Improved premises for cell factory development : An enhanced understanding of established microbial protein production systems

Abstract

The sustainable manufacturing of medicines, materials and chemicals is enabled with biotechnology, and the key to the development of new processes, as well as improvement of existing ones, lies in our fundamental understanding of the biological systems we manipulate. Recombinant protein production is at the core of biotechnology and numerous molecular tools and bacterial strains have been developed over the past four decades for this purpose. Understanding of the genetic code and our ability to manipulate genetic material, paves the way for the microbial cell factory development that enables production of protein in a sustainable, costefficient manner. In this thesis I report the joined efforts of my colleagues and myself, to improve the premises for cell factory development by optimizing the cloning strategies, improving the awareness of unforeseen side-effects in complex bacterial expression systems, and building a platform for enhanced expression of certain plant genes in bacteria. The relevance of the conducted research to the field of biotechnology is covered, as well as necessary scientific background and history. Specifically, the surprisingly minor effects of tRNA overexpression on the production of a large number of membrane proteins in Escherichia coli are reported, and also the subsequent work elucidating two types of side effects: in some cases growth and gene expression are directly impaired by the extra tRNA sequences and in other cases the apparent positive effects are instead caused by a differential expression of the lysozyme gene encoded on the same plasmid. These phenomena seem to have been largely overlooked despite the huge popularity of the T7/pet-based systems for bacterial protein production. Additionally, the paradox that standardization comes at the cost of reduced flexibility is addressed. The development of a cloning strategy is covered, aiming at maximized standardization while maintaining high flexibility in choice of cloning technique. Finally, the systematic optimization of production of six plant-derived cytochrome P450 enzymes in E. coli, using a high-throughput workflow based on C-terminal fusions to the green fluorescent protein is also presented. Our work suggests that there are no inherent limitations in using different standard E. coli strains and expression conditions for exploiting cytochrome P450s for biotechnological applications. The results of this thesis have improved the premises for cell factory development in the future.