Analysis of cellular heterogeneity for enhancing the protein production in industrial scales

Abstract

The ability of microorganisms such as B. subtilis to differentiate into subpopulations with distinct phenotypes has been an important feature for their persistence in the face of environmental fluctuations. This so called heterogeneity provides B. subtilis with a selective advantage in its natural habitat, despite such bet-hedging strategies being energetically unfavourable processes. Unfortunately, heterogeneous gene expression in an industrial setting will reduce process efficiency by diverting substrate and energy consumption away from product formation. This thesis investigates the relationship between cellular heterogeneity and enzyme production by analysing different physiological and developmental pathways during the high level production of the native xylanase XynA and the heterologous amylase AmyM. The potential cellular heterogeneity of the quality control system was investigated using fluorescence microscopy and flow cytometry in different mutants. For the investigation of potential strategies to reduce the heterogeneous expression and optimise protein production in biotechnologically relevant processes, plasmid and integration based systems were compared and analysed by using fluorescent reporter fusions reflecting the enzymes transcription level and induced secretion stress. Furthermore, this thesis investigated the approach of generating a synthetic expression library to discover new expression units demonstrating high expression levels and reduced heterogeneous activities. The library was screened using flow cytometry and modules were assessed using the production of the industrially relevant enzyme XynA. Finally, controlled gene expression levels are of importance for the in silico design of novel pathways (synthetic biology). However, the expression of integrated genes can depend on their chromosomal location. This thesis assessed the impact of genomic location and orientation on gene expression levels by using an unbiased transposon approach for the random integration of an expression cassette comprising a divergent fluorescence and enzyme reporter system. In addition, the gene dosage effect was investigated in an industrially relevant setting using XynA as model enzyme.