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

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.