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

SBIR 第一阶段：工程啤酒酵母用于萜烯的生物合成，用于生产风味决定因素

• 批准号: 1722376
• 负责人: Charles Denby
• 金额: $ 22.5万
• 依托单位: Berkeley Brewing Science Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1722376&HistoricalAwards=false
• 关键词: SBIR; Phase; Engineering; brewer; yeast

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.

该小企业创新研究 (SBIR) 项目更广泛的影响/商业潜力是对啤酒酵母进行工程设计，以实现更一致、更具成本效益和可持续的酿造过程。啤酒花植物的花朵为啤酒提供苦味和“啤酒花”风味。然而，啤酒花是一种水和能源密集型作物，精油含量差异很大，这使得啤酒价格昂贵且难以实现一致的啤酒花味道。 目标是对啤酒酵母进行工程改造，生物合成芳香单萜分子，通过掺入来自酵母、薄荷和罗勒的重组 DNA，为啤酒赋予啤酒花风味。在过去的二十年中，消费者对含有啤酒花风味的啤酒表现出越来越大的偏好。啤酒花是啤酒厂采购的一种昂贵原料（由于需求增加，过去 10 年国内啤酒花总销量增加了两倍），而且是一种需要大量自然资源的作物：每年灌溉国内啤酒花需要约 100 万亿升水。减少酿造业对啤酒花的依赖将通过减少用水量显着提高酿造过程的可持续性，同时带来降低酿造成本和提高啤酒风味一致性的商业利益。 该 SBIR 第一阶段项目建议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）正常的繁殖和发酵特性，2）精确而稳健的单萜大规模生产，以及 3）无异味。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个菌株中满足这些性能要求中的每一个。 最近开发的合成生物学工具将用于创建数百个 DNA 构建体，每个构建体都包含驱动 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 构建体整合到啤酒酵母中因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来开发 Cas9 介导的整合策略，该策略利用比色测定来协助应对识别稳定途径转化体的挑战。 菌株将在随后的两轮中构建和评估。 在第一轮中，将探索一系列不同的设计，在第二轮中，将根据最接近性能要求并避免产生潜在异味的设计构建菌株。