Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.COMET6676761

Sulfolobus acidocaldarius - Etablierung eines neuartigen Produktionswirts für zukunftsweisende Biotechnologie unter extremen Bedingungen

Kumulative Dissertation aus sechs ArtikelnAbweichender Titel nach Übersetzung der Verfasserin/des VerfassersWe live in exciting times! The importance of the once overly dominant production host Escherichia coli is dwindling, while cell culture and more exotic microbial hosts are taking over the playing field. Within this Thesis I developed fundamental methods analytic procedures and process technology alike with the capability to catalyze the adoption of the extreme thermo-acidophilic Archaeon Sulfolobus acidocaldarius in future commercially relevant bioprocesses. S. acidocaldarius grows optimally at 75 C and pH 3. It is amenable to genetic manipulation and characterized by several very unique metabolic pathways, making it a promising candidate for bioconversion processes (e.g. for upcycling of biogenic waste streams or for treatment of process wastewaters), and for the production of unique biological material (e.g. tetraether lipids used in medical applications for the formation of drug delivery vehicles). A further promising field of application is bioleaching for the economic mining of low grade ores in large scale. In general, due to its harsh growth conditions the organism is predestined for processes where sterility is difficult to achieve and/or to maintain. Difficult to sterilize batches and continuous processes which run over extended periods of time are important examples. Important prerequisites for the use of S. acidocaldarius in industrial applications, like characterization of the host s biology, development of genetic tools and clinical safety, can be considered to be met. Nevertheless, some aspects still need to be investigated in much more detail and a selection of remaining challenges were tackled within this Thesis. Firstly, for the investigation of the potential of S. acidocaldarius in the field of waste-to-value applications I performed extensive growth experiments on wheat straw and beech wood hydrolysates with wild type strains and genetically modified S. acidocaldarius mutants to utilize non-food substrate streams for the production of value-added products. Secondly, to push into pharmaceutical applications and the market of high value products I developed a defined cultivation medium adhering to Quality by Design guidelines. The medium was optimized in regards of salt and trace element composition. Complex protein hydrolysates, which can be inhomogeneous in their composition and are prone to formation of inhibiting substances under growth conditions, were replaced by a combination of sodium glutamate and citric acid. Thirdly, for the assessment of cell viability, a critical parameter during process development and control, fluorescence based assays were developed. Finally, I determined critical process parameters during the cultivation of S. acidocaldarius for the direct manipulation of the organisms membrane composition. Thereby it is possible to generate a tailor-made pattern of industrially relevant lipids that might be recoverable to an increased yield via a greatly facilitated downstream procedure.17

Regelung der Proteinexpression in E. coli auf zellulärer Ebene mit Hilfe eines gemischten Glucose/Lactose-Feedsystems

Zusammenfassung in deutscher SpracheAbweichender Titel nach Übersetzung der Verfasserin/des VerfassersKurze Produktionsphase aufgrund von hoher Stoffwechselbelastung und Bildung von unlöslichen Einschlusskörperchen sind bekannte Einschränkungen für die rekombinante Proteinproduktion in Escherichia coli. Zum heutigen Zeitpunkt wird die Lösung dieser Problemen vor allem mittels Methoden der Gentechnik in Angriff genommen. In der vorliegenden Arbeit werden die folgenden Fragestellungen behandelt: (1) die Übertragbarkeit der Beziehung zwischen der Glucose- und Lactoseaufnahme bei Glucoselimitierung und gleichzeitigem Lactoseüberschuss wird geprüft. Dazu werden die spezifischen Substrataufnahmeraten zweier E. coli Stämme BL21(DE3), welche unterschiedliche Zielproteine unter der Kontrolle des T7lac Promoters exprimieren, verglichen; (2) die Regelung der Rate der rekombinanten Proteinproduktion und das Verhältnis von gelöstem Protein zu Protein in Einschlusskörperchen wird untersucht; (3) die Stärke der Induktion bei Lactosefütterung wird auf zellulärer Ebene untersucht. Die Ergebnisse dieser Arbeit zeigen, dass die maximale spezifische Lactoseaufnahmerate stark von der Expression des Zielproteins beeinflusst wird. Weiters kann aus den Resultaten geschlossen werden, dass eine Induktion mit Laktose durch eine limitierende Fütterungsstrategie mit Glucose und Lactose die Rate der rekombinanten Proteinproduktion und das Verhältnis von gelöstem Protein zu Protein in Einschlusskörperchen steigern kann. Gleichzeitig ist es damit möglich die Ausbildung von unterschiedlich stark induzierten Subpopulationen zu vermeiden. Diese Ergebnisse werden den Forderungen der pharmazeutischen Industrie gerecht, effizient hohe Erträge an aktivem Zielprotein zu erreichen und dabei potentiell die Produktionsphase durch Reduktion der Stoffwechselbelastung der produzierenden Zellen auszuweiten.Short production phases due to high metabolic burden and formation of insoluble inclusion bodies are known limitations for recombinant protein production in Escherichia coli. Up to now, these issues are mainly tackled by genetic engineering. In this work the following issues are studied: (1) the transferability of the relationship between the specific uptake rates of glucose and lactose during their concomitant uptake is investigated. Therefore specific substrate uptake rates of two E. coli BL21(DE3) strains producing different target proteins under the control of the T7lac promotor system are compared during limiting glucose concentrations, while lactose was available in excess; (2) the tunability of the recombinant protein production rate and the ratio of soluble protein (SP) to inclusion bodies (IBs) are assessed; (3) the degree of induction during lactose feeding is investigated on the cellular level. The results of this Thesis show that the maximum specific lactose uptake rate is strongly influenced by the expressed target protein. Furthermore it can be concluded that lactose induction via a limiting mixed feeding strategy can increase the recombinant protein production rate and the ratio of SP to IBs while differently induced subpopulation can be avoided. This meets the demands of pharmaceutical bioprocesses to efficiently yield high amounts of active target protein and to extend the production phase by reducing metabolic burden and enhancing cell fitness.8

Physiological Characterization of Sulfolobus acidocaldarius in a Controlled Bioreactor Environment

The crenarchaeal model organism Sulfolobus acidocaldarius is typically cultivated in shake flasks. Although shake flasks represent the state-of-the-art for the cultivation of this microorganism, in these systems crucial process parameters, like pH or substrate availability, are only set initially, but cannot be controlled during the cultivation process. As a result, a thorough characterization of growth parameters under controlled conditions is still missing for S. acidocaldarius. In this study, we conducted chemostat cultivations at 75 °C using a growth medium containing L-glutamate and D-glucose as main carbon sources. Different pH values and dilution rates were applied with the goal to physiologically characterize the organism in a controlled bioreactor environment. Under these controlled conditions a pH optimum of 3.0 was determined. Washout of the cells occurred at a dilution rate of 0.097 h−1 and the optimal productivity of biomass was observed at a dilution rate of 0.062 h−1. While both carbon sources were taken up by S. acidocaldarius concomitantly, a 6.6-fold higher affinity for L-glutamate was shown. When exposed to suboptimal growth conditions, S. acidocaldarius reacted with a change in the respiratory behavior and an increased trehalose production rate in addition to a decreased growth rate

Exploitation of Wheat Straw Biorefinery Side Streams as Sustainable Substrates for Microorganisms: A Feasibility Study

Lignocellulosic agricultural side products, like wheat straw, are widely seen as an important contribution to a future sustainable economy. However, optimization of biorefinery processes and exploitation of all side streams are crucial for an economically viable biorefinery. Pretreatment of lignocellulosic raw material, which is necessary for further processing steps, can generate low-value side streams. In this feasibility study, side streams from a liquid hot water (LHW) pretreatment of wheat straw were utilized for the production of polyhydroxybutyrate (PHB) and highly valuable tetraether lipids (TELs). Additional value created by these products can benefit the biorefinery’s economic operation. The utilized wheat straw was pretreated at 120 °C and 170 °C for up to two hours in laboratory and lab scale. The resulting side stream consists mainly of carbohydrates from hemicelluloses and fermentation inhibitors such as acetic acid. In order to achieve a successful production of both products, an acetic acid separation via distillation was necessary. Subsequently, the acetic acid fraction was utilized for the PHB production using cyanobacteria. The carbohydrate-rich fraction was applied in the cultivation of Sulfolobus acidocaldarius and resulted in the successful production of TELs. Both fractions achieved better fermentation yields compared to their corresponding reference media

Sulfolobus – A Potential Key Organism in Future Biotechnology

Extremophilic organisms represent a potentially valuable resource for the development of novel bioprocesses. They can act as a source for stable enzymes and unique biomaterials. Extremophiles are capable of carrying out microbial processes and biotransformations under extremely hostile conditions. Extreme thermoacidophilic members of the well-characterized genus Sulfolobus are outstanding in their ability to thrive at both high temperatures and low pH. This review gives an overview of the biological system Sulfolobus including its central carbon metabolism and the development of tools for its genetic manipulation. We highlight findings of commercial relevance and focus on potential industrial applications. Finally, the current state of bioreactor cultivations is summarized and we discuss the use of Sulfolobus species in biorefinery applications

A defined cultivation medium for Sulfolobus acidocaldarius

Quehenberger J, Albersmeier A, Glatzel H, Hackl M, Kalinowski J, Spadiut O. A defined cultivation medium for Sulfolobus acidocaldarius. JOURNAL OF BIOTECHNOLOGY. 2019;301:56-67.The thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius is an important model organism for Archaea and genetic systems are well established. To date, the organism is routinely cultivated on complex media based on protein hydrolysates and no common defined medium is established. In this work we address this lack of a standardized defined medium and replaced the complex protein hydrolysate with sodium glutamate as primary substrate. Starting from an existing medium formulation we stepwise managed to improve the medium regarding formation of precipitates, buffer capacity, concentration of basal salts and trace elements, and optimized growth rates. The differences on the cellular level between the original medium and our new formulation, called VD Medium, were investigated by comparative gene expression analysis and significant differences were discussed. The final formulation of the VD Medium contains 1.75 g/L Na-glutamate, 3 g/L D-glucose and 0.5 g/L citric acid as carbon sources. Using the described medium for the cultivation of S. acidocaldarius DSM 639 in shake flasks yields 1.1 g/L dry cell weight (OD600 = 1.7) after a typical incubation time of 95 h with an overall biomass yield of 0.33 g(DCW/)g(substrate)

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable