Abstract:The asymmetric aldol addition reaction mediated by aldolases is recognized as a green and sustainable method for carbon-carbon bond formation. Research in this area has unveiled their unprecedented synthetic potential toward diverse, new chemical structures; novel product families; and even as a technology for industrial manufacturing processes. Despite these advances, aldolases have long been regarded as strictly selective catalysts, particularly for nucleophilic substrates, which limits their broad applicabi… Show more
“…The biocatalytic process in this study is elegant and nicely exploits the power of cascades: (i) it provides a universal, efficient, and renewable catalyst to replace precious transition metals such as Pd 10 , 11 in the modern chemical C–H functionalization-mediated asymmetric assembly of α-functionalized organic acids and (ii) when compared to other enzymatic methods 3 – 5 , 9 , 26 , 28 , 36 , 39 , the universality and practicality of this biocatalytic cascade is more attractive owing to the more diverse product range that it supports, including liphatic, aromatic, heteroaromatic, and heterocyclic products from three classes (α-keto/hydroxy/amino acids). Furthermore, since enantiocomplementary ʟ- and D -specific TAs were identified to accept nucleophilic substrates other than glycine (such as alanine, serine, and cysteine) 42 , 43 , the expansion of this biocatalytic process could enable the synthesis of a vast array of α-functionalized products simply by combinatorically integrating different components such as other catalyst modules and substrate units.…”
The preparation of α-functionalized organic acids can be greatly simplified by adopting a protocol involving the catalytic assembly of achiral building blocks. However, the enzymatic assembly of small amino acids and aldehydes to form numerous α-functionalized organic acids is highly desired and remains a significant challenge. Herein, we report an artificially designed chiral-group-resetting biocatalytic process, which uses simple achiral glycine and aldehydes to synthesize stereodefined α-functionalized organic acids. This cascade biocatalysis comprises a basic module and three different extender modules and operates in a modular assembly manner. The engineered Escherichia coli catalysts, which contained different module(s), provide access to α-keto acids, α-hydroxy acids, and α-amino acids with excellent conversion and enantioselectivities. Therefore, this biocatalytic process provides an attractive strategy for the conversion of low-cost achiral starting materials to high-value α-functionalized organic acids.
“…The biocatalytic process in this study is elegant and nicely exploits the power of cascades: (i) it provides a universal, efficient, and renewable catalyst to replace precious transition metals such as Pd 10 , 11 in the modern chemical C–H functionalization-mediated asymmetric assembly of α-functionalized organic acids and (ii) when compared to other enzymatic methods 3 – 5 , 9 , 26 , 28 , 36 , 39 , the universality and practicality of this biocatalytic cascade is more attractive owing to the more diverse product range that it supports, including liphatic, aromatic, heteroaromatic, and heterocyclic products from three classes (α-keto/hydroxy/amino acids). Furthermore, since enantiocomplementary ʟ- and D -specific TAs were identified to accept nucleophilic substrates other than glycine (such as alanine, serine, and cysteine) 42 , 43 , the expansion of this biocatalytic process could enable the synthesis of a vast array of α-functionalized products simply by combinatorically integrating different components such as other catalyst modules and substrate units.…”
The preparation of α-functionalized organic acids can be greatly simplified by adopting a protocol involving the catalytic assembly of achiral building blocks. However, the enzymatic assembly of small amino acids and aldehydes to form numerous α-functionalized organic acids is highly desired and remains a significant challenge. Herein, we report an artificially designed chiral-group-resetting biocatalytic process, which uses simple achiral glycine and aldehydes to synthesize stereodefined α-functionalized organic acids. This cascade biocatalysis comprises a basic module and three different extender modules and operates in a modular assembly manner. The engineered Escherichia coli catalysts, which contained different module(s), provide access to α-keto acids, α-hydroxy acids, and α-amino acids with excellent conversion and enantioselectivities. Therefore, this biocatalytic process provides an attractive strategy for the conversion of low-cost achiral starting materials to high-value α-functionalized organic acids.
“…A particular success in this area is fructose-6-phosphate aldolase (FSA), an aldolase that accepts dihydroxyacetone as donor but also other ketones and even some aldehydes (Garrabou et al 2009 ; Junker et al 2018 ). This donor or nucleophile promiscuity was recently reviewed (Hernandez et al 2018 ). Scheme 2 Four groups of aldolases according to the functionality of their donor substrates, above the arrows (acetaldehyde, dihydroxyacetone (phosphate), pyruvate dependent or phosphoenolpyruvate, and glycine) …”
2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.
“…The interesting feature is the substrate promiscuity as DERA enzymes have been shown to accept a wide range of aldehydes as acceptor molecules, thus offering a biocatalytic alternative for a (stereo)selective synthesis of C-C bonds. Substrate specificity on the donor substrate side is stricter but recent biodiversity screen has revealed that some DERAs also display nucleophile substrate promiscuity (Hernández et al 2018;Chambre et al 2019). Furthermore, DERA enzymes can carry out a tandem reaction with acetaldehyde as a sole substrate, leading to formation of a C6 product, 2,4,6-trideoxyhexose, which cyclises spontaneously and is removed from the reaction.…”
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.
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