Production Scale-Up and Target Identification of the Antioxidant Ergothioneine

摘要 麦角硫蛋白是人体许多部位含量最丰富的硫醇之一，有几种疾病 与麦角硫蛋白缺乏有关，包括类风湿性关节炎、克罗恩病、神经退行性变 疾病、心血管疾病和糖尿病。动物不会合成麦角硫蛋白，而是会获得 它通过一种麦角硫氨酸特异性转运体(OCTN1)从饮食中释放出来，累积高达mM 在几个器官和组织中的浓度。由于它们的还原电位不同(E0‘=-0.06 V 麦角硫氨酸和E0‘=-0.24V对于谷胱甘肽)，麦角硫氨酸和谷胱甘肽的组合提供 在各种条件下的细胞保护，例如对抗活性氧物种(ROS)和活性物质 氮物种(RNS)。尽管麦角硫蛋白在维持人类健康方面的作用已得到证实，但目前 生产方法已被证明是其广泛使用的瓶颈。具体地说，这种化学物质 合成方法存在立体中心外消旋，副产物异构体有毒。作为一名 因此，大规模生产既具有挑战性，又成本高昂。在我们的初步研究中，我们开发了一种 一种可替代发酵生产10克/升的生物合成方法L 批处理规模。此外，我们还发现了与麦角硫蛋白功能相关的信号通路(S)。这些 在供应和机械理解方面的突破为科学和制造提供了支持 实现麦角硫蛋白的全部商业潜力所必需的，其中包括：其在 化妆品市场，扩展到新市场(例如，作为营养食品的一个组成部分)，或用于药物发现 和发展。在本提案中，我们将解决两个关键的通过/不通过问题，这两个问题将指导 该产品的开发。具体地说，我们将使用我们的 获得专利的麦角硫蛋白生产技术，并精确定位麦角硫蛋白的生物靶点。为此， 目标是： 目的1：实现麦角硫蛋白中试生产(100‘S g~(-1)kg)，纯度99%。 目的2：验证麦角硫蛋白相关信号通路。 结果。在成功完成这些研究后，我们会申请第二期拨款，目的是： 在良好的生产实践下扩大生产规模，以支持更多的市场渗透和扩张。 同时，我们将在体内重新研究麦角硫蛋白对衰老和衰老相关疾病的影响。我们会 还与抗衰老领域的其他科学家联系，寻求合作，包括与 美国国立卫生研究院现有的抗衰老计划(https://www.nia.nih.gov/research/dab/interventions-testing-program-itp).

ABSTRACT Ergothioneine is one of the most abundant thiols in many parts of the human body and several diseases have been linked to ergothioneine insufficiency, including rheumatoid arthritis, Crohn's disease, neurodegenerative diseases, cardiovascular disorders and diabetes. Animals do not synthesize ergothioneine and, instead, obtain it from their diet through an ergothioneine-specific transporter (OCTN1) with accumulation up to mM concentrations in several organs and tissues. Due to their differences in reduction potential (E0' = - 0.06 V for ergothioneine and E0' = - 0.24 V for glutathione), a combination of ergothioneine and glutathione provide cellular protection over a wide range of conditions such as against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Despite ergothioneine's demonstrated role in maintaining human health, the current method used to produce it has proved to be a bottleneck in its widespread use. Specifically, the chemical synthesis method suffers from racemization of a stereo-center with the byproduct isomer being toxic. As a result, production at a large scale is both challenging and costly. In our preliminary studies, we developed an alternative, fermentation-based biosynthetic production method capable of producing 10 grams/liter on a 1 L batch-scale. In addition, we have identified the signaling pathway(s) related to ergothioneine function. These breakthroughs in supply and mechanistic understanding provide the scientific and manufacturing support necessary to realize ergothioneine's full commercial potential, which includes: further growth of its share in the cosmetic market, expansion into new markets (e.g., as a component in nutraceuticals), or for drug discovery and development. In this proposal we will address two key go/no-go questions that will guide the further development of this product. Specifically, we will scale-up the synthesis to pilot scale production using our patented ergothioneine production technology and also pinpoint ergothioneine's biological targets. To this end, the aims are: Aim 1: Achieve pilot-scale (100's g – 1 kg) production of ergothioneine with purity >99%. Aim 2: Verify the ergothioneine-related signaling pathways. Outcomes. Upon successful completion of these studies, we will apply for a Phase II grant with aims of large- scale production under good manufacturing practices to support additional market penetration and expansion. At the same time, we will re-examine ergothioneine's impact on ageing and age-related disease in vivo. We will also reach out to other scientists in the anti-ageing field to seek collaboration, including collaboration with an existing NIH anti-ageing program (https://www.nia.nih.gov/research/dab/interventions-testing-program-itp).