Greening Up Hydroxylations

Manufacturers of pharmaceuticals, agricultural chemicals, flavors and fragrances, and biofuels are eager to exploit enzyme-catalyzed chemical processes. These methods typically are more efficient, safer, cheaper, and environmentally friendlier than classic metal-catalyzed chemical processes. But obtaining the enzymes and designing optimized processes to use them remain underdeveloped.

To help advance the field, an international research team has engineered a bacterium to produce multiple enzymes that work in tandem to convert simple, inexpensive olefin starting materials into high-value chiral diols in a single process (ACS Catal. 2013, DOI: 10.1021/cs400992z).

Led by Zhi Li of the National University of Singapore, the team programmed Escherichia coli to coproduce a styrene monooxygenase enzyme along with either an (S)- or (R)-selective epoxide hydrolase enzyme. The monooxygenase converts styrene or other olefins to epoxides, and the hydrolases selectively open the epoxide rings to form chiral diols.

“This work represents a very convincing example of the power of tandem or cascade biocatalysis, which is emerging as a major theme in biocatalysis,” says Nicholas J. Turner of the University of Manchester, in England, whose group specializes in enzyme catalysis. “It further emphasizes the value of applying biocatalytic retrosynthesis in the construction of molecules for the future.”

Retrosynthesis is a strategy chemists use to envision the best pathway to a desired product. Researchers start with the compound they want to make and then work backward on paper to figure out the steps of the reaction, the reagents needed, and the optimal starting compound. Li and coworkers first had to determine which genes code for the needed enzymes. Then they cut those genes from other bacteria and pasted them into E. coli. The resulting engineered microbes coexpress the enzymes and enantioselectively convert olefins to the desired chiral diols in a bioreactor.

Converting alkenes to diols is widely practiced in academic and industrial circles. The leading method—the Sharpless asymmetric dihydroxylation—requires an osmium catalyst, chiral quinine ligand, and potassium ferricyanide oxidant. These reagents are toxic and expensive.

The new tandem biocatalysis process works on both terminal and internal cis and trans olefins, using oxygen as the oxidant. The (S)- or (R)-selective versions of the engineered E. coli produce each of the possible diol enantiomers in a one-pot process. “Overall, the new whole-cell biocatalysts offer green alternatives to the metal-catalyzed Sharpless reaction,” Li says. He and his colleagues have patented their process and are currently optimizing the system for industrial use and seeking business partners.”