(228bi) De Novo Biosynthesis of 3,4- Dihydroxybutyric Acid in Engineered Escherichia coli.

3-Hydroxy-γ-butyrolactone (3HBL) is a building block widely used in pharmaceutical industry. Many important pharmaceuticals are derived from 3HBL such as anti-cholesterol medicines Lipitor and Crestor, lipid-lowering drug Zetia, nutrition additive L-carnitine and HIV inhibitors. Its market price is about $450 per kilogram. Owing to its great commercial value, US Department of Energy listed 3HBL as one of the top value-added compound. Currently, 3HBL is mainly produced by chemical synthesis, which involves hazardous materials and processing conditions. Only one biosynthetic pathway has been reported that use glucose and glycolate as the substrates and coenzyme A as the activator, leading to 221 mg/L of 3HBL and 555 mg/L of 3,4- dihydroxybutyric acid (3,4-DHBA, hydrolyzed form of 3HBL). However, this pathway involves multiple steps (>10 steps) and needs exogenous supplement of glycolate that limit the production and yield. Here we established a novel and efficient biosynthetic pathway for 3,4-DHBA that can be easily converted to 3HBL by acid treatment using xylose as the substrate in Escherichia coli. The new pathway contains only 4 steps. We first screened efficient enzymes for each step through in vitro enzyme assay. Then we introduced the optimized pathway into E. coli, resulting in production of 3,4-DHBA in vivo. Additional host strain engineering enables the titer of increased up to 2.1 g/L 3,4-DHBA in shake flask experiments, which is the highest titer reported so far. This study demonstrates a novel biosynthetic pathway for the production of 3,4-DHBA from xylose in E. coli, and this engineered pathway indicates the potential for industrial manufacturing of 3,4-DHBA and 3HBL.