(234e) Biobutanol From Yeast: A Synergistic Genome and Protein Engineering Approach

Although bioethanol is currently the leading candidate to replace gasoline as transportation fuel, biobutanol has been recently gaining favor over ethanol. Having better properties as fuel such as higher energy content, higher octane number, and lower latent heat, it promises a better long-term solution to transportation fuel requirements. In spite of its advantages, there are two major issues plaguing economical production of biobutanol. The first issue is that the natural producers of butanol are anaerobic gram-positive bacteria of the genus Clostridia, which are relatively difficult to culture. And while butanol fermentation using Clostridia has been studied extensively for decades, very few genetic tools are available to engineer them for improved titer and productivity. The second issue is that butanol is toxic to bacteria at concentrations over 20 g/L, far below its solubility in water (~70 g/L). This remains an unresolved issue with current Clostridial fermentations. Heterologous butanol production in more congenial organisms such as E. coli or S. cerevisiae (common yeast) could alleviate some problems associated with Clostridial fermentation. Given the choice between the two, S. cerevisiae might be the better option being a far more resilient organism. Butanol production from glucose has been demonstrated in E. coli, but the same has only been shown using galactose as carbon source in yeast. Unlike glucose, galactose is not readily available from plant biomass, and is a relatively expensive substrate. Here we demonstrate fermentative butanol production is possible using recombinant yeast.

Expression of the entire six-step pathway from Clostridia, or of homologs of each enzyme from various organisms ranging from soil bacteria to protists, did not yield any butanol. Analysis revealed the inability to solubly express one key protein impeded butanol production. We performed directed evolution to engineer its solubility and found a mutant that was solubly expressed in yeast. Expression of this mutant with the remainder of the pathway finally yielded a functional butanol biosynthetic pathway. To increase the flux of carbon toward butanol genes encoding for undesired byproducts were deleted. With the synergistic effect of protein engineering to create a functional pathway, and genome engineering to increase carbon flux, butanol productivity was increased. Currently, further metabolic engineering using MIRAGE (mutagenic inverted repeat assisted genome engineering) for gene deletions is underway to decrease byproducts and increase flux toward butanol. In addition, we are overexpressing several heterologous proteins to increase availability of precursor metabolites.