Important Advances in Fuels & Chemicals from Bacteria, Biomass,

Exciting E. Coli Biorefinery from JBEI

GCC

E. coli bacteria normally cannot grow on switchgrass, but JBEI researchers engineered strains of the bacteria to express several enzymes that enable them to digest cellulose and hemicellulose and use one or the other for growth. These cellulolytic and hemicellulolytic strains of E. coli, which can be combined as co-cultures on a sample of switchgrass, were further engineered with three metabolic pathways that enabled the E. coli to produce the fuel substitute or precursor molecules.

The JEBI team chose to implement pathways that produce alcohols, linear hydrocarbons, or branched-chain hydrocarbons to test the integration of the biomass-consumption pathways with the “extensive” biosynthesis capabilities of E. coli. The team chose:

Biodiesel. Biodiesel can be made by E. coli in vivo in the form of fatty-acid ethyl esters (FAEE). They encoded a six-gene FAEE production pathway on a single plasmid and introduced the construct into a strain of E. coli. Using a co-culture of two strains grown in minimal medium containing 5.5% w∕vol IL-treated switchgrass, they produced 71 ± 43 mg∕L of FAEE. This corresponds to 80% of the estimated yield obtainable with this pathway from the amount of sugars anticipated to be released from 5.5% switchgrass by the Cel and Xyn10B enzymes.

Butanol. Butanol has been proposed as a gasoline replacement because it is fully compatible with existing internal combustion engines. Based in part on previous work, they constructed a heterologous butanol pathway encoded on a single plasmid. A co-culture yielded 28 ± 5 mg∕L butanol from defined rich medium containing 3.3% w∕vol IL-treated switchgrass. A control strain produced 8 ± 2 mg∕L butanol from pretreated switchgrass.

Pinene. The monoterpene pinene is an immediate chemical precursor to a potential jet fuel. The pinene synthesis pathway was encoded on a single plasmid. A co-culture yielded 1.7 ± 0.6 mg∕L pinene from pretreated switchgrass.

The pre-treatment of the switchgrass with ionic liquids was essential to this demonstration, according to Gregory Bokinsky, a post-doctoral researcher with JBEI’s synthetic biology group and lead author of the PNAS paper.

If properly optimized, I suspect you could use ionic liquid pre-treatment on any plant biomass and make it readily digestible by microbes. For us it was the combination of biomass from the ionic liquid pretreatment with the engineered E. coli that enabled our success.

—Gregory Bokinsky

The JBEI researchers also attribute the success of this work to the “unparalleled genetic and metabolic tractability” of E. coli, which over the years has been engineered to produce a wide range of chemical products. However, the researchers believe that the techniques used in this demonstration should also be readily adapted to other microbes. _GCC

Another significant advance from the JBEI is a beefing up of the starch content in switchgrass, which will make conventional fermentation of alcohols from switchgrass much easier.

The team of JBEI researchers, working with researchers at the U.S. Department of Agriculture’s Agricultural Research Service (ARS), has demonstrated that introducing a maize (corn) gene into switchgrass, a highly touted potential feedstock for advanced biofuels, more than doubles (250 percent) the amount of starch in the plant’s cell walls and makes it much easier to extract polysaccharides and convert them into fermentable sugars. _BrianWestenhaus

Amyris is raising the stakes in the biomass-to-chemicals market with a significant acceleration of its Biofene (farnesene) product, via expansion of its partnership with oil giant Total. Farnesene is an important precursor for the production of multiple high value chemicals, including fuels.

Amyris has developed advanced microbial engineering and screening technologies that modify the way microorganisms process sugars. Amyris is using this industrial synthetic biology platform to design microbes, primarily yeast, and use them as living factories in established fermentation processes to convert plant-sourced sugars into renewable chemical and transportation fuel products.

...Amyris is scaling its Biofene production in Brazil, Europe and the United States through various production arrangements; the company signed its sixth production agreement in October...

Farnesene is a 15-carbon isoprenoid hydrocarbon molecule that forms the basis for a wide range of products varying from specialty chemical applications to transportation fuels such as diesel. When used as a fuel precursor, farnesene can be hydrogenated to farnesane, which has a high cetane number (58). Amyris modifies farnesene to become renewable diesel. _GCC