Berkeley Lab scientists produce advanced biofuels in ‘one-pot’ recipe

Researchers at the US Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) have engineered a strain of bacteria that enables a “one-pot” method for producing advanced biofuels from a slurry of pre-treated plant material.

The traditional method for producing advanced biofuels is a three-step process: Liquid salts break down plant matter. Enzymes convert the plant matter into sugars. Then, microbes like E.coli convert the sugars to biofuels. Those steps had to be broken up because the enzymes and microbes could not survive the salts. 

Now Berkley Lab researchers have engineered E.coli that can tolerate the liquid salt used to break apart plant biomass into sugary polymers. 

Marijke Frederix, a postdoctoral researcher involved in the biofuels research, established that an amino acid mutation in the gene rcdA, which helps regulate various genes, leads to an E.coli strain that is highly tolerant to ionic liquids, providing an important piece to the puzzle.

They used this strain as the foundation to build on earlier work – including the ionic-liquid-tolerant enzymes – and take the steps further to the one-pot biofuel finishing line.

“Armed with the rcdA variant, we were able to engineer a strain of E.coli that could not only tolerate ionic liquid, but that could also produce ionic-liquid-tolerant enzymes that chew up the cellulose, make sugars, eat it and make biofuels,” said Frederix.

“E.col remains the workhorse microbial host in synthetic biology, and in our study, using the ionic-liquid-tolerant E.colistrain, we can combine many earlier discoveries to create an advanced biofuel in a single pot.”

While ethanol may be one of the more common products to emerge from this process, researchers have looked to more advanced biofuels that can pack more energy punch. In this case, they used production pathways also developed at Joint BioEnergy Institute (JBEI) previously, and produced d-limonene, a precursor to jet fuel.

“Ultimately, we at JBEI hope to develop processes that are robust and simple where one can directly convert any renewable plant material to a final fuel in a single pot,” said study principal investigator Aindrila Mukhopadhyay, vice president of the Fuels Synthesis Division at the JBEI, a DOE Bioenergy Research Center at Berkeley Lab.

“This study puts us one step closer to this moonshot,” Mukhopadhyay added.