Rational engineering of a multi-step biocatalytic cascade for the
conversion of cyclohexane to polycaprolactone monomers in P.
The current industrial production of polymer building blocks such as
ε-caprolactone (ε-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step
process associated with critical environmental issues such as the
generation of toxic waste and high energy consumption. Consequently,
there is a demand for more eco-efficient and sustainable production
routes. This study deals with the generation of a platform organism that
converts cyclohexane to such polymer building blocks without the
formation of byproducts and under environmentally benign conditions.
Based on kinetic and thermodynamic analyses of the individual enzymatic
steps, we rationally engineered a 4-step enzymatic cascade in
Pseudomonas taiwanensis VLB120 via stepwise biocatalyst
improvement on the genetic level. We found that the intermediate product
cyclohexanol severely inhibits the cascade and optimized the cascade by
enhancing the expression level of downstream enzymes. The integration of
a lactonase enabled exclusive 6HA formation without side products. The
resulting biocatalyst showed a high activity of 44.8 ± 0.2 U
gCDW-1 and fully converted 5 mM
cyclohexane to 6HA within 3 h. This platform organism can now serve as a
basis for the development of greener production processes for
polycaprolactone and related polymers.