In Vitro Reconstitution of Metabolic Pathways: Insights into Nature’s Chemical Logic

Abstract

In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature’s core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway’s constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature’s biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques.

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

“…Additionally, robust heterologous expression of biosynthetic proteins opens the possibility of rapid design-build-test cycles to re-engineer pathways to produce useful scaffolds beyond those found in nature. 3 …”

Cell-free synthetic biology for in vitro biosynthesis of pharmaceutical natural products

“…Full reconstitution of natural product biosynthetic pathways with purified enzymes in both PKs and NRPs contexts has allowed the elaboration of remarkable complexity from simple building blocks as aforementioned cases. This strategy not only significantly contributes to decipher novel chemical logic and enzymatic machinery of PKS and NRPS assembly lines, but also provides efficient platforms for in vitro total biosynthesis of natural products [ 18 , 19 ]. In addition, due to the flexibility of open cell-free systems, precursor analogues could be easily added to the reaction, allowing the formation of natural product analogues with potential novel bioactivities as shown in an example of the NRP beauvericin [ 55 ].…”

“…In vitro reconstruction of purified enzymes has been widely used to characterize individual enzymes, identify intermediates, and elucidate entire natural product biosynthetic pathways [ 18 , 19 ]. While there are many studies on individual enzymes, we primarily focus on the in vitro reconstitution of PK and NRP biosynthetic pathways with a set of purified enzymes to generate core scaffolds and final products, which include the PKs 6-deoxyerythronolide B and enterocin, the NRPs terrequinone A and pacidamycin, as well as the PK/NRP hybrids yersiniabactin and ikarugamycin.…”

Modules for in vitro metabolic engineering: Pathway assembly for bio-based production of value-added chemicals

“…Several studies have investigated the synthesis of complicated compounds from other key intermediates by reconstituting native biosynthesis pathways [34] . The understanding of reaction cascades in nature can be used in in vitro metabolic engineering for the design of modules.…”