Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

Abstract

Cytochrome P450 monooxygenases (Cyps) effectively catalyze the regiospecific oxyfunctionalization of inert C-H bonds under mild conditions. Due to their cofactor dependency and instability in isolated form, oxygenases are preferably applied in living microbial cells with Pseudomonas strains constituting potent host organisms for Cyps. This study presents a holistic genetic engineering approach, considering gene dosage, transcriptional, and translational levels, to engineer an effective Cyp-based whole-cell biocatalyst, building on recombinant Pseudomonas taiwanensis VLB120 for cyclohexane hydroxylation. A lac-based regulation system turned out to be favorable in terms of orthogonality to the host regulatory network and enabled a remarkable specific whole-cell activity of 34 U g CDW −1. The evaluation of different ribosomal binding sites (RBSs) revealed that a moderate translation rate was favorable in terms of the specific activity. An increase in gene dosage did only slightly elevate the hydroxylation activity, but severely impaired growth and resulted in a large fraction of inactive Cyp. Finally, the introduction of a terminator reduced leakiness. The optimized strain P. taiwanensis VLB120 pSEVA_Cyp allowed for a hydroxylation activity of 55 U g CDW −1. Applying 5 mM cyclohexane, molar conversion and biomass-specific yields of 82.5% and 2.46 mmol cyclohexanol g biomass −1 were achieved, respectively. The strain now serves as a platform to design in vivo cascades and bioprocesses for the production of polymer building blocks such as ε-caprolactone.

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

“…The construction of an efficient biocatalytic in vivo cascade necessitates a balanced expression of the cascade genes to avoid side product accumulation. Besides the well-characterized initiating Cyp, [24,25] CDH, and CHMO have been employed for PCL monomer synthesis from cyclohexane, [23] but nothing is known about respective reaction kinetics and possible inhibitions by pathway intermediates as they have been observed before, for example, for Baeyer-Villiger monooxygenases. [26,27] Consequently, CDH and CHMO were characterized as the first step in this study to support the rational engineering of a productive 3-step cascade based on the optimized Cyp-containing whole-cell biocatalyst.…”

“…[26,27] Consequently, CDH and CHMO were characterized as the first step in this study to support the rational engineering of a productive 3-step cascade based on the optimized Cyp-containing whole-cell biocatalyst. [24]…”

“…CDH and CHMO were cloned separately into the pSEVA244_T vector [ 24 ] to characterize their in vivo activity. As CDH catalyzes an equilibrium reaction, the kinetic parameters were assayed for both reaction directions ( Table 1 ).…”

“…In a separate study, the activity of cells containing only the Cyp could be more than doubled by expression system engineering. [24] In this study, we set out to amend the latter system with CDH and CHMO genes and thereby achieve high respective activities, utilize glucose instead of citrate as carbon and energy source, prevent the accumulation of intermediates, and keep the expression related metabolic burden reasonably low to allow for stable catalysis. Explicitly, oxygenases such as Cyps or Baeyer-Villiger monooxygenases are prone to form reactive oxygen species via uncoupling reactions, which may hamper the catalytic prowess of the cells.…”

“…In a separate study, the activity of cells containing only the Cyp could be more than doubled by expression system engineering. [ 24 ]…”