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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Microbial Biocatalysis

Background:

• Baeyer-Villiger monooxygenases (BVMOs) are crucial enzymes for synthesizing valuable chemicals like ε-caprolactone.
• Pseudomonas biofilms offer a robust platform for immobilized biocatalysis.
• Optimizing BVMO performance within biofilms requires addressing challenges in biofilm development and reactor conditions.

Purpose of the Study:

• To investigate the biocatalytic performance of a Pseudomonas-based Baeyer-Villiger monooxygenase (BVMO) system for ε-caprolactone production.
• To evaluate the impact of engineered diguanylate cyclase (DGC) on Pseudomonas biofilm growth and BVMO activity.
• To optimize reactor conditions in drip flow reactors (DFRs) and rotating bed reactors (RBRs) for enhanced biocatalysis.

Main Methods:

• Two Pseudomonas taiwanensis VLB120 variants, Ps_BVMO and an engineered Ps_BVMO_DGC strain, were cultivated in DFRs and RBRs.
• Biofilm growth, surface coverage, and product formation rates were monitored.
• Reactor parameters, including oxygen supply and substrate feeding strategy, were manipulated to improve performance.

Main Results:

• The engineered Ps_BVMO_DGC strain exhibited faster biofilm growth in DFRs compared to the control.
• Mature biofilms achieved high product formation rates (up to 92 g m⁻² d⁻¹).
• Reactor challenges in RBRs, such as low surface coverage and oxygen limitation, were mitigated, leading to stable production (14 g m⁻² d⁻¹) with 60% substrate conversion at 4 mM cyclohexanone.

Conclusions:

• Engineering Pseudomonas with enhanced DGC activity improves biofilm formation for biocatalysis.
• Optimized reactor configurations and operational strategies are essential for efficient BVMO-catalyzed production in biofilms.
• Controlling substrate concentration and mitigating inhibitory byproducts are key to achieving stable and high yields.