UC Berkeley

The fermentation of simple sugars to ethanol has been the most successful biofuel process to displace fossil fuel consumption worldwide thus far. However, the physical properties of ethanol and automotive components limit its application in most cases to 10-15 vol% blends with conventional gasoline. Fermentative co-production of ethanol and acetone coupled with a catalytic alkylation reaction could enable the production of gasoline blendstocks enriched in higher-chain oxygenates. Here we demonstrate a synthetic pathway for the production of acetone through the mevalonate precursor hydroxymethylglutaryl-CoA. Expression of this pathway in various strains of Escherichia coli resulted in the co-production of acetone and ethanol. Metabolic engineering and control of the environmental conditions for microbial growth resulted in controllable acetone and ethanol production with ethanol:acetone molar ratios ranging from 0.7:1 to 10.0:1. Specifically, use of gluconic acid as a substrate increased production of acetone and balanced the redox state of the system, predictively reducing the molar ethanol:acetone ratio. Increases in ethanol production and the molar ethanol:acetone ratio were achieved by co-expression of the aldehyde/alcohol dehydrogenase (AdhE) from E. coli MG1655 and by co-expression of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Z. mobilis. Controlling the fermentation aeration rate and pH in a bioreactor raised the acetone titer to 5.1 g L(-1) , similar to that obtained with wild-type Clostridium acetobutylicum. Optimizing the metabolic pathway, the selection of host strain, and the physiological conditions employed for host growth together improved acetone titers over 35-fold (0.14-5.1 g/L). Finally, chemical catalysis was used to upgrade the co-produced ethanol and acetone at both low and high molar ratios to higher-chain oxygenates for gasoline and jet fuel applications. Biotechnol. Bioeng. 2016;113: 2079-2087. © 2016 Wiley Periodicals, Inc.