Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

Autor: d'Oelsnitz S; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA. simonsnitz@gmail.com.; Synthetic Biology HIVE, Department of Systems Biology, Harvard Medical School, Boston, MA, 02115, USA. simonsnitz@gmail.com., Diaz DJ; Department of Chemistry, University of Texas at Austin, Austin, TX, 78712, USA.; Institute for Foundations of Machine Learning, University of Texas at Austin, Austin, TX, 78712, USA., Kim W; McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, 78712, USA., Acosta DJ; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Dangerfield TL; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Schechter MW; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Minus MB; Department of Chemistry, Prairie View A&M University, 100 University Dr, Prairie View, TX, 77446, USA., Howard JR; Department of Chemistry, University of Texas at Austin, Austin, TX, 78712, USA., Do H; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Loy JM; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Alper HS; McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, 78712, USA., Zhang YJ; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA., Ellington AD; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2024 Mar 07; Vol. 15 (1), pp. 2084. Date of Electronic Publication: 2024 Mar 07.
DOI: 10.1038/s41467-024-46356-y
Abstrakt: A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50  = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.
(© 2024. The Author(s).)
Databáze: MEDLINE
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