Characterization of recombinant protein production in Escherichia coli and its influence on host cell metabolic activity

  • Charakterisierung der rekombinanten Proteinproduktion in Escherichia coli und deren Einfluss auf die Stoffwechselaktivität des Wirts

Rahmen, Natalie; Büchs, Jochen (Thesis advisor); Jaeger, Karl-Erich (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen, 2015

Abstract

Biotechnological processes are environmentally friendly, often require less resources and energy compared to conventional chemical processes and are therefore economically feasible. Hence, they are of increasing importance for the production of industrially relevant products. In particular, the production of proteins and enzymes plays an important role since they have versatile possible applications in the field of medicine, pharmaceutical, food or chemical industry. The bacterium Escherichia coli is commonly used in academia and industry for the biotechnological production of recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and host strains. Due to the great diversity of genetically modified expression strains, selecting an appropriate host strain for the biotechnological production of a desired recombinant protein is often laborious and time-consuming.One further important issue during recombinant protein production is the so-called ‘metabolic burden’: the material and energy normally reserved for the microbial metabolism which is sapped from the bacterium for plasmid replication, heterologous gene expression and recombinant protein production. This material and energy drain harms biomass formation and modifies respiration. Thereby, the abundance of rare codons in a heterologous gene may be a further drawback. The overall aim of this thesis was to characterize the recombinant protein production in E. coli and its influence on the metabolic activity of the host cell metabolism. First, an easy approach to systematically characterize industrially relevant and to identify well-suited T7-based E. coli host strains for recombinant protein production was investigated. Thereby, the qualification of online respiration activity measurement for selecting appropriate expression hosts was assessed. The Oxygen Transfer Rate (OTR) was determined online to monitor the host cell metabolic burden during the production of plasmid-encoded proteins using the Respiration Activity MOnitoring System (RAMOS). Seven commonly applied T7-based E. coli host strains expressing the genes encoding for two recombinant enzymes, namely a lipase from the mesophilic bacterium Bacillus subtilis (BSLA) and an oxidoreductase from the thermophilic bacterium Thermus thermophiles, were compared during cultivation under non-inducing and inducing conditions. For further characterization of the recombinant protein production in E. coli it was investigated whether smallest differences within the heterologous lipA gene encoding the BSLA target enzyme affect the metabolic activity of the host organism. Therefore, E. coli BL21(DE3) clones containing small differences within the lipA gene sequence leading either to single amino acid exchanges within the recombinant protein or to even smaller differences in form of single codon exchanges within the gene were analyzed regarding their effects on the host cell metabolic activity. The amino acid exchanges were randomly distributed over the entire amino acid sequence of the target protein lipase A from Bacillus subtilis (BSLA). Silent mutations were introduced into the coding sequence of the BSLA to introduce all synonymous arginine or leucine codons at two randomly defined positions, as well as substitutions leading to identical amino acid exchanges with different synonymous codons. The respective E. coli clones were compared during cultivation in a mineral autoinduction medium using specialized online and offline measuring techniques. To quantitatively analyze the metabolic state of the recombinant organisms, the RAMOS device was applied. Biomass and product formation were determined using the microtiter plate based BioLector system. Further cultivation parameters like e.g. carbon source consumption or plasmid copy number were determined to deeper analyze the underlying molecular relations. In this thesis, it could be shown that online measurement of the respiration activity using the RAMOS device allows for a simple estimation of recombinant protein production without laborious offline protein determination as well as for a first identification of suitable T7-based E. coli host strains. In mineral cultivation media, a qualitative as well as a quantitative correlation between respiration activity and protein production could be verified for the investigated E. coli host strains under IPTG- as well as under autoinduction. Under the chosen cultivation conditions, E. coli BL21(DE3) was found to be an appropriate host strain for the recombinant BSLA production using IPTG- or autoinduction. The investigated small modifications within the lipA gene sequence resulted in strong, highly reproducible differences in respiration activity, biomass formation and target protein production of the respective E. coli clones. A quantitative evaluation of the respiration activity allowed a classification of all investigated clones into two types named Type A and Type B. With respect to the respiration activity, five characteristic cultivation phases could be identified, providing information about the time points of the depletion of the different carbon sources as well as about biomass and product formation. Type A clones indicated higher product formation, Type B clones were identified as clones with stronger biomass formation. A large number of potential factors causing the observed two patterns of respiration behavior was assessed to gain a deeper understanding of the observed phenomenological differences. The availability of intracellular nucleobases as well as codon usage, metabolic costs for the amino acid biosynthesis, enzyme activity, the formation of inclusion bodies as well as the ratio of insoluble to soluble protein neither showed any impact on heterologous gene expression and protein production nor did they cause the differences observed between Type A and Type B clones. However, the small differences in the heterologous lipA gene led to a reduced initial growth of Type B clones resulting in a lower biomass concentration at the time point of induction. As a consequence, plasmid copy number and expression level remained lower compared to Type A clones, and the consumption pattern of the carbon sources lactose and glycerol changed. Even though the underlying molecular mechanisms are not yet identified, in this thesis, the astonishing phenomenon was observed that smallest differences within a heterologous gene leading to single amino acid or silent codon exchanges tremendously affect host cell metabolism and recombinant protein production. In future, this knowledge could have enormous impact on the codon optimization of heterologous genes, screening procedures for improved enzyme variants, and biotechnological protein production processes.