Improving production of bMdVL in E. coli by directed evolution
Yi-Shu Tai, Jilong Wang, Kechun Zhang
b-Methyl-d-valerolactone (bMdVL) is an attractive monomer which can be polymerized to amorphous polyester with a low Tg at -58 C. Previously, we developed a total biosynthetic pathway of bMdVL from glucose in engineered E. coli. However, the low activities of downstream enoate reductase, OYE2 or YqjM, in the pathway have limited the production titer. To address this challenge, we have developed directed evolution platforms to screen enzymes with improved activities. The first platform exploits a Ralstonia strain which we isolated from soil samples. This strain can use bMdVL as the carbon source to grow, but cannot use the precursor, anhydro-bMdVL (AMVL), for growth. Therefore, when anhydro-bMdVL was fed to the Ralstonia strain containing a library of enzyme mutants, the improved variants can be selected. In the second platform, we use toxicity of AMVL for screening. We found that 15 g/L of AMVL would significantly inhibit cell growth. Cells harboring more active enoate reductase can consume excess AMVL and restore cell growth. Overall, these selection platforms enable us to improve the activity of the pathway enzymes for bMdVL. This can further reduce the production cost of this novel biobased monomer used for soft polymer.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division