Production Scale-Up and Target Identification of the Antioxidant Ergothioneine

摘要 麦角硫因是人体许多部位中最丰富的硫醇之一，并且有几种疾病具有 与麦角硫因不足有关，包括类风湿性关节炎、克罗恩病、神经退行性疾病 疾病、心血管疾病和糖尿病。动物不合成麦角硫因，而是获得 它通过麦角硫因特异性转运蛋白（OCTN 1）从饮食中摄取，累积量高达mM 在几个器官和组织中的浓度。由于它们在还原电位上的差异（对于 麦角硫因和谷胱甘肽的E0' =-0.24V），麦角硫因和谷胱甘肽的组合提供 在广泛的条件下的细胞保护，例如对抗活性氧（ROS）和活性氧（ROS）。 氮物种（RNS）。尽管麦角硫因在维持人类健康方面的作用已得到证实， 用于生产它的方法已被证明是其广泛使用的瓶颈。具体来说，化学 合成方法的缺点是立体中心的外消旋化，副产物异构体是有毒的。作为 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， 一种替代的基于发酵的生物合成生产方法，其能够在1 L发酵罐上生产10克/升， 批量生产此外，我们还鉴定了与麦角硫因功能相关的信号通路。这些 供给和机理认识上的突破，提供了科学和制造支撑 实现麦角硫因的全部商业潜力所必需的，其中包括： 化妆品市场，拓展新市场（例如，作为营养保健品中的组分）或用于药物发现 发展先行者的要求在本提案中，我们将解决两个关键的去/不去问题，这将指导进一步的 这个产品的发展。具体而言，我们将使用我们的合成方法将合成扩大到中试规模生产。 专利麦角硫因生产技术，并确定麦角硫因的生物靶点。为此目的， 其目的是： 目标1：实现纯度> 99%的麦角硫因的中试规模（100 μ g - 1 kg）生产。 目的2：验证麦角硫因相关的信号通路。 结果。在成功完成这些研究后，我们将申请第二期补助金，目的是： 根据良好生产规范进行规模生产，以支持进一步的市场渗透和扩张。 与此同时，我们将重新研究麦角硫因对体内衰老和年龄相关疾病的影响。我们将 我们也会与抗衰老领域的其他科学家寻求合作，包括与 现有的NIH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).