SBIR Phase I: Engineering brewer's yeast for biosynthesis of terpenes for the production of flavor determinants

这个小型企业创新研究(SBIR)项目的更广泛影响/商业潜力是设计啤酒酵母，以实现更一致、更具成本效益和更可持续的酿造过程。啤酒花植物的花朵为啤酒提供了苦涩和“跳跃”的味道。然而，啤酒花既是一种水分密集型作物，也是一种能源密集型作物，而且精油含量差异很大，这使得啤酒价格昂贵，很难在啤酒中保持稳定的啤酒花味道。其目标是改造啤酒酵母，通过整合来自酵母、薄荷和罗勒的重组DNA，生物合成芳香族单萜烯分子，使啤酒具有啤酒花的味道。在过去的二十年里，消费者越来越喜欢含有啤酒花味道的啤酒。啤酒花是啤酒厂采购的一种昂贵的原料(由于需求增加，国内啤酒花总销量在过去10年里增长了两倍)，而且是一种需要大量自然资源的作物：国内啤酒花每年需要大约100万亿升水来灌溉。减少酿造行业对啤酒花的依赖将通过减少用水量显著提高酿造过程的可持续性，还会带来降低酿造成本和提高啤酒风味一致性的商业好处。这个SBIR第一阶段项目建议在啤酒酵母中设计酒花衍生单萜烯的生物合成，以满足以下工业性能要求：1)正常的繁殖和发酵特性，2)精确和稳健的规模化生产单萜烯，以及3)没有异味。拟议的研究旨在测试5种关键生物合成途径酶的表达是否可以在单一菌株中满足这些性能要求。最近开发的合成生物学工具将被用来创建数百个DNA构建体，每个构建体包含驱动4个相应基因(设计)表达的不同启动子组合。将遗传稳定的DNA结构引入酿酒酵母中，由于其多倍体基因组而变得复杂。为了克服这一困难，我们进行了初步工作，以开发一种由Cas9介导的整合策略，该策略利用比色分析来帮助识别稳定的途径转化子。菌株将在随后的两轮中进行构建和评估。在第一轮中，将探索一套多样化的设计，在第二轮中，将基于最符合性能要求并避免产生潜在异味的设计来构建菌株。

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.