Rational Engineering of a Multi‐Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis. Issue 11 (6th September 2020)

• Record Type: Journal Article
• Authors: Schäfer, Lisa; Bühler, Katja; Karande, Rohan; Bühler, Bruno
• Is Part Of: Biotechnology journal. Volume 15:Issue 11(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-09-06
• Subjects: biocatalysis -- genetic engineering -- in vivo cascade -- polycaprolactone -- Pseudomonas; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202000091
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: 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, a 4‐step enzymatic cascade in Pseudomonas taiwanensis VLB120 is rationally engineered via stepwise biocatalyst improvement on the genetic level. It is found that the intermediate product cyclohexanol severely inhibits the cascade which could be optimized by enhancing the expression level of downstream enzymes. The integration of a lactonase enables exclusive 6HA formation without side products. The resulting biocatalyst shows a high activity of 44.8 ± 0.2 U gCDW −1 and fully converts 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. Abstract : A whole‐cell biocatalyst based on Pseudomonas taiwanensis VLB120 efficiently catalyzing the 4‐step conversion of cyclohexane to the polycaprolactone (PCL) building block 6‐hydroxyhexanoic acid was developed. Rational catalyst engineering allows for 100% conversion and selectivity as well as the highest activity for biocatalytic PCL monomer synthesis reported so far. This biocatalyst represents a promising basis for efficient and sustainable monomer production.