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

这一小型企业技术转移项目的更广泛的影响/商业潜力将是开发一种微生物，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天然产品，在世界范围内已被用作抗抑郁剂、助眠剂和食欲抑制药。此外，5-HTP还可用于预防牛乳热，这是一种在农场动物中发生的常见疾病，会造成重大经济损失。目前，5-羟色胺的唯一商业生产是通过从单叶灰树的种子中提取出来的，灰树是一种生长在西非和中非的木质攀援灌木。但由于原料供应不足且不稳定，导致5-羟色胺价格居高不下，极大地制约了其市场的发展。该技术有望使5-HTP的生产成本比目前的植物提取方法降低90%，保证5-HTP的稳定生产，满足市场需求。该菌株预计将具有以下预期特征：1)不使用抗生素；2)不使用昂贵的诱导剂IPTG；3)提供优化的辅因子产生和回收系统。该公司在摇瓶中实现了色氨酸微生物生产5-HTP，产量超过1g/L，具有很大的商业潜力。然而，这种方法仍然使用抗生素和昂贵的诱导剂IPTG，这可以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发环境问题。为了解决这些问题并实现STTRI期工程的目标，我们将致力于以下三个方面的研究：1)构建无抗生素的质粒选择系统；2)发展无诱导子的表达系统；3)优化辅因子MH4的产生和循环。

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.