Total New Energies Inc.

Emeryville, CA, United States

Total New Energies Inc.

Emeryville, CA, United States
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Frederix M.,Joint BioEnergy Institute | Frederix M.,Lawrence Berkeley National Laboratory | Mingardon F.,Total New Energies Inc. | Hu M.,Joint BioEnergy Institute | And 16 more authors.
Green Chemistry | Year: 2016

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) for pretreatment of switchgrass biomass. [C2C1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C2C1Im]-tolerant E. coli strain, where [C2C1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C2C1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C2C1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C2C1Im]-tolerant E. coli strain so as to secrete this [C2C1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process. © 2016 The Royal Society of Chemistry.


Mingardon F.,Total New Energies Inc. | Clement C.,Total New Energies Inc. | Hirano K.,Joint BioEnergy Institute | Hirano K.,Lawrence Berkeley National Laboratory | And 6 more authors.
Biotechnology and Bioengineering | Year: 2015

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha-olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production. © 2015 Wiley Periodicals, Inc.

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