Development of aldolase-based catalysts for the synthesis of organic chemicals

Aldolases catalyze condensation reactions between donor ketones and acceptor aldehydes by forming C–C bonds to produce aldol chemicals with high selectivity. Aldolases must be engineered, or de novo aldolases must be designed, to enhance their activity, stability, and selectivity, or to catalyze novel reactions. High-throughput screening techniques, such as phage display screening and microfluidic droplet screening, can be applied to rapidly acquire desired aldolases. Aldolase-containing catalysts are used to synthesize not only aldol chemicals but also complex organic chemicals via multiple sequence reactions using simple materials. Synthetic chemistry can be applied to develop novel artificial aldolases, and chemo- and biocatalysis can be combined for the efficient synthesis of organic chemicals for industrial applications. Aldol chemicals are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse organic chemicals requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-containing catalysts, which catalyze the aldol reaction combined with other enzymatic and/or chemical reactions, can efficiently synthesize diverse complex organic chemicals using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solutions for aldolase-based catalysts. Aldol chemicals are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse organic chemicals requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-containing catalysts, which catalyze the aldol reaction combined with other enzymatic and/or chemical reactions, can efficiently synthesize diverse complex organic chemicals using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solutions for aldolase-based catalysts. aldolase-mimicking molecules with α-amino groups and polymers combined by hydrogen-bond linkages using linkers. aldolase-mimicking molecules created by ligand-selective recognition sites such as metal-binding sites in synthetic polymers using the molecular imprinting technique. condensation reactions between ketones and aldehydes to produce C–C bonds. biocatalysts with biosynthetic pathways that are generated by inserting genes encoding exogenous enzymes into cells that use endogenous pathways. biocatalysts with constructed de novo designed pathways in cells that are evaluated, selected, and optimized using an in silico retrosynthetic pathway design. biocatalysts with exogenous enzymes that participate in multiple reactions by using enzymes in cells, but not endogenous biosynthetic pathways. chemical catalysts combined with enzyme catalysts that reduce reaction steps, simplify processes, and improve the yield as well as stereoselectivity compared to chemical catalysts. de novo designed enzymes that are created by synthesizing the whole amino acid sequences of the framework structures of entire aldolase scaffolds. random mutagenesis that consists of error-prone PCR and DNA shuffling and is similar to natural evolution. random mutagenesis performed by fragmentation of parent genes by DNase, annealing and reassembling of DNA fragments to each other, followed by extension to the size of parental genes by DNA polymerase. PCR that generates errors during nucleotide incorporation during DNA copying. a screening method that dramatically reduces the screening time because each aldolase variant requires only a small reaction space, and many reactions can be performed simultaneously. aldolases attached to insoluble membrane films, polymers, or nanotubes. a HTS method that controls the formation and manipulation of nano- to femtoliter droplets of a single fluid phase through compartmentalization into micro-sized droplets, and is used to effectively and rapidly select the desired aldolases through fast droplet sorting by fluorescence-activated detection. a multi-protein complex formed by sequential enzymes in a metabolic pathway. biocatalysts with several enzymes that mediate enzyme cascade reactions; they are used in cases where the intermediates are unstable or expensive. a HTS method that rapidly selects a target peptide with high selectivity by detecting the interaction between the displayed binding domain of the protein and its substrate. genetic modification to improve the catalytic properties of enzymes by altering several amino acids using computational structure design. random mutagenesis that generates multiple and random single-amino acid mutations in each gene sequence. specific mutagenesis that substitutes a specific amino acid residue with one of the other possible amino acids, thus altering protein properties by accurate point mutation.