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

Lisa Schäfer,Rohan Karande,Bruno Bühler

doi:10.3389/fbioe.2020.00140

Lisa Schäfer, Rohan Karande + Show 1 more

Open Access

https://doi.org/10.3389/fbioe.2020.00140

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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 gCDW–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 gCDW–1. Applying 5 mM cyclohexane, molar conversion and biomass-specific yields of 82.5% and 2.46 mmolcyclohexanol gbiomass–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.

Highlights

Realizing aerobic oxidation of thermodynamically stable and kinetically inert C–H bonds in cyclohexane under sustainable and environmentally safe conditions remains a major challenge in current academic and industrial research (Schuchardt et al, 2001; Cavani and Teles, 2009)
Respective genes have been isolated from Acidovorax CHX100 and functionally expressed in Pseudomonas taiwanensis VLB120, enabling a specific whole-cell activity of 20 U gCDW−1 for cyclohexane oxidation
CHX100 can be expressed in P. taiwanensis VLB120 under the control of the alk regulatory system from P. putida GPo1, enabling a resting cell activity of 20 U gCDW−1

Summary

Introduction

Realizing aerobic oxidation of thermodynamically stable and kinetically inert C–H bonds in cyclohexane under sustainable and environmentally safe conditions remains a major challenge in current academic and industrial research (Schuchardt et al, 2001; Cavani and Teles, 2009). Owing to the high demand, but low price, alternative production pathways need to be developed for an economical viable production process (Schuchardt et al, 1993; Van Beilen et al, 2003; Weissermel and Arpe, 2003) With their high selectivity and catalytic effectiveness, biocatalysts often constitute a promising alternative to chemical catalysts. A class I cytochrome P450 monooxygenase (Cyp)-based whole-cell biocatalyst has been reported to perform selective cycloalkane (C5–C8) oxyfunctionalization under ambient conditions (Salamanca et al, 2015; Karande et al, 2016) To this end, respective genes have been isolated from Acidovorax CHX100 and functionally expressed in Pseudomonas taiwanensis VLB120, enabling a specific whole-cell activity of 20 U gCDW−1 for cyclohexane oxidation. The higher growth rate obtained with the moderate RBS (Figure 5A) indicates a lower metabolic burden for the cells

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