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

这个小型企业技术转让（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-羟色氨酸（5-HTP），用作膳食补充剂和动物饲料补充剂。5-羟色胺是一种多功能的天然产物，在世界范围内被用作抗抑郁药、助眠药和食欲抑制剂。此外，5-HTP可用于预防奶热，这是农场动物中常见的一种疾病，会造成巨大的经济损失。目前，5-HTP的唯一商业生产是从生长在西非和中非的木本攀缘灌木格里菲斯（Griffonia simplicifolia）的种子中提取出来的。然而，由于原料供应的不足和不稳定，导致5-HTP价格居高不下且波动较大，极大地制约了其市场的发展。与目前的植物提取方法相比，该技术有望将生产成本降低90%，确保5-HTP的稳定生产，满足市场需求。这个STTR一期项目建议创建一个最佳的5- htp生产菌株，可以很容易地用于大规模生产。预计该菌株具有以下期望特征：1)无需使用抗生素；2)省去了昂贵的诱导剂IPTG的使用；3)提供了一个优化的辅因子生成和回收系统。公司在摇瓶中实现了以色氨酸为原料微生物生产1 g/L以上的5-HTP，显示出巨大的商业化潜力。然而，这种方法仍然使用抗生素和昂贵的诱导剂IPTG，这可能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，实现STTR一期项目的目标，将实现以下三个研究目标：1)构建无抗生素质粒选择系统；2)构建无诱导剂的增量表达体系；3)优化辅因子MH4的生成和循环利用。

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.