Development of a cannabinoid yeast factory: improving geranyl pyrophosphate production through metabolic engineering

摘要 随着人口老龄化，人们越来越担心神经系统疾病对老年人的影响 医疗保健系统。神经系统疾病(如阿尔茨海默病)是#年增长最快的死亡原因 而美国新药的开发一直非常缓慢。天然产品是强大的 新药发现的来源--最近，大麻素已被确定为一类普通药物 具有广泛的治疗效果的天然化合物，包括治疗 神经退行性疾病。事实上，对大麻素的研究导致了内源性大麻素的发现 系统(ECS)，被认为是神经系统过程中最重要的调节器之一，并 神经系统疾病的靶向系统。从那时起，大麻类物质被发现对 治疗疼痛、神经炎症和骨丢失等多种疾病。大麻类物质可以在 许多植物；最著名的是大麻，从大麻中分离出各种大麻类物质 并被证明与医学相关。然而，很难获得高纯度的大麻素，抑制了 研究并使大规模商业化生产成为问题。一种生物合成的生产方法 这些天然产品不仅解决了这些问题，而且使人们能够详细地了解 产品化学，进一步推动医学研究和药物开发进入ECS。 为了能够开发天然大麻素作为治疗剂，利布雷德开发了一种 选育酵母生物合成大麻素生产/药物发现平台 大麻代谢途径的一部分。在酵母中生产大麻素是一种理想的方法。 平台，因为发酵和基因工程是成熟的，低成本，可扩展的。利布雷德氏病 生物合成大麻素生产平台是模块化的；通过添加或移除不同的表达 酶，不同的大麻素可以根据需要产生，甚至是低丰度的大麻素。 当然。使用这种方法，在初步工作中，我们已经在酵母中生产了大麻二酚酸(CBDA)， 世界上第一个在植物外通过生物合成产生的大麻素。尽管这一概念验证工作 结果表明，酵母可以产生大麻素，CBDA的产量受到酵母的限制 一种特定的化学中间体--焦磷酸香叶醇(GPP)的可用性。为了让我们的平台 对于研究和大麻素生产是可行的，我们将通过减少将其转移到 通过敲除或下调分流代谢途径以及增加GPP来替代产品 制作。

Abstract As the population ages there is a growing concern about the impact of neurological diseases on the healthcare system. Neurological diseases (e.g. Alzheimer’s Disease) are the fastest growing cause of death in the United States and the development of new drugs has been very slow. Natural products are powerful sources for the discovery of new medications—recently, cannabinoids have been identified as a general class of natural compounds with a wide range of proven therapeutic effects, including treatment of neurodegenerative diseases. In fact, studies of cannabinoids led to the discovery of the endocannabinoid system (ECS), regarded as one of the most important modulators of nervous system processes and a targetable system for neurological diseases. Since then, cannabinoids have been found to have efficacy in treating pain, neuroinflammation, and bone loss, among many other conditions. Cannabinoids can be found in many plants; the most well-known is Cannabis sativa, from which a variety of cannabinoids have been isolated and shown to be medically relevant. However, it is difficult to source cannabinoids in high purity, inhibiting research and making large scale commercial production problematic. A biosynthetic approach to producing these natural products not only addresses these problems but allows a detailed understanding of the natural product chemistry, furthering medical research and pharmaceutical development into the ECS. To enable the development of natural cannabinoids as therapeutic agents, Librede has developed a biosynthetic cannabinoid production/drug discovery platform by genetically engineering yeast with selected portions of the Cannabis sativa metabolic pathways. The production of cannabinoids in yeast is an ideal platform because fermentation and genetic engineering are well-established, low cost, and scalable. Librede’s biosynthetic cannabinoid production platform is modular; by adding or removing expression of different enzymes, different cannabinoids can be produced as desired, even cannabinoids produced in low abundance naturally. Using this approach, in preliminary work we have produced Cannabidiolic acid (CBDA) in yeast, the world’s first biosynthetically-produced cannabinoids outside of plants. Although this proof-of-concept work showed that cannabinoids can be produced by yeast, the yield of CBDA production was limited by the availability of a specific chemical intermediate, geranyl pyrophosphate (GPP). In order to make our platform feasible for research and cannabinoid production, we will improve GPP levels by reducing its diversion to alternative products by knocking out or down-regulate diverting metabolic pathways, as well as increasing GPP production.