(629f) Improving the Production of Mvl in E. coli By Directed Evolution

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 T_g 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.