A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology

Centralized
facilities for genetic engineering, or “biofoundries”,
offer the potential to design organisms to address emerging needs
in medicine, agriculture, industry, and defense. The field has seen
rapid advances in technology, but it is difficult to gauge current
capabilities or identify gaps across projects. To this end, our foundry
was assessed via a timed “pressure test”, in which 3
months were given to build organisms to produce 10 molecules unknown
to us in advance. By applying a diversity of new approaches, we produced
the desired molecule or a closely related one for six out of 10 targets
during the performance period and made advances toward production
of the others as well. Specifically, we increased the titers of 1-hexadecanol,
pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne
warhead underlying powerful antimicrobials, established a cell-free
system for monoterpene production, produced an intermediate toward
vincristine biosynthesis, and encoded 7802 individually retrievable
pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran
and barbamide were designed and constructed, but toxicity or analytical
tools inhibited further progress. In sum, we constructed 1.2 Mb DNA,
built 215 strains spanning five species (Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces
albovinaceus), established two cell-free systems, and performed
690 assays developed in-house for the molecules.