Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry

Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 μm, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 μm without and 106 μm with glass beads, diameters from flow cytometry were 170 and 100 μm, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability

Morphological PAT tools for filamentous bioprocesses : monitoring and control enhanced for fungal morphology

Kumulative Dissertation aus sieben ArtikelnBiomass is one of the most essential process variables in bioprocesses. Process performance, process controls strategies and productivity in filamentous processes highly depend on cellular aspects, such as morphological elements of the biomass which calls for a segregated view of biomass. In filamentous bioprocesses, quality of the biomass is of prime importance. Hereby one strives to ensure high biomass viability and productivity. Filamentous fungi display a large variety of morphological forms in submerged cultures. These range from dispersed hyphae to denser hyphal aggregates, the so-called pellets. Morphology, viability and productivity are tightly interlinked. Depending on the objective function of the bioprocess, different characteristics of morphology are favourable and need to be quantified accordingly. Morphology and viability can be determined via a variety of methods, each limited to specific areas of application. The most common method to characterize morphology is image analysis based on microscopy, which is work intensive and time consuming. Typical methods to determine viable biomass encompass di-electric spectroscopy or staining reagents, which is troublesome regarding the complex fungal morphology. Therefore, our objective was to develop several alternative, robust and statistically sound methods based on flow cytometry and fluorescent staining for at-line application capable of assessment of morphology and viability. Furthermore, novel morphological characteristics describing pellet biomass are assessable via these methods: ‘pellet compactness and ‘viable pellet layer. Additionally, spatially resolved mass spectrometry was used in a novel technique to study metabolism and productive zones in fungal pellets. Our superior goal was to apply said analytical methods to study novel morphological responses and use this knowledge to determine a process design space in fermentation ensuring optimal morphology, viability and productivity. To achieve this, we employed and developed additional control strategies to regulate the growth rate or specific substrate uptake during Penicillium chrysogenum fermentations. In a design of experiments (DOE) approach, fermentation factors power input, dissolved oxygen concentration and specific substrate uptake rate were varied to feed a data-driven model including novel morphological responses. Thereby an optimised process design could be obtained which resulted in enhanced biomass viability and specific productivity. We envision that the methods compiled in the comprehensive ‘Analytics and ‘Control chapters of this Thesis will serve as process analytical technology (PAT) tools specifically enhanced for complex fungal morphology and providing novel morphological descriptors. Additionally, we have successfully tested our methodology on other agglomerate-forming organisms like yeast to demonstrate versatility and transferability.17

The filamentous fungus Penicillium chrysogenum analysed via flow cytometry—a fast and statistically sound insight into morphology and viability

Filamentous fungi serve as production host for a number of highly relevant biotechnological products, like penicillin. In submerged culture, morphology can be exceptionally diverse and is influenced by several process parameters, like aeration, agitation, medium composition or growth rate. Fungal growth leads to several morphological classes encompassing homogeneously dispersed hyphae and various forms of hyphal agglomerates and/or clump structures. Eventually, the so-called pellet structure can be formed, which represents a hyphal agglomerate with a dense core. Pellet structures can hinder oxygen and substrate transport, resulting in different states of viability, which in turn affects productivity and process control. Over the years, several publications have dealt with methods to either gain morphological insight into pellet structure or determine biomass viability. Within this contribution, we present a way to combine both in a flow cytometry–based method employing fluorescent staining. Thereby, we can assess filamentous biomass in a statistically sound way according to (i) morphology and (ii) viability of each detected morphological form. We are confident that this method can shed light on the complex relationship between fungal morphology, viability and productivity—in both process development and routine manufacturing processes.Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesse

Verbesserung der Löslichkeit von rekombinantem Protein produziert durch das pET Expressionssystem

Zusammenfassung in deutscher SpracheAbweichender Titel nach Übersetzung der Verfasserin/des VerfassersThis Thesis deals with the production of an antibody fragment in Escherichia coli in preferably soluble form. Full length antibodies feature glycosylation and thus are typically produced in mammalian cells which usually involves low space time yields, risk of contamination and considerable cost for equipment and media. Since desired antigen-binding properties are located in the fragment antigen binding (Fab) region, recombinant expression of only a Fab or single chain fragment variable (scFv) instead of a whole antibody is promising and can be accomplished in E. coli. The gut bacteria E. coli is one of the most widely used expression hosts for the production of recombinant proteins. It is superior to mammalian cells in several regards such as easier genetic manipulation and higher productivity in high cell density fermentations. The pET expression system features an inducible lac promotor which enables optimal cell growth in the Upstream Process (USP) until induction of gene expression, usually with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) as inducer. Induction by IPTG is used in most industrial fermentation processes as it cannot be metabolized by the cells and thus can be added in a simple one pulse addition. It however imposes a strong metabolic load onto the cells and is toxic at higher concentrations. Furthermore over-expression of recombinant protein in the cytoplasm results in inclusion body (IB) formation, containing the target protein in wrongly folded, non-functional form. Alternatively the lac operon's natural inducer lactose can induce expression of recombinant proteins equally effective. Regarding IB formation it was found that solubility of recombinant protein can be increased by modification of fermentation parameters like decreasing the growth rate (µ). Also studies indicate that lactose induction of the lac operon results in enhanced solubility (SP) of recombinant protein. The aim of this Thesis was to develop a mixed substrate feeding strategy for the production of a novel single chain fragment variable (scFv) using glucose and lactose in high density fermentation cultures and thereby increase the amount of soluble product as opposed to IPTG. Advantages of this strategy include a higher fraction of soluble protein providing significant improvement in Downstream Process (DSP) complexity. Furthermore knowledge on the link between substrate uptake and product formation as IB or SP leads to tunability of protein production rate and form. Such a fermentation setup is challenging because lactose needs to be fed continuously since it serves both as substrate and inducer. Consequently concomitant substrate uptake must be taken into account to avoid over-feeding, as in our experience glucose presence is necessary for lactose uptake. We found that lactose not only favors the recombinant production of soluble scFv when compared to IPTG, but furthermore that formation of soluble product can be tuned by the specific uptake rate of glucose during induction. On this basis a mechanistic correlation between specific uptake rates of lactose and glucose was determined in order to develop a corresponding model to be potentially valid for other E. coli strains as well.6

The filamentous fungal pellet—relationship between morphology and productivity

Filamentous fungi are used for the production of a multitude of highly relevant biotechnological products like citric acid and penicillin. In submerged culture, fungi can either grow in dispersed form or as spherical pellets consisting of aggregated hyphal structures. Pellet morphology, process control and productivity are highly interlinked. On the one hand, process control in a bioreactor usually demands for compact and small pellets due to rheological issues. On the other hand, optimal productivity might be associated with less dense and larger morphology. Over the years, several publications have dealt with aforementioned relations within the confines of specific organisms and products. However, contributions which evaluate such interlinkages across several fungal species are scarce. For this purpose, we are looking into methods to manipulate fungal pellet morphology in relation to individual species and products. This review attempts to address (i) how variability of pellet morphology can be assessed and (ii) how morphology is linked to productivity. Firstly, the mechanism of pellet formation is outlined. Subsequently, the description and analysis of morphological variations are discussed to finally establish interlinkages between productivity, performance and morphology across different fungal species

A Chemometric Tool to Monitor and Predict Cell Viability in Filamentous Fungi Bioprocesses Using UV Chromatogram Fingerprints

Monitoring process variables in bioprocesses with complex expression systems, such as filamentous fungi, requires a vast number of offline methods or sophisticated inline sensors. In this respect, cell viability is a crucial process variable determining the overall process performance. Thus, fast and precise tools for identification of key process deviations or transitions are needed. However, such reliable monitoring tools are still scarce to date or require sophisticated equipment. In this study, we used the commonly available size exclusion chromatography (SEC) HPLC technique to capture impurity release information in Penicillium chrysogenum bioprocesses. We exploited the impurity release information contained in UV chromatograms as fingerprints for development of principal component analysis (PCA) models to descriptively analyze the process trends. Prediction models using well established approaches, such as partial least squares (PLS), orthogonal PLS (OPLS) and principal component regression (PCR), were made to predict the viability with model accuracies of 90% or higher. Furthermore, we demonstrated the platform applicability of our method by monitoring viability in a Trichoderma reesei process for cellulase production. We are convinced that this method will not only facilitate monitoring viability of complex bioprocesses but could also be used for enhanced process control with hybrid models in the future

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

Abstract Background The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce. Results A Man5GlcNAc2 glycosylating and a Man8–10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8–10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C. Conclusion This study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

The final publication is available at Springer via https://doi.org/10.1007/s00253-016-7620-7.Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (qs,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (qs,lac) and qs,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying qs,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between qs,lac and qs,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli