## Researchers progress with engineering E. coli to produce pinene for biosynthetic alternative to rocket fuel

##### 16 March 2014
 Energy density of petroleum-based fuels and advanced biofuels. Shown is the heating value of petroleum-based fuels (black) and advanced biofuels (green) as a function of density. Pinene dimers (red) have density and heating value similar to that of JP-10. Credit: ACS, Sarria et al. Click to enlarge.

Recent progress in engineering microbes has resulted in the production of biosynthetic alternatives to gasoline, diesel, and diesel precursors. However, the development of microbial platforms for the production of high-energy density fuels—i.e., tactical fuels for use in aircraft and aircraft-launched missiles—has lagged behind. Existing biosynthetic jet fuels lack the volumetric energy content required to replace high-energy density fuels such as the tactical fuels JP-10, tetrahydrodicy-clopentadiene, and RJ-5.

A team from Georgia Tech, University of California, Berkeley, and the Joint BioEnergy Institute at Lawrence Berkeley National Laboratory has now engineered Escherichia coli bacteria to produce pinene, the immediate precursor to pinene dimers, a biosynthetic alternative to JP-10. Although their work produced a significant increase in yield from earlier attempts, the yield will need to be some 26-times larger for commercial viability, they calculated.

Attaining the volumetric energy content necessary for tactical fuels requires mimicking the strained ring systems found in JP-10 and RJ-5. Recently, pinene dimers have been show to contain high volumetric energy similar to that found in JP-10. Pinene dimers are synthesized via chemical dimerization of pinene, a bicyclic terpene.… Today, the major source of pinene is turpentine, a byproduct of the wood pulp industry

… Given the large quantities of pinene dimers needed for use as a biofuel, engineering microorganisms to produce pinene from inexpensive sugars may be the most convenient and cost-effective approach to obtaining the necessary quantities of this advanced biofuel precursor.

—Sarria et al.

The researchers had previously demonstrated pinene production of ∼1 mg/L from ionic liquid-treated switchgrass. More recently, others engineered E. coli to produce pinene at titers of ∼5 mg/L. Microbial pinene titers have been orders of magnitude lower than those of sesquiterpenes (bisabolene) and diterpenes (taxadiene). Limonene, a different monoterpene, has been produced microbially at a titer of ∼400 mg/L.

The team suggested likely reasons for the low microbial production of pinene may be the toxicity of pinene or GPP to E. coli and enzymatic inhibition.

The researchers combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. A further combinatorial construction produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli.

Using flux balance analysis, we calculated the theoretical yield of pinene produced from E. coli using the mevalonate pathway to be 0.270 g pinene/g glucose. Experimentally, we produced pinene at ∼1.2% of the pathway-dependent calculated theoretical yield, that is, assuming only glucose in the EZ-rich media is used for pinene production. Assuming a break-even price of glucose at the mill to be close to US $0.10/ lb, we calculate that the raw material cost of pinene production, ignoring non-sugar costs, to be approximately$68/kg of pinene.

Assuming raw material costs to be only 50% of the final cost, the final price of pinene would be $136/kg, or$443/gal, at current production levels. Assuming that we could produce pinene at the theoretical yield, the final price would be $1.63/kg, or$5.31/gal of pinene, resulting in a final price of ∼$6.42/gal for pinene dimers (assuming 90% yield and negligible conversion cost), a significant savings over the current price of JP-10 of ∼$25/gal. Commercial viability of microbial pinene for use as tactical fuel therefore requires reaching 26% theoretical yield.

—Sarria et al.

Future engineering of microbial pinene production to reduce inhibition will require addressing the pathway problems at the enzyme level, the team suggested.

Resources

• Stephen Sarria, Betty Wong, Hector García Martín, Jay D. Keasling, and Pamela Peralta-Yahya (2014) “Microbial Synthesis of Pinene,” ACS Synthetic Biology, doi: 10.1021/sb4001382

• Harvey, B., Benjamin, G., Wright, M., and Quintana, R. (2010) “High-density renewable fuels based on the selective dimerization of pinenes,” Energy Fuels 24, 267−273 doi: 10.1021/ef900799c