ALBERT

Description: Abstract In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. Key points • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.

Description: Abstract Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C–C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning–guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. Key points • DERA aldolases are versatile biocatalysts able to make new C–C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts. Graphical abstract