Cell-Free Production of Terpenoid Chemical Astaxanthin Using Crude Cofactor Lysates

Replacing petroleum products with renewable alternatives is important to mitigate pollution and reduce our carbon footprint. Production of renewable products using engineered microorganisms have largely not achieved the yields, titers and productivities required for large scale market adoption. Invizyne Technologies has developed cell-free enzymatic systems that have the potential to increase the overall efficiency of the conversion process and bring down the cost of renewable products. A Techno Economic Analysis identified the cost of using purified cofactors as a main driver of cost of cell-free bioconversions.Bringing down the cost of cofactors will be a critical improvement that will allow for the widespread adoption of this technology. We will bring down the cost of cofactors by developing an alternative process to incorporate crude cofactor mixes from inexpensive waste streams. In phase I we quantified the cofactor pools in industrial strains of yeast and E. coli and demonstrated that they can be effectively integrated into a cell-free enzymatic reaction to make terpenes. We produced a variety of terpenes using crude cofactor lysates as potential products including the long chain carotenoid lycopene which serves as a proof of concept for expansion into other related carotenoids in the future. In phase II we plan to use crude cofactors sourced from industrial yeast to produce an initial carotenoid product called astaxanthin. Our end goal is to produce astaxanthin at industrial relavent titers and scales (10g/L at a 10L scale). Cell free enzymatic bioconversions have the potential to revolutionize the green chemicals industry by lowering the overall cost of production. This cell free approach can also make bio- based chemicals that are impossible to produce in organisms because of their toxicity and therefore can be used to make rare and expensive chemicals more accessible.