STTR Phase I: Bio-based Manufacturing of An Anticoagulant Precursor 4-Hydroxycoumarin

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project is to establish a bio-based manufacturing process for the production of the commodity chemical 4-hydroxycoumarin (4-HC) in an economical and renewable way. 4-HC is a direct synthetic precursor used to manufacture widely used oral anticoagulants, such as warfarin, acenocoumarol, and phenprocoumon. In addition, 4-HC-derived anticoagulants are commonly used as rodenticides to kill rodents. Traditionally, 4-HC is commercially manufactured through chemical synthesis using petro-related aromatic chemicals as starting materials, which is neither economical nor environmentally friendly. The proposed technology to be developed in this project is expected to dramatically lower the production cost, and reduce the reliance on petro chemicals. This STTR Phase I project proposes to create an optimal 4-HC-producing microbial strain that can be readily used for large-scale production. The strain is expected to have high genetic stability without the need for antibiotics or an inducer for production. Preliminary research has shown the microbial production of 4-HC from a renewable carbon source, glycerol, in shake flasks, which demonstrates great commercialization potential. However, the approach at this stage still uses host-plasmid systems that involve antibiotics and the inducer IPTG. This will not only increase the production cost but also will raise environmental concerns. Additionally, host-plasmid systems may cause instability in genetic properties, which is undesirable for large-scale production of commodity chemicals. To address these issues and achieve the goal of this STTR Phase I project, the following research objectives will be pursued: Integration of constitutively expressed 4-HC biosynthetic pathway genes into E. coli chromosomal DNA and its optimization; and inactivation of the E. coli endogenous enzymes responsible for unwanted degradation of 4-HC biosynthetic intermediates.