SBIR Phase I: Engineering biocatalysts for biomanufacturing of medicinal opioids

这一小型企业创新研究(SBIR)项目的更广泛影响/商业潜力是加强来自自然的复杂药物分子的供应。目前超过60%的药物是天然产品，或者是从天然产品中提取的，其中大约一半来自植物。特定植物分子的治疗活性编码在其化学结构中，通过特殊的代谢途径进行生物合成。尽管这些生物合成的效率很高，但植物在特定的细胞和组织类型中积累了相对较少的有效代谢物，从而限制了从植物中获得基本药物。阿片类药物体现了植物疗法的这一局限性；阿片类药物的结构复杂，无法进行商业规模的化学合成，在没有这种合成来源的情况下，唯一可行的替代办法是从鸦片罂粟中提取天然阿片剂。该项目建议使在微生物宿主中生物合成药物阿片类药物成为可能。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，并释放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当地、可扩展的、安全的药用阿片类药物供应。SBIR第一阶段项目提议开发一种用于医用阿片类药物的微生物生产系统，该系统将取代罂粟的现有供应。最近在贝克酵母中构建了一个完整的阿片生物合成途径，表明这项技术在供应价值20亿美元的阿片活性药物成分(API)市场方面具有巨大的潜力。该SBIR项目解决的关键技术障碍是提高限速酶的活性，这些限速酶催化构建五环阿片类药物支架的关键步骤。植物酶的靶标在异源宿主如酵母中的表达很低，必须定位在膜上。这项研究采取了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)构建具有更高稳定性的N末端嵌合蛋白，以及3)寻找支持这些酶的催化功能的伙伴酶。其目标是消除现有生产菌株中的瓶颈步骤，使与商业相关的效价超过1 g/L。结果将是一个新的生产系统，它以更低的成本提供活性药物成分，具有更多的可获得性和分子的多样性，更短的交货期，并对阿片类药物的医疗需求做出快速反应。

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.