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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...

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ⌬pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp 2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.