Metabolic Engineering of an Endogenous Pathway for n-Butanol Production in Saccharomyces Cerevisiae

Increasing concerns about depleting crude reserves have renewed interests in the biological production of n-butanol, due to its desirable fuel properties and its role as a platform chemical. Currently, the global market of n-butanol has been estimated over $5 billion, with a predicted 4.7 % expansion per year (Mascal 2012). n-Butanol can be produced commercially from fossil fuels, through generally expensive and environmentally unfriendly routes. The biological production of n-butanol is traditionally through ABE (acetone-butanol-ethanol) process by Clostridia species. The limitation of this process includes lack of genetic tools, undesired byproducts, and low alcohol tolerance of Clostridia species. To overcome these hurdles, various attempts have been made to transfer the Clostridial n-butanol pathway to more suitable industrial organisms including Escherichia coli and Saccharomyces cerevisiae. This CoA-dependent pathway enables an impressive production titer (30 g/L) in E. coli (Shen et al. 2011). However, much lower productions have been reported for S. cerevisiae, with reports of only 2.5 mg/L (Steen et al. 2008) and 16.3 mg/L (Krivoruchko et al. 2013) from glucose.  At the same time, n-butanol production at grams per liter in E. coliby the amino-acid metabolic pathway (Atsumi et al. 2008) and the reversed β-oxidation pathway (Dellomonaco et al. 2011) were reported.

Although the current production level of n-butanol in S. cerevisiae is far less promising, there are advantages to utilize S. cerevisiae as an n-butanol producer, due to its high n-butanol tolerance, phage resistance, well-established genetic tools and compatibility to current industrial infrastructure (Si et al. 2014). A recent paper suggested a metabolic route for n-butanol production from l-glycine in S. cerevisiae (Branduardi et al. 2013). The production relies on the addition of substrate, L-glycine. In addition, the pathway cannot avoid the production of isobutanol, and n-butanol and isobutanol accumulate simultaneously.

Here we report the discovery, characterization and engineering of an endogenous n-butanol pathway in S. cerevisiae. The pathway was switched on to produce a large amount of n-butanol from glucose (120 mg/L) by introduction of a single gene deletion adh1Δ (Si et al. 2014). Little isobutanol (below 12 mg/L) was produced in this pathway. In addition to the deletion of ADH1 for n-butanol production, we engineered yeast to have an increased flux toward threonine, the precursor metabolite for n-butanol biosynthesis. Elimination of competing pathways could increase the n-butanol titer by up to 106%. The pathway downstream of threonine has been over expressed in mitochondria or cytosol resulting in a 87 % increase in the final titer when the pathway is in mitochondria and a 40 % increase when the same pathway is in cytosol. Construction of a cimA mediated pathway further increased the n-butanol titer to 349 mg/L, which is the highest n-butanol titer ever reported and represents a 21-fold improvement compared to previous values reported in S. cerevisiaefrom glucose (Krivoruchko et al. 2013). A combination of beneficial manipulations and strain engineering is still in progress for further strain improvement.

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