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Chemists hijack whole cell to build new-to-nature compounds

Introduced metalloenzyme produces unnatural terpenoid in E. coli

by Leigh Krietsch Boerner
October 17, 2021 | A version of this story appeared in Volume 99, Issue 38


The Escherichia coli cell produces (-)-limonene by a natural pathway. This can then react with the Ir-porphyrin metalloenzyme and ethyl diazoacetate in the buffer to form the unnatural cyclopropanation terpenoid product.

Researchers have made an unnatural terpenoid compound inside an Escherichia coli cell by coupling an engineered metalloenzyme with a natural terpene production system (Nat. Chem. 2021, DOI: 10.1038/s41557-021-00801-3). Chemists commandeering enzymes to construct molecules is nothing new. But John Hartwig, who co-led the research with colleagues at the University of California, Berkeley, and Lawrence Berkeley National Laboratory, says the new approach lets chemists combine the catalysis offered by an engineered metalloenzyme with the one-pot simplicity of synthesis by microbes.

This pathway is similar to one typically found in nature, Hartwig says. Cells use terpene building blocks—10-, 15-, or 20-carbon molecules with a functionalizable double bond—to make a variety of terpenoid natural products, he says. The team’s biosynthetic reaction is analogous to the functionalization that happens in the organisms, but it catalyzes a different reaction than any known in nature, Hartwig says.

The group knew that they could induce E. coli to make limonene, a terpene compound, by introducing the right genes from other organisms and that their engineered enzyme catalyzes a cyclopropanation reaction with limonene. “But what we didn’t know is how to assemble the enzyme in the cytoplasm” so that it would be part of the biosynthetic pathway, Hartwig says. The team targeted the cytochrome P450 (CYP) family of enzymes, which bind iron porphyrins. Working with a CYP from Sulfolobus acidocaldarius, a single-celled organism found growing in high-temperature, acidic conditions such as volcanic springs, the team found they could change the enzyme’s activity by adding an iridium mesoporphyrin, allowing the enzyme to cyclopropanate a variety of alkenes (ACS Cent. Sci. 2017, DOI: 10.1021/acscentsci.6b00391). The Ir mesoporphyrin is too big to move through cell membranes on its own, so the group engineered the E. coli cell to express a porphyrin-specific transporter protein; the protein ferries the porphyrin into the cell, where it encounters the S. acidocaldarius enzyme, which the team had engineered E. coli to produce.

Once inside the cell, the metalloenzyme can meet up with the limonene made in the cell. Combined with a buffer containing the reagent ethyl diazoacetate, the enzyme converts limonene into a cyclopropyl terpenoid not found in nature (shown).

It’s a dream of synthetic chemists to be able to synthesize molecules with the efficiency of nature, Hartwig says. But what they’re really after is the ability to build unnatural complex molecules, marrying the chemistries evolved in nature with reactions that chemists have invented, he says.

This research demonstrates that this idea is actually possible, says Hans Renata, an enzymatic chemist at Scripps Research in Florida. “I anticipate that this work would inspire other researchers to think about how to incorporate more non-natural enzymatic transformations into existing biosynthetic pathways to produce a wide range of novel small molecules,” he says.



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