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Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction


Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon–carbon bond-forming transformations in traditional organic chemistry and nature, relatively few C–C bond-forming enzymes have found their way into the biocatalysis toolbox. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally guided engineering. The enzyme that emerged, UstDv2.0, is efficient in a whole-cell biocatalysis format. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on the gram scale. The X-ray crystal structure of UstDv2.0 at 2.25 Å reveals the active site and provides a foundation for probing the UstD mechanism.

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Fig. 1: Relevance and mechanism of enzymatic C–C bond formation.
Fig. 2: Directed evolution of UstD and the evaluation of variants.
Fig. 3: Engineering UstD for an increased crystallizability and activity in whole-cell catalysis.

Data availability

The structure of UstDv2.0 is available through the Protein Data Bank ID 7MKV. The sequence-activity data used for linear regression modelling is available through GitHub42. All the other data are available from the authors upon reasonable request.

Code availability

The linear regression modelling code used during the final round of protein engineering is available through GitHub42 under the MIT License.


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We thank I. Guzei for small-molecule X-ray structure determinations and S.H. Gellman and members of the Buller group for critical reading of the manuscript. The crystal mounting and data collection were mediated by the Collaborative Crystallography Core, Department of Biochemistry, UW–Madison, and data were collected at the Life Sciences Collaborative Access Team beamline 21ID-D at the Advanced Photon Source, Argonne National Laboratory, and we thank Z. Wawrzak for technical assistance during data collection. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). This work was supported by the Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison, Wisconsin Alumni Research Foundation, National Institute of Health (grant DP2-GM137417, A.R.B.), Morgridge Institute for Research—Metabolism Theme Fellowship (P.K.) and the NIH Biotechnology Training Grant (T32-GM008349, J.M.E.). The Bruker AVANCE III-500 NMR spectrometers were supported by the Bender Fund. The Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. The Bruker D8 VENTURE Photon III X-ray diffractometer was partially funded by a NSF Award (no. CHE-1919350) to the UW–Madison Department of Chemistry.

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Authors and Affiliations



A.R.B. and J.M.E. conceptualized the goals and aims of the project. J.M.E., M.E.C., P.K., E.P.G., C.A.B. and A.R.B. carried out the development of the chemistry and enzymes. J.M.E. developed the code for data analysis and developed the linear regression model. J.M.E. and M.E.C. verified the results. J.M.E., M.E.C., P.K. and A.R.B. prepared the figures and data visualizations. A.R.B. secured funding for the project that led to this publication. A.R.B. coordinated team members for the development of the chemistry and enzyme evolution. C.A.B. supervised the data acquisition of protein crystals that led to the resolved crystal structure. A.R.B. supervised the research activity planning and execution. J.M.E., M.E.C. and A.R.B. prepared the initial manuscript. J.M.E., M.E.C., P.K. and A.R.B. reviewed and edited the initial manuscript and provided critical commentary and revisions.

Corresponding author

Correspondence to Andrew R. Buller.

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Competing interests

A.R.B., J.M.E. and P.K. have a patent pending on the use of engineered UstD for the synthesis of nsAAs, US Patent application no. 20210115480A1. All other authors declare no competing interests.

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Ellis, J.M., Campbell, M.E., Kumar, P. et al. Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction. Nat Catal 5, 136–143 (2022).

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