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JBEI team engineers E. coli for one-pot production of bio-jet fuel precursor from ionic-liquid-pretreated biomass

A team led by researchers at the DOE’s Joint BioEnergy Institute (JBEI) in Emeryville, CA, has engineered E. coli bacteria for the one-pot production of the monoterpene bio-jet fuel precursor D-limonene from ionic-liquid-pretreated cellulose and switchgrass. A paper on their work is published in the RSC journal Green Chemistry.

The ionic liquid 1-ethyl-3-methylimidazolium acetate is highly effective in deconstructing lignocellulose, but leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. The JBEI researchers discovered a strain of E. coli that is tolerant to that ionic liquid due to a specific mutation. They engineered this strain to express a D-limonene production pathway.

They also expressed a cellulase also tolerant to the ionic liquid in the engineered bacteria. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel precursor D-limonene in a one-pot process.

Terpene-based compounds provide a range of candidates with energy content and combustion properties that make them suitable for gasoline, diesel as well as jet fuel needs.

Monoterpenes are 10-carbon (C10) terpenes derived from two C5 isoprene units.

Recent progress in consolidated bioprocessing addresses the cost of the saccharification enzymes and has led to the development of a variety of strains that express cellulase enzymes and in some examples are also coupled to a production pathway. However, these engineered strains will also be hampered by growth inhibitory compounds that are routinely present in pretreated hydrolysates, making the use of industrially relevant hydrolysates challenging. In the case of IL pretreated hydrolysates, the toxicity of residual ILs to the microbial production host is the major 8 challenge to optimizing an integrated process, and has led to studies that focus on discovery of tolerance bestowing mechanisms and strain engineering to optimize the expression of such genes.

A necessary next step in the area of biofuel production is to consolidate the findings from studies that have examined and found solutions to different segments of the bioconversion process, from cellulases that may allow efficient saccharification in IL-containing hydrolysate, strains that can withstand residual ILs and synthetic metabolic pathways that convert the biomass-derived sugars to desirable final products.

—Frederix et al.

The engineered E. coli produced D-limonene from IL-pretreated biomass at final titer of 150 mg/L. These levels are lower than the best reported yields obtained using glucose as a carbon source; however, the control E. coli production strain is unable to produce any D-limonene in IL-containing medium.

To our knowledge this is the first demonstration of one-pot microbial D-limonene production from IL-containing saccharified hydrolysate as the substrate.

—Frederix et al.


  • Marijke Frederix, Florence Mingardon, Matthew Hu, Ning Sun, Todd Pray, Seema Singh, Blake Simmons, Jay D Keasling and Aindrila Mukhopadhyay (2016) “Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass” Green Chem. doi: 10.1039/C6GC00642F


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