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

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力是设计啤酒酵母，以实现更一致，更具成本效益和可持续的酿造过程。啤酒花植物的花为啤酒提供苦味和“啤酒花”风味。然而，啤酒花是一种水和能量密集型作物，并且精油含量变化很大，这使得啤酒中难以获得一致的啤酒花味道。 其目标是通过整合来自酵母、薄荷和罗勒的重组DNA，使啤酒酵母生物合成赋予啤酒花风味的芳香单萜分子。在过去的二十年里，消费者对含有啤酒花风味的啤酒表现出越来越多的偏好。啤酒花对于啤酒厂来说是一种昂贵的原料（由于需求增加，国内啤酒花总销售额在过去10年中增加了两倍），而且这种作物需要大量的自然资源：每年灌溉国内啤酒花需要约100万亿升水。减少酿造工业对啤酒花的依赖将通过减少用水量来显著提高酿造工艺的可持续性，还导致降低酿造成本和提高啤酒风味一致性的商业效益。 该SBIR第一阶段项目提出在啤酒酵母中设计啤酒花衍生单萜的生物合成，以满足以下工业性能要求：1）正常繁殖和发酵特性，2）大规模精确和稳健地生产单萜，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中的每一种。 最近开发的合成生物学工具将用于创建数百个DNA构建体，每个构建体包含驱动4个相应基因表达的启动子的不同组合（设计）。 将遗传稳定的DNA构建体转化到啤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，进行了初步工作以开发Cas9介导的整合策略，其利用比色测定法来协助鉴定稳定途径转化体的挑战。 菌株将在随后的两轮中构建和评价。 在第一轮中，将探索一组不同的设计，在第二轮中，将根据最符合性能要求并避免产生潜在异味的设计构建菌株。

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.