Engineering of an Escherichia coli Strain for the Production of 3-Methyl-1-Butanol

Abstract

3-Methyl-1-butanol is a potential fuel additive or substitute. Previously this compound was identified in small quantities in yeast fermentation as one of the fusel alcohols. In this work, we engineered an Escherichia coli strain to produce 3-methyl-1-butanol from glucose via the host's amino acid biosynthetic pathways. Strain improvement with the removal of feedback inhibition and competing pathways increased the selectivity and productivity of 3-methyl-1-butanol. This work demonstrates the feasibility of production of 3-methyl-1-butanol as a biofuel and shows promise in using E. coli as a host for production.Energy and environmental concerns have resulted in an increased interest in the production of alternative fuels from renewable sources. The primary focus of such research thus far has been on the production of bioethanol, with more than 4.8 billion gallons being produced in the United States in 2006 (22). Ethanol, however, cannot completely replace existing petroleum-based fuels, since the high vapor pressure and water content may present problems in engine performance and the supply infrastructure of the current fuel economy. The use of longer-chain alcohols can compensate for some of these issues. Five-carbon alcohols, such as 3-methyl-1-butanol, possess properties that can increase the potential for a biomass-derived replacement for gasoline. With a vapor pressure more than 20-fold lower than that of ethanol and an energy density calculated from the heat of combustion (16) that is more than 80% (28.2 MJ/liter) of that of gasoline (34.8 MJ/liter) (10), 3-methyl-1-butanol can offer advantages over ethanol as a supplement to or replacement for gasoline.3-Methyl-1-butanol is a natural, although minor, product in fungus and yeast fermentation. Recent work with yeast has focused on identifying gene targets, mutations, and pathways for increased 3-methyl-1-butanol production (1,11,21). 3-Methyl-1-butanol is also used as a precursor for synthesis of various chemicals, such as isoamyl acetate, which has been successfully produced from 3-methyl-1-butanol in Escherichia coli (15,23). Fusel oil, a by-product of ethanol distillation which contains a fraction of 3-methyl-1-butanol, has also been shown to be an effective substrate for the biocatalysis of triolein to biodiesel (20). With a wide variety of uses and its potential role as a fuel, the development of a process for the production of 3-methyl-1-butanol is desirable. Here we demonstrate the first design for the production of 3-methyl-1-butanol in Escherichia coli from glucose.Our laboratory has previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway to alcohols (4). In order to produce 3-methyl-1-butanol, the valine and leucine biosynthesis pathways can be used to generate 2-ketoisocaproate (KIC), the precursor to leucine. KIC can then be converted to 3-methyl-1-butanol via a decarboxylation and reduction step. The entire pathway to 3-methyl-1-butanol from glucose is diagrammed in Fi...

Engineering alternative butanol production platforms in heterologous bacteria

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Advanced biofuel production in microbes

“…Continuing with the theme of using amino acid biosynthetic pathways to produce higher alcohols, Connor and Liao [20] improved the production of 3MB, a C 5 alcohol with an energy density of 30.5 MJ/L close to that of gasoline (32 MJ/L). The production of 3MB builds onto previous work by Atsumi et al [17] to produce isobutanol from 2-ketoisovalerate.…”

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

“…Clostriduim were recently expressed in E. coli. Aside from producing ethanol during fermentation, S. cerevisiae is also known to produce higher alcohols and esters from amino acids [19][20][21]. Recently, a similar pathway for higher alcohol production was expressed in E. coli to yield six different straight and branched-chain alcohols, and the same group has demonstrated production of 1.28 g/L of isopentanol by increasing the flux through the desired pathway [ 22].…”