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

Cellular heterogeneity is a major bottleneck for the development of stable and reproducible microbial processes on industrial scale. Most studies on cellular heterogeneity investigated its role and underlying sources in the context of metabolic and regulatory network structure and functionality, but only few studies took account of this field in the context of industrial bioprocessing. In this thesis, relevant aspects were identified and analyzed with the goal to minimize cell-to-cell heterogeneity and establish reproducible whole-cell bioprocesses with Pseudomonas-based asymmetric styrene epoxidation catalyzed by the styrene monooxygenase StyAB from Pseudomonas taiwanensis VLB120 as prime example. Different plasmid-based expression systems and inducers were evaluated regarding styAB expression stability and reproducibility in Pseudomonas strains differing regarding phenotypic characteristics. Using an eGFP-StyA fusion construct, variability in specific activity among individual isogenic cultures was found to rely on variations in recombinant gene expression levels and subpopulation structure. Furthermore, the cellular capacity to support NADH-dependent StyAB catalysis in two-liquid phase bioreactors was investigated in more detail, revealing significantly reduced process efficiencies under energy source limitation (fed-batch cultivation). Applying the constitutively solvent-tolerant regulatory mutant P. taiwanensis VLB120ΔCΔttgV in batch instead of the typically preferred fed-batch cultivation mode resulted in increased styAB expression increased specific activity (up to 180 U/gCDW). The knowledge gained on factors influencing cell-to-cell heterogeneity and productivity of isogenic Pseudomonas cultures will help to establish economically attractive bioprocesses.