Interleukin-3 is a cytokine, which acts on many target cells within the haemopoietic system, often in synergy with the other cytokines. Streptomyces lividans NCIMB 11416/IL3 p002 secreting human interleukin-3 was used as the host organism in this study of improving target protein production. Streptomyces also produces several proteases including extracellular endoprotease that truncate the N-terminus of the recombinant protein. Federal guidelines and regulations banning animal-derived medium components necessitate the refinement or redevelopment of industrial medium formulations. The development of a defined medium without animal products is most desirable for the production of pure and safe biological products. The objective of the proposed research was the development and application of engineering methodology for the development of a defined medium and the analysis and optimization of a bacterial bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. During the first phase of this study the task was to develop a systematic procedure for the design and optimization of a chemically defined medium. The study aimed at replacing casein peptone in conventional medium for S. lividans with essential amino acids and determining the optimum proportion of the amino acids. To accomplish this, starvation trials with growth limiting amino acids were performed to establish the baseline for the nutritional requirement. The starvation trials revealed that essential amino acids for growth and product formation are amongst the following eight amino acids: Arg, Asn, Asp, Glu, Leu, Met, Phe, and Thr. Following these preliminary experiments, a statistically based experimental method called mixture experiments along with distance-based multivariate analysis revealed that Asp, Leu, Met, and Phe were the essential amino acids. Then, another mixture experiment design known as simplex lattice design was performed and artificial neural networks were employed to obtain the optimum proportions of the essential amino acids. The optimal medium was found to be composed of 56% Asp, 5% Met, and 39% Phe. It was found in previous studies that in complex media, several types of protease are produced during fermentation. Using the defined medium no proteolytic activity was detected in the fermentation broth. The second optimization method was based on metabolic flux analysis. A comprehensive metabolic network was developed for S. lividans. The metabolic network included carbohyderate and amino acid metabolism in both anabolic and catabolic reactions. According to the experimental results, the time course of the fermentation was divided into two phases, Phase E1 and Phase E2. In the first phase amino acids were used as a nitrogen source and in the second phase ammonia was the nitrogen source for growth and product formation. The metabolic network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consisted of 62 intracellular metabolites and 91 biochemical reactions. Two different objective functions were considered for optimization: maximizing the specific growth rate and minimizing the redox equivalent. A linear programming approach was used for optimizing the objective functions. The proposed model was able to predict the specific growth rate very accurately with a maximum error of 10%. The oxygen uptake rate and carbon dioxide evolution rate were evaluated with maximum error of 27% and 35%, respectively. Sensitivity analysis revealed that amino acid uptake was the growth limiting flux during the Phase E1 of the fermentation. During Phase E2 the uptake rate of ammonia had a significant effect on the specific growth rate. Sensitivity analysis of the specific growth rate and redox potential with respect to the biomass components showed that any additional supply of biomass building blocks (amino acids, nucleotides) would not significantly affect the specific growth rate and redox potential production as well as the calculated flux pattern.

Interleukin-3 is a cytokine, which acts on many target cells within the haemopoietic system, often in synergy with the other cytokines. Streptomyces lividans NCIMB 11416/IL3 p002 secreting human interleukin-3 was used as the host organism in this study of improving target protein production. Streptomyces also produces several proteases including extracellular endoprotease that truncate the N-terminus of the recombinant protein. Federal guidelines and regulations banning animal-derived medium components necessitate the refinement or redevelopment of industrial medium formulations. The development of a defined medium without animal products is most desirable for the production of pure and safe biological products. The objective of the proposed research was the development and application of engineering methodology for the development of a defined medium and the analysis and optimization of a bacterial bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. During the first phase of this study the task was to develop a systematic procedure for the design and optimization of a chemically defined medium. The study aimed at replacing casein peptone in conventional medium for S. lividans with essential amino acids and determining the optimum proportion of the amino acids. To accomplish this, starvation trials with growth limiting amino acids were performed to establish the baseline for the nutritional requirement. The starvation trials revealed that essential amino acids for growth and product formation are amongst the following eight amino acids: Arg, Asn, Asp, Glu, Leu, Met, Phe, and Thr. Following these preliminary experiments, a statistically based experimental method called mixture experiments along with distance-based multivariate analysis revealed that Asp, Leu, Met, and Phe were the essential amino acids. Then, another mixture experiment design known as simplex lattice design was performed and artificial neural networks were employed to obtain the optimum proportions of the essential amino acids. The optimal medium was found to be composed of 56% Asp, 5% Met, and 39% Phe. It was found in previous studies that in complex media, several types of protease are produced during fermentation. Using the defined medium no proteolytic activity was detected in the fermentation broth. The second optimization method was based on metabolic flux analysis. A comprehensive metabolic network was developed for S. lividans. The metabolic network included carbohyderate and amino acid metabolism in both anabolic and catabolic reactions. According to the experimental results, the time course of the fermentation was divided into two phases, Phase E1 and Phase E2. In the first phase amino acids were used as a nitrogen source and in the second phase ammonia was the nitrogen source for growth and product formation. The metabolic network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consisted of 62 intracellular metabolites and 91 biochemical reactions. Two different objective functions were considered for optimization: maximizing the specific growth rate and minimizing the redox equivalent. A linear programming approach was used for optimizing the objective functions. The proposed model was able to predict the specific growth rate very accurately with a maximum error of 10%. The oxygen uptake rate and carbon dioxide evolution rate were evaluated with maximum error of 27% and 35%, respectively. Sensitivity analysis revealed that amino acid uptake was the growth limiting flux during the Phase E1 of the fermentation. During Phase E2 the uptake rate of ammonia had a significant effect on the specific growth rate. Sensitivity analysis of the specific growth rate and redox potential with respect to the biomass components showed that any additional supply of biomass building blocks (amino acids, nucleotides) would not significantly affect the specific growth rate and redox potential production as well as the calculated flux pattern.

Frankfurt (Main), Universiẗat, Diss., 2008. / Erscheinungsjahr an der Haupttitelstelle: 2007.

Techn. Universiẗat, Diss., 2004--Berlin.

Das lineare Chromosom von S.lividans zeichnet sich durch eine hohe Variabilität insbesondere der chromosomalen Endbereiche aus. Hier finden sich unter anderen auch verschiedene Gene, die bisher einzigartig sind. Nach Klonierung der Gene in E.coli wurden die entsprechenden Genprodukte als His-Tag Fusionsproteine überproduziert, aufgereinigt und zur Herstellung von Antikörpern verwendet. Der untersuchte Abschnitt, als Ganzes und in Unterabschnitten, wurde auf einem Hoch Kopien Vektor in S.lividans transformiert. In extra hierfür konstruierten Vektorsystemen erfolgte die Produktion von His-Tag Proteinen in S.lividans. Nach Fusion von potentiellen Promoterbereichen mit dem promoterlosen EGFP-Gen, gelang deren Identifizierung in enhanced green fluorescent protein (EGFP) produzierenden S.lividans Transformanten. Mit Hilfe eines Vektors, der ein Temperatur sensitives Replikon besitzt, wurden Gene durch die Integration eines Hygromycin-Resistenzgenes ersetzt, bzw. als Fusionsgen mit dem EGFP-Gen erstellt. Ein Flavoprotein wurde zur Homogenität gereinigt. Es wurde nachgewiesen, dass in S.lividans pro Monomer ein FAD-Molekül interagiert. Physiologische Studien zeigen, dass die Synthese des chromosomal determinierten Proteins in S.lividans nur erfolgt, wenn dieser Stamm ein Plasmid- oder chromosomal- kodiertes Thiostrepton Resistenzprotein (23S rRNA Methylase) enthält. Es muss geschlussfolgert werden, dass die Methylierung der 23S rRNA die Translation verschiedener mRNAs beeinflusst. Die Synthese dieses Proteins ist des Weiteren abhängig von hohen Konzentrationen an NaCl und KCl im Medium, wie auch die zweier Aldo-Keto Reduktasen. Disruptionsmutanten eines dieser zwei Aldo-Keto Reduktase-Gene zeigen jeweils eine erhöhte und verfrühte Produktion eines rot gefärbten Mycel-assoziierten Antibiotikums (Undecylprodigiosin), während die eines weiteren (Actinorhodin) unbeeinflusst blieb.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression systems and vector systems, providing the basis for successful expression of all kinds of complex proteins. Based on the evaluation of expression systems in this review, such toolbox can be obtained.