SBIR Phase I: Engineering biocatalysts for biomanufacturing of medicinal opioids

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力是提高从自然界复杂药物分子的供应。目前超过60%的药物是天然产品，或来自天然产品，其中约一半来自植物。给定植物分子的治疗活性编码在其化学结构中，其通过专门的代谢途径生物合成。尽管这些生物合成效率很高，但植物在专门的细胞和组织类型中积累了相对少量的有效代谢物，从而限制了从植物中获得基本药物。类阿片克服了植物疗法的这一局限性;类阿片的结构复杂性排除了商业规模的化学合成，在缺乏这种合成来源的情况下，唯一可行的替代方法是从罂粟中提取天然阿片剂。该项目旨在使药用阿片类药物的生物合成在微生物宿主中成为可能。这种合成生物学方法将把阿片类药物的生产转移到发酵设施中，并腾出每年用于种植罂粟的10万公顷耕地。这项研究产生的颠覆性技术将首次提供本地，可扩展，安全的药用阿片类药物供应。 该SBIR第一阶段项目提议开发一种药用阿片类药物的微生物生产系统，以取代现有的罂粟供应。最近在贝克酵母中构建了一个完整的阿片类生物合成途径，表明该技术具有巨大的潜力，可为20亿美元的阿片类活性药物成分（API）市场提供支持。在这个SBIR项目中解决的关键技术障碍是增强限速酶的活性，这些酶催化五环阿片支架构建中的关键步骤。植物酶的目标类别在异源宿主如酵母中表达很差，并且必须是膜定位的。拟议的研究采用三种方法来支持这些酶：1）调节表达以保护内膜环境并促进活性，2）构建具有增强稳定性的N-末端嵌合蛋白，以及3）鉴定支持这些酶的催化功能的伴侣酶。目标是消除现有生产菌株中的瓶颈步骤，以实现大于1 g/L的商业相关滴度。其结果将是一个新的生产系统，以更低的成本提供活性药物成分，具有更大的可用性和分子种类，更短的交货时间，以及对阿片类药物治疗的医疗需求的急性反应。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to enhance the supply of complex pharmaceutical molecules from nature. Over 60% of current drugs are natural products, or are derived from natural products, and of these approximately half are from plants. The therapeutic activity of a given plant molecule is encoded in its chemical structure, which is biosynthesized by a specialized metabolic pathway. Despite the efficiency of these biosyntheses, plants accumulate relatively small amounts of the potent metabolites in specialized cells and tissue types, thereby restricting the availability of essential medicines from plants. The opioids exemplify this limitation of plant therapeutics; the structural complexity of the opioids precludes chemical synthesis at commercial scale, and in the absence of this synthetic source the only viable alternative is to extract natural opiates from opium poppy. This project proposes to make the biosynthesis of medicinal opioids possible in a microbial host. This synthetic biology approach will move opioid production into fermentation facilities and free up the 100,000 hectares of arable land used each year for poppy crops. The disruptive technology resulting from this research will for the first time provide for a local, scalable, secure supply of medicinal opioids. This SBIR Phase I project proposes to develop a microbial production system for medicinal opioids that will displace the existing supply from opium poppies. A complete opioid biosynthesis pathway was recently constructed in Baker's yeast, demonstrating that this technology holds enormous potential for supplying the $2B opioid active pharmaceutical ingredient (API) market. The key technical hurdle addressed in this SBIR project is to enhance the activity of rate-limiting enzymes that catalyze key steps in the construction of the five-ring opioid scaffold. The target class of plant enzymes is poorly expressed in heterologous hosts such as yeast and must be membrane localized. The proposed research takes three approaches to support these enzymes: 1) tuning expression to conserve the endomembrane environment and promote activity, 2) constructing N-terminal chimeric proteins with enhanced stability, and 3) identifying partner enzymes that support the catalytic function of these enzymes. The goal is to remove the bottleneck steps in existing production strains to allow for commercially-relevant titers of greater than 1 g/L. The outcome will be a new production system that offers active pharmaceutical ingredients at lower cost, with greater availability and variety of molecules, shorter lead times, and acute responsiveness to the medical demand for opioid therapeutics.