Opportunities for enzyme engineering in natural product biosynthesis

Abstract

Organisms from all kingdoms of life produce a plethora of natural products that display a range of biological activities. One key limitation of developing these natural products into pharmaceuticals is the inability to perform effective, fast, and inexpensive structure-activity relationship studies (SAR). Recently, enzyme engineering strategies have allowed the exploration of metabolic engineering of biosynthetic pathways to create new 'natural' products that can be used for SAR. The enzymes that enable the biosynthesis of natural products represent a largely untapped resource of potential biocatalysts. A challenge for the field is how to harness the wealth of reaction types used for natural product metabolism to obtain useful biocatalysts for industrial biotransformations.

Figure 1

Biosynthetic pathways fall into two categories depending on how the core structure of a natural product is assembled. A. Megasynthases are modular structures where the growing substrate is shuttled between different domains and protein-protein interactions are key for activity. B. Dissociated pathways are arrays of individual enzymes that turn over a set of given substrates. Substrate specificity is often tightly regulated, since enzyme-substrate complementarity determines the order of biosynthetic events.

Figure 2

Simplified schematics illustrating a few of the reactions catalyzed by megasynthase domains. A. KR domains use NADPH to reduce carbonyl groups to alcohols; exemplified here by an acetyl extender unit (malonyl group). Altering the stereoselectivity of KR domains could alter the product outcome. B. After KR and DH domain action on a propionyl extender unit (methyl-malonyl group), the resulting olefin can be reduced with NADPH by the action of stereoselective ER domains. Reengineering the ER stereoselectivity is also a potential means to alter product outcome. C. Illustration of NRPS domain actions on an amino acyl extender unit. A typical module is minimally composed of A (loading an amino acid onto the PCP domain), C (catalyzing the peptide bond formation), and PCP (carrying the amino acyl chain). The TE domain hydrolyzes the thioester linkage to release the natural product. KR: ketoreductase; DH: dehydratase; ER: enoyl-reductase; A: adenylation domain; C: condensation domain; PCP: peptidyl carrier protein domain; TE: thioesterase domain.

Figure 3

Examples of enzyme engineering work on dissociated pathways. A. Strictosidine synthase (STS) catalyzes the Pictet-Spengler reaction between tryptamine and secologanin. STS variants, found by screening site-directed and saturation mutagenesis libraries, accept substrates not accepted by wild-type STS. B. Isoprenoids are synthesized by four coupling reactions: chain elongation, branching, cyclopropanation, and cyclobutanation. By replacing the sequences of a chain-elongating enzyme with the corresponding homologous sequences of a cyclopropanation enzyme, all four coupling reactions were observed. C. OleD catalyzes the C-O bond formation between the C-2 hydroxyl group on oleandomycin (acceptor) and the anomeric carbon of UDP-glucose (donor). OleD is 300-fold less efficient in transferring UDP-glucose onto 4-methylumbelliferone, a fluorescent surrogate substrate. Variants of OleD with enhanced activity were found by mutagenesis and screening the libraries in spectrophotometric assays for the loss of fluorescence of the acceptor starting material.