Pseudomonas and heterogeneity - benefits and challenges for strain and process engineering

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Cellular heterogeneity poses a significant challenge for the development of stable and reproducible microbial processes at an industrial scale. While most research has focused on its role in metabolic and regulatory networks, few have explored its implications for industrial bioprocessing. This thesis identifies and analyzes key aspects to minimize cell-to-cell heterogeneity and establish reproducible whole-cell bioprocesses, using Pseudomonas-based asymmetric styrene epoxidation catalyzed by the styrene monooxygenase StyAB from Pseudomonas taiwanensis VLB120 as a prime example. Various plasmid-based expression systems and inducers were assessed for their stability and reproducibility in Pseudomonas strains with differing phenotypic traits. An eGFP-StyA fusion construct revealed that variability in specific activity among isogenic cultures stemmed from differences in recombinant gene expression levels and subpopulation structure. Additionally, the study examined the cellular capacity for NADH-dependent StyAB catalysis in two-liquid phase bioreactors, highlighting reduced process efficiencies under energy source limitations during fed-batch cultivation. Notably, using the solvent-tolerant regulatory mutant P. taiwanensis VLB120ΔCΔttgV in batch cultivation led to increased styAB expression and specific activity (up to 180 U/gCDW). Insights gained on factors affecting cell-to-cell heterogeneity and productivity in isogenic Pseudo