The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to engineer brewer's yeast for a more consistent, cost-effective, and sustainable brewing process. The flowers of hop plants provide both bitterness and "hoppy" flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it expensive and difficult to achieve a consistent hoppy taste in beer. The goal is to engineer brewer's yeast to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Over the last two decades, consumers have displayed an increasing preference for beers that contain hoppy flavor. Hops are an expensive ingredient for breweries to source (total domestic hop sales have tripled over the last 10 years due to heightened demand) and a crop that requires a large amount of natural resources: ~100 trillion liters of water is required for annual irrigation of domestic hops. Reducing the reliance of the brewing industry on hops would significantly increase the sustainability of the brewing process by decreasing water usage, also leading to the commercial benefits of decreased brewing costs and greater consistency of beer flavor. This SBIR Phase I project proposes to engineer the biosynthesis of hop-derived monoterpenes in brewer's yeast to meet the following industrial performance requirements: 1) Normal propagation and fermentation characteristics, 2) precise and robust production of monoterpenes at scale, and 3) the absence of off-flavors. The proposed research sets out to test whether the expression of 5 key biosynthesis pathway enzymes can be expressed in a way that each of these performance requirements is met in a single strain. Recently developed synthetic biology tools will be used to create hundreds of DNA constructs, each containing a different combination of promoters driving expression of the 4 corresponding genes (designs). Incorporating genetically stable DNA constructs into brewer's yeast is complicated by their polyploid genome. To overcome this difficulty, preliminary work was conducted to develop a Cas9-mediated integration strategy, which leverages a colorometric assay to assist with the challenge of identifying stable pathway transformants. Strains will be constructed and evaluated in two subsequent rounds. In the first round, a diverse set of designs will be explored, and in a second round, strains will be constructed based on the designs that most closely meet with performance requirements and avoid production of potential off-flavors.
