Advancements towards the production of biofuels from loblolly pine: Increases in terpenoid content and development of innovative and high-throughput extractions and analysis of terpenoids

Loblolly Pine (Pinus taeda L.), an important feedstock in the pulp and paper industry, synthesizes and exudes terpene oleoresin that contains terpenoids as a defense mechanism against stem boring insects. Pine monoterpenes and diterpenoids, derived from isoprene units, are renewable alternatives to petroleum-based products including solvents, fragrances and flavors and have shown potential for use as a biofuel. We have employed several strategies to increase terpene production in pine stems, including engineering the DXP pathway, introducing a novel pathway from ribulose phosphate to DXP, and overexpression of prenyl transferases, terpene synthases, and key regulatory genes. Several extraction and analysis methods have been tested to efficiently characterize the terpenoid content of pine samples. A high-throughput method has been developed, using a 1:1 hexane/acetone solvent system coupled with a low thermal mass modular accelerated column heater fast-GC/FID, to efficiently extract and quantify the terpenoids present in samples obtained from loblolly pine saplings. At least 200 pine samples/week can be extracted and injected on the fast-GC for analysis of total terpenoid content. Additionally, ionic liquids, such as 1-ethyl-3-methylimidazolium acetate, which are capable of disrupting the intricate network of polymer constituents in the wood, have been evaluated for their utility in extraction of terpenes from pine samples while generating a fermentable sugar stream suitable for bioconversion into renewable fuels and chemicals.