Metabolic and genetic switches for stable long-term fermentative production of anabolic products

The strategic partnership between Total and Amyris is focused on producing biomolecules for renewable fuels and chemicals, and in particular for blending with petroleum-derived products to create diesel and jet fuel with reduced carbon footprint and pollution. Production and marketing of the flagship molecule farnesene (Biofene®) has already been commercialized with production scale in some markets. Farnesene has many potential applications as a renewable feedstock for the aforementioned diesel and jet fuels, as well as polymers, lubricants, solvents, and cosmetics. To reduce the production cost of farnesene, at Amyris we engineer strains using state-of-the art industrial synthetic biology platform to have high titer, yield, and productivity, and we perform fermentations in 200 m3 vessels over the course of many days or weeks. The challenge is that high-producer cells grow more slowly than spontaneous mutant low- or non-producer cells, especially in the nutrient-unlimited conditions of the seed train expansion, and yet must comprise the vast majority of the population. We have successfully addressed this challenge using metabolic and genetic switches that turn off production in the seed train. As a metabolic switch, we found that omission of pantothenate reduced product formation and increased growth of high-producer cells, while decreasing growth of non-producer cells. Removal of pantothenate from seed train eliminated the growth advantage of low-producing spontaneous mutants, and thereby increased purity of the inoculum and improved performance in bioreactors. We also have developed an industrially scalable genetic switch and used other approaches to successfully maintain high performance throughout lengthy fermentations.