In vitro reconstitution guide for targeted synthetic metabolism of chemicals, nutraceuticals and drug precursors

With the developments in metabolic engineering and the emergence of synthetic biology, many breakthroughs in medicinal, biological and chemical products as well as biofuels have been achieved in recent decades. As an important barrier to traditional metabolic engineering, however, the identification of rate-limiting step(s) for the improvement of specific cellular functions is often difficult. Meanwhile, in the case of synthetic biology, more and more BioBricks could be constructed for targeted purposes, but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge. Owing to the lack of steady-state kinetic data for overall flux, balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials. A strategy called targeted engineering is proposed in an effort to solve this problem. Briefly, a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors, substrates and each enzyme will be analyzed systematically. Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays. To demonstrate its practical application, biosynthesis pathways for the production of important products, e.g. chemicals, nutraceuticals and drug precursors, have been engineered in Escherichia coli and Saccharomyces cerevisiae. These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.