(54g) Engineering a Synthetic Consortium for Bioconversion of Algal Carbohydrate and Protein to Sesquiterpenes As Advanced Biofuel

The existing strategies for algae-based biofuels have primarily focused on biodiesel production through exploiting high algal lipid yields under nutrient stress conditions. However, under conditions supporting robust algal biomass accumulation, carbohydrate and proteins typically comprise up to ~80 of the ash-free dry weight of microalgae biomass. Therefore, the comprehensive utilization of algal biomass to add high energy density fuel compounds with “fit for purpose” properties into the fuel production process will not only diminish the process cost but also improve overall fuel yield. Terpenes are hydrocarbon and hydrocarbon-like (C:O>10:1) compounds with high energy density, and potentially promising candidates for the next generation of biofuel as “drop-in” replacements for petroleum-based fuels. In this study, we demonstrated the feasibility of bioconversion of protein into sesquiterpene compounds as well as comprehensive bioconversion of algal carbohydrate and protein into biofuels, simultaneously. To achieve this, the mevalonate pathway was reconstructed into an E. coli chassis with six different terpene synthases (TSs). Strains containing each TS produced a wide spectrum of sesquiterpene compounds under minimal medium containing an amino acid mixture as the sole carbon source. The sesquiterpene production was optimized through three different regulation strategies using chamigrene synthase as an example. The highest total terpene titer reached 166 mg/L, by applying a strategy to minimize mevalonate accumulation in vivo.  The strain produced the highest yields of total terpene under reduced IPTG induction levels (0.25 mM), lower induction temperature (25·C), and a higher substrate concentration (20 g/L amino acid mixture).  A synthetic bioconversion consortium consisting of two engineering strains (DH1-TS and YH40-TS) with reconstructed terpene biosynthetic pathways was designed for comprehensive single-pot conversion of algal carbohydrate and protein to sesquiterpene. At an inoculum ratio 2:1 YH40-TS:DH1-TS, the consortium yielded the highest total terpene concentration up to 187 mg/L, corresponding to  was 31 mg fuel/g algae biomass ash free dry weight. This study demonstrated a feasible process for algal biofuel production.