Caryophyllene, a natural bicyclical sesquiterpene compound, and its alcohol are widely used in citrus flavors, spice blends, soaps, detergents, creams, lotions as well as in various food and beverage products. Recent studies have revealed that beta-caryophyllene exhibits a wide range of biological activities including anti-inflammatory, anti-cancer, anti-genotoxic capacity, neuroprotection…etc. Besides the biological activities, recent studies suggested blending of hydrogenated sesquiterpanes (carophyllanes, in particular, which have a moderate cetane number and only moderately high viscosity) with synthetic branched paraffins to raise cetane and reduce viscosity. Therefore, caryophyllene and its isomers have been deemed to be among the top three most promising jet fuel compounds with increased energy density. In this study, caryophyllene, caryolan-1-ol, and other terpenes were significantly produced by heterologous expressing a mevalonate pathway with a geranyl pyrophosphate synthase (GPPS), a caryophyllene synthase, and a caryolan-1-ol synthase into an E.coli strain. With the optimization of metabolic flux through four different pathway constructs and fermentation parameters, the engineered strains yielded 448.7mg/L total terpene including 405.9 mg/L sesquiterpene, 42.7 mg/L monoterpene,100 mg/L of caryophyllene, 10 mg/L of caryolan-1-ol. Furthermore, an algal hydrolysate was used by the engineered strain as solo carbon source for the production of caryophyllene and other terpene compounds. Under optimal fermentation conditions, the total terpene, sesquiterpene, and caryophyllene reached 360.3-, 322.5-, and 75.2 mg/L, respectively. The highest yields achieved were 47.9 mg total terpene/ g algae and 10.0 mg caryophyllene/ g algae, respectively, which is about ten times higher than essential oil yield extracted from plant tissue. This study was the first report of caryophyllene production using algae biomass as feedstock. The study provides a sustainable alternative for caryophyllene and its alcohol production as potential candidates for next generation aviation fuels and pharmaceutical applications.
The feasibility of converting algal protein to mixed alcohols has recently been demonstrated with an engineered E. coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the four highest activity YqhD mutants were selected for combination with two IlvC mutants, both accepting NADH as a redox cofactor, for modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E. coli strain, subtype AY3, with increased fusel alcohol yield of ~ 60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.
Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.
Recent strategies for algae-based biofuels have primarily focused on biodiesel production by 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 algae biomass. Therefore, comprehensive utilization of algal biomass for production of multipurpose intermediate- to high-value bio-based products will promote scale-up of algae production and processing to commodity volumes. Terpenes are hydrocarbon and hydrocarbon-like (C:O>10:1) compounds with high energy density, and are therefore potentially promising candidates for the next generation of value added bio-based chemicals and “drop-in” replacements for petroleum-based fuels. In this study, we demonstrated the feasibility of bioconversion of proteins into sesquiterpene compounds as well as comprehensive bioconversion of algal carbohydrates and proteins into biofuels. To achieve this, the mevalonate pathway was reconstructed into an E. coli chassis with six different terpene synthases (TSs). Strains containing the various TSs produced a spectrum of sesquiterpene compounds in minimal medium containing amino acids 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, and was achieved by applying a strategy to minimize mevalonate accumulation in vivo. The highest yields of total terpene were produced under reduced IPTG induction levels (0.25 mM), reduced induction temperature (25°C), and elevated substrate concentration (20 g/L amino acid mixture). A synthetic bioconversion consortium consisting of two engineering E. coli strains (DH1-TS and YH40-TS) with reconstructed terpene biosynthetic pathways was designed for comprehensive single-pot conversion of algal carbohydrates and proteins to sesquiterpenes. The consortium yielded the highest total terpene yields (187 mg/L) at an inoculum ratio 2:1 of strain YH40-TS: DH1-TS, corresponding to 31 mg fuel/g algae biomass ash free dry weight. This study therefore demonstrates a feasible process for comprehensive algal biofuel production.
Increasing energy costs, the dependence on foreign oil supplies, and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. The strategy for producing next-generation biofuels must include efficient processes for biomass conversion to liquid fuels and the fuels must be compatible with current and future engines. Unfortunately, biofuel development generally takes place without any consideration of combustion characteristics, and combustion scientists typically measure biofuels properties without any feedback to the production design. We seek to optimize the fuel/engine system by bringing combustion performance, specifically for advanced next-generation engines, into the development of novel biosynthetic fuel pathways. Here we report an innovative coupling of combustion chemistry, from fundamentals to engine measurements, to the optimization of fuel production using metabolic engineering. We have established the necessary connections among the fundamental chemistry, engine science, and synthetic biology for fuel production, building a powerful framework for co-development of engines and biofuels.