Complete integration of carbene-transfer chemistry into biosynthesis

Jing Huang; Andrew Quest; Pablo Cruz-Morales; Kai Deng; Jose Henrique Pereira; Devon Van Cura; Ramu Kakumanu; Edward E K Baidoo; Qingyun Dan; Yan Chen; Christopher J Petzold; Trent R Northen; Paul D Adams; Douglas S Clark; Emily P Balskus; John F Hartwig; Aindrila Mukhopadhyay; Jay D Keasling

doi:10.1038/s41586-023-06027-2

Complete integration of carbene-transfer chemistry into biosynthesis

Nature. 2023 May;617(7960):403-408. doi: 10.1038/s41586-023-06027-2. Epub 2023 May 3.

Authors

Jing Huang^{1

2

3}, Andrew Quest^{4

5}, Pablo Cruz-Morales^{1

2

6}, Kai Deng^{2

7}, Jose Henrique Pereira^{2

8}, Devon Van Cura⁹, Ramu Kakumanu^{1

2}, Edward E K Baidoo^{1

2}, Qingyun Dan^{1

2}, Yan Chen^{1

2}, Christopher J Petzold^{1

2}, Trent R Northen^{2

10}, Paul D Adams^{2

8

11}, Douglas S Clark^{12

13}, Emily P Balskus^{9

14}, John F Hartwig^{15

16}, Aindrila Mukhopadhyay^{17

18

19}, Jay D Keasling^{20

21

22

23

24

25}

Affiliations

¹ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
² Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA.
³ Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA.
⁴ Department of Chemistry, University of California, Berkeley, CA, USA.
⁵ Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁶ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
⁷ Department of Biomaterials and Biomanufacturing, Sandia National Laboratories, Livermore, CA, USA.
⁸ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
¹⁰ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
¹¹ Department of Bioengineering, University of California, Berkeley, CA, USA.
¹² Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA. dsc@berkeley.edu.
¹³ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. dsc@berkeley.edu.
¹⁴ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
¹⁵ Department of Chemistry, University of California, Berkeley, CA, USA. jhartwig@berkeley.edu.
¹⁶ Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. jhartwig@berkeley.edu.
¹⁷ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. amukhopadhyay@lbl.gov.
¹⁸ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA. amukhopadhyay@lbl.gov.
¹⁹ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. amukhopadhyay@lbl.gov.
²⁰ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. keasling@berkeley.edu.
²¹ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA. keasling@berkeley.edu.
²² Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA. keasling@berkeley.edu.
²³ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark. keasling@berkeley.edu.
²⁴ Department of Bioengineering, University of California, Berkeley, CA, USA. keasling@berkeley.edu.
²⁵ Synthetic Biochemistry Center, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China. keasling@berkeley.edu.

PMID: 37138074
DOI: 10.1038/s41586-023-06027-2

Abstract

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions¹. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis^2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural

MeSH terms

Azaserine* / biosynthesis
Azaserine* / chemistry
Biocatalysis
Biological Products / chemistry
Biological Products / metabolism
Coenzymes / chemistry
Coenzymes / metabolism
Cyclopropanes / chemistry
Cytochrome P-450 Enzyme System / genetics
Cytochrome P-450 Enzyme System / metabolism
Multigene Family / genetics
Styrene / chemistry

Substances

Azaserine
carbene
Biological Products
Styrene
Cyclopropanes
Coenzymes
Cytochrome P-450 Enzyme System

Supplementary concepts

Streptomyces albus

Grants and funding

S10 OD024998/OD/NIH HHS/United States