(66a) 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. When other logistical concerns about long distance conveyance in pipelines are considered, butanol still comes out a winner over ethanol as it has lower solubility in water, is less corrosive, and has higher vapor pressure. Finally, unlike ethanol, butanol can be directly used in the current design of internal combustion engines.

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, although butanol production has already been demonstrated in E. coli. Having been evolved for high alcohol tolerance for hundreds of years, S. cerevisiae is a far more resilient organism than bacteria and therefore it may be possible to engineer it for higher tolerance to butanol than may be possible for any bacteria.

Expression of the entire six-step pathway from either Clostridia, or of homologs and orthologs of each enzyme from various organisms ranging from soil bacteria to protists, did not yield any butanol. Analysis revealed the inability to solubly express a few key proteins impeded butanol production. Directed evolution experiments were implemented to engineer soluble, functional mutants, which finally yielded a functional butanol biosynthetic pathway. In order to enhance productivity, a new method called MIRAGE (mutagenic inverted repeat assisted genome engineering) was developed to perform efficient, one-step genome modifications in yeast. Metabolic engineering using MIRAGE decreased byproduct formation and increased butanol productivity. Further engineering of enzymes by directed evolution and metabolic engineering by MIRAGE is currently underway to improve fermentative butanol yield.