (a) The use of an E. coli strain RP523 with a mutation that renders the membrane heme-permeable. (b) Co-expression of an outer-membrane transporter ChuA with the hemoprotein of interest. (c) This work: in vivo assembly of Ir-CYP119 within E. coli Nissle 1917 that contains a native uptake system for metalloporphyrins.

Figure 2.

(a) The cell growth curves of EcN in the presence of 0, 0.1, 0.5, and 1 ppm of Ir(Me)MPIX. 1 ppm = 1 mg/L. The curves were fitted to a Gompertz growth model using Prism 8. (b) The percentages of iridium added to the growth medium that were recovered in the total cell lysates and the percent within the supernatants of cell lysates and the insoluble fractions of cell lysates from the two E. coli strains expressing CYP119. The data are shown as the average from three biological replicates, with error bars indicating 1 standard deviation.

Figure 3.

(a) The turnover numbers for the reaction of phthalan 1 with EDA vs time (h) in whole cells. Reaction conditions: EcN expressing CYP119 mutant, OD₆₀₀ ~30, phthalan 1 (2 μmol), EDA (16 μmol), DMSO (20 μL), M9-N (300 μL), 30 °C, 1–7 h. The cells were cultivated with 0.1 ppm of Ir(Me)MPIX during protein expression. (b) The C–H activation of 4-chlorophthalan 3b catalyzed by resuspended pellets. The age of cells refers to the length of time for which the cells had been used for catalysis before resuspension. Reaction conditions: resuspended EcN cell pellets, OD₆₀₀ ~30, 4-chlorophthalan 3b (1 μmol), EDA (8 μmol), DMSO (10 μL), M9-N (300 μL), 30 °C, 2 h. All data are shown as the average from three biological replicates, with error bars indicating 1 standard deviation.

Figure 4.

Directed evolution of Ir-CYP119 for diastereoselective cyclopropanation of (+)-nootkatone 5 with EcN as a screening platform. Reaction conditions: EcN expressing CYP119 mutant, OD₆₀₀ ~30, 5 (2 μmol), EDA (16 μmol), DMSO (20 μL), M9-N (300 μL), 30 °C, 4 h. The cells were cultivated with 0.1 ppm of Ir(Me)MPIX during protein expression. P₁–P₄ are the four diastereomeric products numbered in the order of elution by gas chromatography. The selectivity of each diastereomer corresponds to its percent in the total four diastereomeric products. The relative yield is calculated by normalizing the yield of the reaction catalyzed by the final mutant as 100%. All data from the reactions in EcN are shown as the average from three biological replicates, with error bars indicating 1 standard deviation.

Figure 5.

C–H bond functionalization of phthalan 1 catalyzed by BL21(DE3) cells co-expressing the potential transporter and CYP119 mutant. The cells were cultivated with 0.1 ppm of Ir(Me)MPIX during protein expression. Reaction conditions: BL21(DE3) expressing CYP119 mutant (C317G, A152L, T213G, P252S, V254A), OD₆₀₀ ~20, phthalan (2 μmol), EDA (16 μmol), DMSO (20 μL), M9-N (300 μL), 30 °C, 4 h. The relative yield is calculated by normalizing the yield of the reaction in cells co-expressing the E7 transporter and CYP119 as 100%. All data are shown as the average from three biological replicates, with error bars indicating 1 standard deviation.

Scheme 1.. Site-Selective C–H Activation Catalyzed by EcN Cells Harboring Ir-CYP119^a

^aReaction conditions: EcN expressing CYP119 mutant, OD₆₀₀ ~30, phthalan derivatives (2 μmol), EDA (16 μmol), DMSO (20 μL), M9-N (300 μL), 30 °C, 4 h. The cells were cultivated with 0.1 ppm of Ir(Me)MPIX during protein expression. The heights of bars correspond to the relative amounts of para (blue) and meta (red) products. All data are shown as the average from three biological replicates.