Cell-free biosynthesis of isoprenoids using Escherichia coli crude lysatesPublic Deposited
Metabolic engineering of microorganisms to produce useful compounds from renewable substrates is a promising means for sustainable, on-demand production of chemicals. However, efforts to design and engineer microbial cell factories are constrained by costly and slow “build” times in which each genetic variation requires re-engineering a new strain for each iteration. To alleviate this challenge, we have built a plug-and-play prototyping system for isoprenoid biosynthesis. Isoprenoids are a promising class of target molecules with over 40,000 known structures and potential uses as pharmaceuticals, flavors, fragrances, pesticides, disinfectants, and chemical feedstocks. By mixing together multiple crude extracts, each enriched in a single pathway enzyme, we can synthesize the fragrance and flavoring monoterpenoid limonene. To further minimize the time required to test enzyme homologs, we have used cell-free protein synthesis (CFPS) to synthesize pathway enzymes directly in the lysate by simply adding the appropriate DNA template along with energy molecules, cofactors, and amino acid substrate. By running nine separate CFPS reactions and mixing them together with a glucose substrate, 28 mg/L limonene is generated over 24 hours. This approach shortens the time from ordering genes to characterizing active enzymes to ~3 days and allows precise measurement and control of enzyme concentration. Forty-four enzyme homologs were characterized and a new optimized “set” of enzymes generates 610 mg/L limonene over 24 hours. In parallel, two new techniques were developed including CoA-SAMDI, a method for high-throughput, parallelized measurement of CoA metabolites such as acetyl-coA, and PRAT, a strategy for removing essential enzymes from crude lysates. In sum, this cell-free limonene production platform opens the possibility of extensive testing of enzymes levels and physiochemical conditions in order to prototype and accelerate in vivo metabolic engineering efforts.