STTR Phase I: Microbial Production of a Multifunctional Natural Product 5-Hydroxytryptophan

这个小企业技术转让（STTR）项目的更广泛的影响/商业潜力将是开发一种微生物来生产5-羟基色氨酸（5-HTP），用作膳食补充剂和动物饲料补充剂。5-HTP是一种多功能的天然产物，在世界范围内被用作抗抑郁剂，睡眠辅助剂和食欲抑制剂。此外，5-HTP可用于预防乳热，这是一种在农场动物中发生的常见疾病，会造成巨大的经济损失。目前，5-HTP的唯一商业化生产是通过从生长在西非和中非的木本攀缘灌木Griffonia simplicifolia的种子中提取。然而，由于原料供应不足且不稳定，导致5-HTP价格高企且波动较大，极大地制约了其市场的发展。与现有的植物提取方法相比，该技术有望降低生产成本达90%，并确保5-HTP的稳定生产，满足市场需求。本STTR I期项目旨在创建一种可用于大规模生产的最佳5-HTP生产菌株。该菌株预期具有以下期望的特征：1）消除抗生素的使用; 2）消除昂贵的诱导剂IPTG的使用;以及3）提供优化的辅因子产生和再循环系统。该公司已经实现了在摇瓶中从色氨酸中以超过1 g/L的微生物生产5-HTP，这显示了巨大的商业化潜力。然而，这种方法仍然使用抗生素和昂贵的诱导剂IPTG，这会使大规模生产的成本增加约44%。此外，在大规模生产中广泛使用抗生素可能会引起环境问题。为了解决这些问题并实现STTR I期项目的目标，将追求以下三个研究目标：1）构建无寄生虫的质粒选择系统; 2）开发具有增量强度的无诱导剂的表达系统; 3）优化辅因子MH 4的产生和回收。

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project will be to develop a microbe to produce 5-hydroxytryptophan (5-HTP) for use as a dietary supplement, and as an animal feed supplement. 5-HTP is a multifunctional natural product that has been used as antidepressant, sleep aid, and appetite suppressant worldwide. In addition, 5-HTP can be used to prevent milk fever, a common disease occurring among farm animals, which causes substantial economic losses. Currently, the only commercial production of 5-HTP is through the extraction from the seeds of Griffonia simplicifolia, a woody climbing shrub grown in west and central Africa. However, the insufficient and unstable supply of the raw material has resulted in a high and volatile price of 5-HTP, which has greatly constrained the development of its market. The technology to be developed is expected to lower the production cost by up to 90% compared with the current botanical extraction method, secure the stable production of 5-HTP, and satisfy the market demands.This STTR Phase I project proposes to create an optimal 5-HTP-producing strain that can be readily used for large scale production. The strain is expected to have the following desired features: 1) Eliminates the use of antibiotics; 2) eliminates the use of the expensive inducer IPTG; and 3) provides an optimized co-factor generation and recycling system. The company has achieved the microbial production of 5-HTP at over 1 g/L from tryptophan in shake flasks, which demonstrates great commercialization potential. However, this approach still uses antibiotics and the expensive inducer IPTG, which can increase the cost of large scale production by about 44%. In addition, extensive use of antibiotics in large scale production may raise environmental concerns. To address these issues and achieve the goal of this STTR Phase I project, the following three research objectives will be pursued: 1) Construction of an antibiotic-free plasmid selection system; 2) development of an inducer-free expression system with incremental strengths; and 3) optimization of the generation and recycling of cofactor MH4.