Engineering ketoreductases for a new-to-nature photobiotransformation

Engineering enzymes with new-to-nature reactivity remains a great challenge in biocatalysis. Combining the merits of biocatalysis and photocatalysis represents a powerful approach to address this challenge.

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To expand the synthetic applications of biocatalysts, our group has been using directed evolution to engineer enzymes for transformations of interest for decades. In recent years, we focused on steering biocatalysis with visible light for new reactivity of enzymes. We summarized the strategies developed by our group and others for combining biocatalysis with photocatalysis, i.e. photobiocatalysis, in a recent review (1). 

Our contributions to this photobiocatalysis area can be traced back to year 2018. In collaboration with the Hartwig group from the University of California at Berkeley, we reported a stereoconvergent reduction of the mixtures of alkenes by cooperating a photo-energy-transfer-enabled isomerization and a selective enzymatic reduction (Fig. 1a) (2). Later, we evolved the synergistic system by merging a third photoredox catalysis for the FMN cofactor reduction to avoid the use of native GDH/NADP+/Glucose based regeneration system (3). In 2020, for the first time we pushed the boundary of photo-induced enzymatic catalysis to achieve an unprecedent cross-coupling of two molecules (Fig. 1b). Illumination of ene-reductases led to an abiological intermolecular stereocontrolled radical hydroalkylation of terminal alkenes with α-halo carbonyls (4). This represents a rare example of biocatalytic enantioselective electron-deficient radical addition to electron-rich alkenes. The herein newest work reported a complementary radical addition to C=C bonds pathway, namely radical conjugate addition to electron-deficient alkenes.

The driving force for the project came from two aspects: first, we noticed that catalytic asymmetric radical conjugate addition to α,α-disubstituted terminal alkenes remained rare, which is a challenge in radical chemistry; second, as a part of U.S. Department of Energy‘s Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), we were eager to design new photoenzymatic catalysis for bioproduct upgrading, such as using fatty acids as starting materials.

With these in mind, we first tested the direct photoinduced single electron oxidation of fatty acids to generate electron-rich radicals. But our ene-reductases failed to give desired transformation, although the photoexcited flavin is known to be able to oxidize carboxylic acids (5). We then turned to the redox-active ester, N-(acyloxy)phthalimide, which is easily synthesized from carboxylic acid and is widely used as electron-rich radical precursor. In particular, it has been reported to undergo single electron transfer (SET) reduction with Hantzsch esters, a NADPH mimic. This inspired us to test NADPH-dependent ketoreductases (KREDs). Indeed, screening a library of KREDs commercially available from Codexis led to the discovery of a ketoreductase capable of catalyzing the desired intermolecular conjugate addition of N-(acyloxy)phthalimides derived benzylic radicals with methyl methacrylate. To improve its enantioselectivity, we engineered the selected ketoreductase using the FRISM strategy initially developed by Wu et al. (6) and the crystal structures resolved by the Jiahai Zhou group from Shanghai Institute of Organic Chemistry. We were able to finish the directed evolution and condition optimizations within approximately 5 months. After the substrate scope investigation, we conducted the mechanistic studies, including computational simulations by the Binju Wang group from Xiamen University, and crystallographic studies by the Jiahai Zhou group. It took almost one year from the initial submission of manuscript to the final acceptance. Although the review process was long and arduous, we were happy to see that both the editor and the reviewers were enthusiastic about the novelty of this work and our contributions in the field of photobiocatalysis. We felt strongly encouraged to continually explore new photoenzyme-catalyzed reactions to address long-standing challenges in synthetic chemistry.


Fig. 1. Our effort in developing abiological transformations by steering the enzymes with light.

(1) Harrison, W., Huang, X. & Zhao, H. Photobiocatalysis for Abiological Transformations. Acc. Chem. Res., 55, 1087-1096, doi:10.1021/acs.accounts.1c00719 (2022).

(2) Litman, Z. C., Wang, Y., Zhao, H. & Hartwig, J. F. Cooperative asymmetric reactions combining photocatalysis and enzymatic catalysis. Nature 560, 355-359, doi: 10.1038/s41586-018-0413-7 (2018).

(3) Wang, Y. J., Huang, X. Q., Hui, J. S., Vo, L. T. & Zhao, H. M. Stereoconvergent Reduction of Activated Alkenes by a Nicotinamide Free Synergistic Photobiocatalytic System. ACS Catal. 10, 9431-9437, doi:10.1021/acscatal.0c02489 (2020).

(4) Huang, X. et al. Photoenzymatic Enantioselective Intermolecular Radical Hydroalkylation. Nature 584, 69-74, doi:10.1038/s41586-020-2406-6 (2020).

(5) Sorigue, D. et al. Mechanism and Dynamics of Fatty Acid Photodecarboxylase. Science 372, eabd5687, doi:10.1126/science.abd5687 (2021).

(6) Xu, J. et al. Stereodivergent Protein Engineering of a Lipase to Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters. J. Am. Chem. Soc. 141, 7934-7945, doi:10.1021/jacs.9b02709 (2019).


Huimin Zhao

Professor, University of Illinois at Urbana-Champaign