Synthetic Biology for Heterotrophic Production of High-Value Chemicals

In addition to the best feedstock sugarcane, industrial beets are of great potential to become another ideal feedstock for production of advanced biofuels and high-value chemicals. Beets produce readily-fermentable sugars (especially sucrose), eliminating the cost for starch hydrolysis required for corn-based biofuel production. However, the whole beet has very high-water content (75% water), which is a big challenge for transportation and storage. Additionally, the energy-intensive process to make table sugar (sucrose) using beet juice is unnecessary for biofuel/biochemical production. To overcome this challenge, this study aims to address alternative ways to produce highly demanded bio-products directly from the raw beet juice. With advancements in the biofuel sector it has become possible to apply synthetic biology of bacteria to produce compounds not solely used in fuel or power generation. Linalool is a monoterpene which sees use in a wide variety of commercial and industrial compounds and potentially medicine. Ethylene is a primary component in the synthesis of plastics, antifreeze, and synthetic fibers. Both of these products are produced in nature in very small quantities, making it economically non-viable to extract these directly from the producing plants, so they must be synthesized using costly processes which have a high carbon efflux into the environment. Through the genetic engineering of E. coli W (ATCC9637), a recently sequenced E. coli strain capable of utilizing sucrose as sole carbon source, it is possible to use a high sucrose-containing beet juice (Beta. vulgaris L.) to produce both linalool and ethylene, respectively. This project successfully engineered E. coli W to produce and secrete perfume linalool and bio-ethylene using the beet juice as sole carbon source. The linalool productivity of E. coli W was 324.05 ± 87.07 nmol L-1 Day-1 OD600 -1. The maximum linalool productivity of E. coli W had an average rate over first three days of 553.47 ± 74.1 nmol L-1 Day-1. The maximum conversion efficiency of sucrose to linalool was between 24 and 48 hours producing 94.34 nmol linalool/mol sucrose. E. coli W with pZR1429 grown in 25 mL BG11 medium containing 7.4% sucrose from beet juice produced 12.68 ± 0.86 nmols within the first 24 hours. The max conversion efficiency of sucrose to ethylene was 20.42 ± 5.02 nmol ethylene/mol sucrose during the first 9 hours