Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors |
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| Institution: | 1. Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA;2. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA;3. Department of Chemistry, University of California, Berkeley, CA 94720, USA;4. Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA;1. Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA;2. Center for Bioenergy and Bioproducts, Auburn University, Auburn, AL 36849, USA;1. Department of Biosciences & Bioengineering, Indian Institute of Technology, Guwahati, Assam 781039, India;2. DBT-PAN IIT Centre for Bioenergy, Indian Institute of Technology, Guwahati, Assam 781039, India;1. School of Bioscience and Biotechnology, Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia;2. Department of Chemical and Process Engineering, Faculty of Engineering, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia;3. Department of Applied Microbiology, Faculty of Applied Sciences, Taiz University, 6803 Taiz, Yemen;1. Laboratory of Environmental Technology, INET, Tsinghua University, Beijing, 100084, PR China;2. Beijing Key Laboratory of Radioactive Waste Treatment, INET, Tsinghua University, Beijing, 100084, PR China |
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| Abstract: | Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AADD485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aadD485G) ABE products resulted in a blend with nearly 50 wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L?1 while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8 g L?1 of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production. |
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| Keywords: | Biodiesel Solvent tolerance Heterogeneous catalysis Butanol Metabolic engineering |
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