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

该小企业技术转让（STTR）项目的更广泛影响/商业潜力是建立一种生物基制造工艺，以经济和可再生的方式生产商品化学品4-羟基香豆素（4-HC）。4-HC是一种直接合成前体，用于生产广泛使用的口服抗凝剂，如华法林、醋硝香豆素和苯丙香豆素。此外，4-HC衍生的抗凝剂通常用作灭鼠剂以杀死啮齿动物。传统上，4-HC是通过使用石油相关的芳香族化学品作为起始材料的化学合成进行商业化生产的，这既不经济也不环保。该项目拟开发的技术有望大幅降低生产成本，减少对石化产品的依赖。该STTR第一阶段项目旨在创建一种可用于大规模生产的最佳4-HC生产微生物菌株。预期该菌株具有高遗传稳定性，而不需要抗生素或诱导剂用于生产。初步研究表明，4-HC的微生物生产的可再生碳源，甘油，摇瓶，这表明了巨大的商业化潜力。然而，该阶段的方法仍然使用涉及抗生素和诱导剂IPTG的宿主质粒系统。这不仅会增加生产成本，而且会引起环境问题。此外，宿主质粒系统可能导致遗传特性的不稳定性，这对于大规模生产商品化学品是不期望的。为了解决这些问题并实现STTR I期项目的目标，将追求以下研究目标：将组成型表达的4-HC生物合成途径基因整合到E.大肠杆菌染色体DNA及其优化;大肠杆菌内源性酶负责不必要的降解4-HC生物合成中间体。

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.