Rational Engineering of a Multi‐Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis

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.

Pseudomonas taiwanensis biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors

“…The observed substrate and product degradation are depicted in Figure 3C. Cyclohexanol is known to severely inhibit the here applied BVMO at high concentrations [37]. In the current experiments, CHOL levels were low and stayed below 0.1 mM during the entire biotransformation.…”

“…A BVMO derived from Acidovorax sp. CHX100 shows an exceptionally high whole‐cell specific activity of 160–180 U g CDW –1 [36, 37] towards cyclohexanone oxidation and was thus genetically introduced into the Pseudomonas strain as the model reaction system (Ps_BVMO). In addition, a plasmid harboring a diguanylate cyclase (DGC), which catalyzes the reaction of two guanosine‐5′‐triphosphate (GTP) molecules to form c‐di‐GMP, was introduced (Ps_BVMO_DGC).…”

“…For the BVMO used in the present study, inhibition of the isolated enzyme was observed already at 0.1–0.2 mM CHOL (data not shown here). Also, resting‐cell activities of Ps_BVMO cells dropped by 50% upon the presence of 0.4–0.5 mM CHOL [37]. To minimize the inhibitory effects of substrate and product on the BVMO, Hilker and coworkers demonstrated an in situ substrate feeding and product removal strategy using an adsorbent resin [49].…”

One‐pot synthesis of 6‐aminohexanoic acid from cyclohexane using mixed‐species cultures

“…In our previous study, P. taiwanensis VLB120 was established as a platform organism to produce the polycaprolactone monomers e-CL and 6HA from cyclohexane (Sch€ afer et al, 2020). In the present work, we aimed to extend this biocatalytic reaction cascade for the production of 6AHA, the building block of Nylon 6 serving an even larger market (4.2 million tons per annum) than polycaprolactone (25,000 tons per annum) (Ritz et al, 1986;Weissermel and Arpe, 2008).…”

“…In this study, we aimed at 6AHA production from cyclohexane in a one-pot approach with high conversion and yield. For this purpose, previously established Pseudomonas taiwanensis VLB120 strains containing cascades for the conversion of cyclohexane to єcaprolactone (є-CL) and/or 6-hydroxyhexanoic acid (6HA) (Sch€ afer et al, 2020) served as the basis. The respective biosynthetic pathways involve a cytochrome P450 monooxygenase (Cyp), a cyclohexanol dehydrogenase (CDH), a cyclohexanone monooxygenase (CHMO), and, facultatively, a lactonase (Lact).…”