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Rational engineering of a multi-step biocatalytic cascade for the conversion of cyclohexane to polycaprolactone monomers in P. taiwanensis
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  • Lisa Schäfer,
  • Katja Bühler,
  • Rohan Karande,
  • Bruno Bühler
Lisa Schäfer
Helmholtz Centre for Environmental Research

Corresponding Author:[email protected]

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Katja Bühler
Helmholtz-Centre for Environmental Research - UFZ
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Rohan Karande
Helmholtz Centre for Environmental Research
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Bruno Bühler
Helmholtz Centre for Environmental Research
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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.
22 May 2020Submitted to Biotechnology Journal
25 May 2020Submission Checks Completed
25 May 2020Assigned to Editor
29 May 2020Reviewer(s) Assigned
02 Jul 2020Editorial Decision: Revise Major
13 Jul 20201st Revision Received
13 Jul 2020Submission Checks Completed
13 Jul 2020Assigned to Editor
13 Jul 2020Reviewer(s) Assigned
21 Jul 2020Editorial Decision: Accept