In Vitro Reconstitution of Mevalonate Pathway Guide to Build the Terpenoids Overproduction Cell Factories

Terpenoids are very important natural products, like fuel chemical farnesene, nutritional compounds lycopene, β-carotene, astaxanthin, and drugs like artemisinin and taxol are belong to this big family. However, due to the lack of understanding of the pathway and enzymes, to build a high efficient synthetic pathway is still a huge challege. In order to sovle this problem to accelerate the progress of pathway engineering, we have develop a strategy called "Targeted Synthetic Metabolism“. In this strategy, the biosynthetic pathway will be fully reconstituted or partially reconstituted in vitro firstly, and then the contributions of cofactors, substrates and each component and best ratio of components will be analyzed systematically. Second, we will choose the right promoters to control each gene in vivo and also prepare the chasis host with the guidance from in vitro study to generate our first generation mutant. Third, we used our Mass-spec platform to quantitatively analyze the intermediates accumulation. And then, we will adjust the targeted point by replacing of different genes or different promoters to improve the titer step by step under our predictions. Here, we have successfully in vitro reconstituted the mevalonate pathway, and these informations guided us to build the cell factories for farnesene, lycopene and astaxanthin in E. coli, and also we can copied the success for lycopene, β-carotene, astaxanthin in Saccharomyces cerevisiae and other important plant natural products in microbial system. The robust IPP and DMAPP chassis can be used for key enzymes evaluation since most of intermediates are not stable. Furthermore, this strategy also can be applied into fatty acid derived compound overproduction and polyketide compounds overproduction and we have successfully increased the performances of some current industrial strains by using this strategy.