Protein Engineering for Improving and Diversifying Natural Product Biosynthesis

Abstract

Proteins found in nature have traditionally been the most frequently used biocatalysts to produce numerous natural products ranging from commodity chemicals to pharmaceuticals. Protein engineering has emerged as a powerful biotechnological toolbox in the development of metabolic engineering, particularly for the biosynthesis of natural products. Recently, protein engineering has become a favored method to improve enzymatic activity, increase enzyme stability, and expand product spectra in natural product biosynthesis. This review summarizes recent advances and typical strategies in protein engineering, highlighting the paramount role of protein engineering in improving and diversifying the biosynthesis of natural products. Future prospects and research directions are also discussed.

Keywords: engineered biosynthesis; natural products; protein engineering.

Figure 1. General protein engineering processes.

The library can be established through either rational design aided by computational simulation and/or crystal structures, or random mutagenesis or directed evolution. With the assist of efficient screening methods, variants with different characteristics, such as enhanced catalytic activity and increased stability towards heat, acid or alkaline, can be selected and analyzed. If no candidate shows the desired feature, it may be necessary to regenerate the library and repeat the process until the desired variant is obtained.

Figure 2. Typical strategies for protein colocalization.

a) Enzymes fused via protein linkers, generating a “substrate channel”. b) Enzymes colocalized on a protein scaffold. c) Enzymes assembled on a nucleic acid scaffold. For DNA scaffolds, enzymes were fused to zinc finger (ZF) or TALE domains that bind DNA; for RNA scaffold, enzymes were fused to protein adaptor that binds RNA aptamer region on the scaffold. d) Encapsulation of enzymes in microcompartments, for instance, carboxysomes.

Figure 3. Protein engineering aids the optimization of genetically encoded biosensors.

a) Engineering biosensors for responding to novel inducers. Through protein engineering, variant regulators with novel binding functions can be generated and applied in metabolic engineering of natural products biosynthesis. b) Engineering biosensors for expanded dynamic range. The enhanced regulators usually have stronger binding affinities and thus can result in more obvious output.

Figure 4. Protein engineering helps to diversify biosynthesis of natural products.

Through engineering existing enzymes, variants with novel catalytic function or expanded substrate spectrum can be obtained. These new enzymes can then be applied in constructing novel pathways for synthesizing natural products or producing analogs of natural products.