Metabolic Engineering of Saccharomyces Cerevisiae to Produce Butanol

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Microbial Cell Factories

BioMed Central

Open Access

Research

Metabolic engineering of Saccharomyces cerevisiae for the
production of n-butanol
Eric J Steen1,2, Rossana Chan1,3, Nilu Prasad1,3, Samuel Myers1,3, Christopher J Petzold1,3, Alyssa Redding1,3, Mario Ouellet1,3 and Jay D Keasling*1,2,3,4
Address: 1Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, USA, 2Department of Bioengineering, University of California, Berkeley, CA 94720, USA, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and 4Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA Email: Eric J Steen - esteen@berkeley.edu; Rossana Chan - rossana18@gmail.com; Nilu Prasad - nilu_prasad12@yahoo.com; Samuel Myers - smyers84@gmail.com; Christopher J Petzold - CJPetzold@lbl.gov; Alyssa Redding - ARedding@lbl.gov; Mario Ouellet - MOuellet@lbl.gov; Jay D Keasling* - keasling@berkeley.edu * Corresponding author

Published: 3 December 2008
Microbial Cell Factories 2008, 7:36

doi:10.1186/1475-2859-7-36

Received: 14 October 2008
Accepted: 3 December 2008

This article is available from: http://www.microbialcellfactories.com/content/7/1/36 © 2008 Steen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol.
Results and conclusion: Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

Background
Soaring energy costs and increased awareness of global
warming have motivated production of renewable, biomass-derived fuels and chemicals. The reasons for producing alternatives to ethanol, the current biofuel standard, are numerous and clear: ethanol suffers from low energy

density, it is hydroscopic, it cannot be piped, and it is

costly to distill, an aspect that detracts from the total
energy output of its production. Ideally, biofuels will
require minimal energy to separate from fermentation
broths, be non-toxic to the host micro-organism, and be
efficiently produced from a variety of feedstocks [1]. Compared to ethanol, n-butanol is more hydrophobic, has a higher energy density, can be transported through existing
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Microbial Cell Factories 2008, 7:36

pipeline infrastructure, and can be mixed with gasoline at
any ratio. Thus, n-butanol is a substantially better biofuel than ethanol.
n-Butanol can be produced either chemically from petroleum or fermentatively in a variety of Clostridial species. Advances in biotechnology and increased...
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