Combining Metabolic Pathway Design and Retrosynthetic Planning for the Design of a Novel Semisynthetic Manufacturing Scheme for Paclitaxel

Abstract

Using the example of paclitaxel, this paper expounds how retrosynthetic analysis can be combined with metabolic pathway design to devise novel semisynthetic schemes for manufacturing natural products. The analytical framework presented herein leverages the latest developments in chem-and bioinformatics, metabolic engineering, and retrosynthetic planning, and the proposed schemes commence with the microbially aided synthesis of an advanced intermediate that is then recruited for target-oriented synthesis (TOS). A technoeconomic analysis of the scheme devised using bioretrosynthetic analysis for manufacturing paclitaxel suggests that the new process competes favorably with the current route for producing paclitaxel. Additionally, since TOS can access precise regions of chemical space, either a single molecule or a small assortment of molecules exhibiting minor variations on a chemical theme, bioretrosynthesis also doubles as a tool for drug discovery.

Biosynthonics: Charting the Future Role of Biocatalysis and Metabolic Engineering in Drug Discovery

“…32,73 For instance, the taxane biosynthetic pathway that is expressed by trees of the Taxus genus synthesizes well over a hundred products, including paclitaxel, and strains of E. coli that were genetically engineered to express a few enzymes of the taxane pathway also produced a comparable diversity of molecules. 74 In this study, when the cytochrome P450 monooxygenase, taxadiene 5α-hydroxylase, was introduced into a strain of E. coli that had been previously engineered to overproduce taxadiene, it was observed that the cultures synthesized as many as 16 eicosanoid molecules (Figure 6).…”

“…Once the combination of terpenoid biosynthetic enzymes that can or could produce the molecule of interest has been identified using the methodology that has been outlined in previous sections, they can be expressed without any modifications to their active site to generate a family of closely related structures that can then be subjected to hydroxyl hydrogen substitution reactions in vitro to yield an even larger library of highly “drug-like” molecules. Alternatively, if conventional TOS is desired (Figure ), the individual active sites can be re-engineered in order to enhance substrate specificity, product selectivity, and activity . For both, DOS and TOS schemes, the enzymes must be combined into a pathway, and enzyme expression must be suitably toggled in order to adjust their concentrations.…”

“…Accordingly, bio- and retrosynthetic planning for the production of paclitaxel identified (1,2α,5α,7β,10β)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax-4,11-diene as the product of microbial metabolic engineering . Considering that taxadiene has already been produced at titers of 1 g/L in a 1 L bioreactor, it is not unreasonable to expectespecially in light of the sterling progress that has been made in bioprocess engineering in the past few decadesthat (1,2α,5α,7β,10β)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax-4,11-diene could also be produced at similar titers.…”

“…A natural products database with validated structural and biosynthetic information, too, must be established. With regard to generating natural product chemical diversity reliably and rapidly, combinatorial biosynthesis has already been applied and optimized for the production of several polyketides 69,82 and nonribosomal peptides; 83 and promiscuous and multispecific enzymes have been expressed in genetically engineered microorganisms 74,84 to produce a collection of natural product structures. Enzyme engineering has also been successfully employed to alter the polymerization and cyclization reactions involved in terpenoid assembly to synthesize altogether novel structures.…”