Zielgerichtete genetische Weiterentwicklung von Bacillus megaterium für Anwendungen in der rekombinanten Proteinproduktion

The work presented in this thesis focused on the directed genetic optimisation of the Gram-positive bacterium Bacillus megaterium for recombinant protein production. For this purpose gene expression was systematically analysed and improved on transcriptional and translational level. A novel gene expression system was developed based on the highly processive RNA polymerase of the bacteriophage T7. Using this system, the green fluorescent protein (GFP) was successfully produced intracellularly to significant higher amounts (49.7 mg l-1) compared to the reference using the well-established xylose-inducible promoter system (7.2 mg l-1). Interestingly, the application of the T7 RNA polymerase dependent gene expression system for the secretory production of the exoenzyme levansucrase resulted in significantly lower protein yields possibly due to an overload of the secretion machinery and the subsequent induction of stress responses. Furthermore, a versatile codon test system was developed and individual codons were experimentally identified which are limiting the efficiency of the translational process in B. megaterium. The subsequent identification of a rare tRNA gene and its coexpression significantly elevated the rate of translation of corresponding codons into the respective polypeptide chain as estimated by the formation of the model protein GFP. First insights into the molecular basis for the development of natural competence in B. megaterium were obtained. Overproduction of the putative central transcription factor ComK for competence development resulted in partial cell lysis. Nevertheless, for the first time the ability of B. megaterium to develop a functional machinery for DNA-uptake and -integration was demonstrated. As a proof of concept two eukaryotic proteins of industrial importance, a human keratin-binding domain and a fungal hydrophobin, were produced with B. megaterium in cooperation with the BASF SE. Multiple parameters like codon usage optimisation, the nature of the signal peptide or the cultivation conditions were analysed in order to improve the yield in secretory production of the respective proteins. It was shown that intracellular aggregation and degradation severely hampered quantitative production of the keratin-binding domain. The fungal hydrophobin on the other hand could successfully be produced, secreted and purified to homogeneity and its surface active properties were demonstrated.Im Rahmen dieser Arbeit wurde das Gram-positive Bakterium Bacillus megaterium gezielt für Anwendungen in der rekombinanten Proteinproduktion optimiert. Hierfür wurde der Prozess der rekombinanten Genexpression auf den Ebenen der Transkription und Translation analysiert und verbessert. Ein neuartiges T7 RNA Polymerase-basiertes Genexpressionssystem wurde für B. megaterium etabliert. Mit diesem System konnte das grün fluoreszierende Protein (GFP) erfolgreich intrazellulär produziert werde, wobei die Ausbeute um den Faktor 7 im Vergleich zum etablierten Xylose-induzierbaren Promotorsystem gesteigert werden konnte. Interessanterweise resultierte die vergleichende Produktion des heterologen Exoenzyms Levansucrase mit Hilfe des T7 RNA Polymerase basierten Genexpressionssystems in einer geringeren Proteinausbeute. Diese Beobachtung deutete auf eine mögliche Überlastung der Sekretionsmaschinerie und einer daraus resultierenden Stressantwort hin. Weiterhin wurde ein Codon-Testsystem etabliert, mit dessen Hilfe einzelne Codons experimentell identifiziert wurden, die die Translationseffizienz in B. megaterium limitieren. Die Coexpression einer seltenen tRNA konnte im Folgenden als Strategie zur Steigerung der Translationseffizienz genutzt werden. Zusätzlich konnten erste Einblicke in die molekularen Grundlagen einer möglichen Kompetenzentwicklung bei B. megaterium gewonnen werden. Überproduktion des zentralen putativen Transkriptionsfaktors zur Entwicklung der natürlichen genetischen Kompetenz resultierte in einer partiellen Zelllyse. Dennoch konnte zum ersten Mal die Fähigkeit von B. megaterium zur Ausbildung einer funktionellen DNA Transformationsmaschinerie aufgezeigt werden. In Kooperation mit der BASF SE wurden des Weiteren zwei industriell relevante Proteine von eukaryotischem Ursprung in B. megaterium produziert. Hierbei handelte es sich um eine Keratin-bindende Domäne und um ein Hydrophobin. Zahlreiche Parameter wie die Codon Usage Anpassung, die Wahl des Signalpeptids oder die Kultivierungsparameter wurden variiert, um eine hohe Ausbeute an sekretiertem Zielprotein zu erzielen. Jedoch zeigte sich, dass intrazelluläre Aggregation und Degradation der Keratin-bindenden Domäne einer quantitativen Produktion entgegenstanden. Das Hydrophobin hingegen konnte erfolgreich sekretorisch produziert, gereinigt und die oberflächenaktiven Eigenschaften evaluiert werden

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

BACKGROUND: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. RESULTS: For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. CONCLUSION: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations

Hepatocyte Nuclear Factor 3beta is Involved in Pancreatic Beta-Cell-Specific Transcription of the PDX-1 Gene

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdx-1 is expressed principally within insulin-secreting pancreatic islet b cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb 24.5 to 18.2 was used to drive a b-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp 22560 and 21880 (site 1), bp 21330 and 2800 (site 2), and bp 2260 and 1180 (site 3), were identified within the 5*-flanking region of the endogenous pdx-1 gene. Pancreatic b-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdx-1–herpes simplex virus thymidine kinase promoter expression constructs in transfected b-cell and non-b-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven b-galactosidase reporter construct was directed to islet b-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in b-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides 22007 and 21996. Gel shift analysis indicated that HNF3b present in islet b cells binds to this element. Immunohistochemical studies revealed that HNF3b was present within the nuclei of almost all islet b cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3b, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37°C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation

Study of an unusually high level of N-glycolylneuraminic acid (NGNA) sialylation on a monoclonal antibody expressed in Chinese hamster ovary cells

Sialic or neuraminic acids of recombinant therapeutic glycoproteins produced in mammalian cells, including monoclonal antibodies, have significant impact on the half-life, stability, and biological activity of these proteins (Hossler et al., 2009; Ghaderi et al., 2012). The predominant sialic acid N-acetylneuraminic acid (NANA or Neu5Ac) is added from precursor CMP-NANA to galactose residues of N-linked glycoproteins by sialytransferases. In most mammals CMP-NANA can also be modified to its hydroxylated derivative CMP-NGNA by CMP-N-acetylneuraminic acid hydroxylase (CMAH). NGNA can then be added from CMP-NGNA to galactose residues of the N-linked glycoproteins, also by sialytransferases. However, humans cannot make functional CMAH due to an inactivating exon deletion mutation in CMAH gene (Okerblom and Varki, 2017), and therefore cannot convert CMP-NANA to CMP-NGNA. Consequently, when injected into human patients, NGNA sialic acid containing mAbs or other recombinant glycoproteins may induce immune responses, which could negatively impact pharmacokinetics or efficacy. Therefore high levels of NGNA on therapeutic mAbs or other recombinant glycoproteins are an undesirable product quality attribute. The level of total sialic acids of recombinant glycoproteins produced in Chinese hamster ovary (CHO) cells is dictated largely by the selected cell lines, upstream process, and to a lesser degree, downstream process. NGNA sialylation is generally rare in CHO cells (Könitzer et al., 2015). Hence, therapeutic glycoproteins manufactured in these cells are considered safe for human use. However, during a first-in-human (FIH) upstream process development for a novel mAb, an initially selected desirable cell line (A) was found to produce the mAb with an unexpectedly high level of the NGNA sialic acid (\u3e30%). To the best of our knowledge such high level of NGNA sialylation on a mAb produced by CHO cells has not been reported. To mitigate potential risks associated with high NGNA in human patients, a new cell line (B) that produces the mAb with very low NGNA was selected as the manufacturing cell line for this project. In order to understand the molecular mechanism causing the high NGNA content in cell line A, we initiated comprehensive genetic gap analyses using next-generation sequencing technologies and determined the differences in genomic, transcriptomic, and miRnomic profiles of the two cell lines. The results indicate spontaneous upregulation of CMAH mRNA expression, at least 10 fold higher in cell line A compared to cell line B. In this talk we will summarize the results of our studies of this unusual sialylation behavior in CHO cells

Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast

Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration

Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.Fil: Rodrigo Albors, Aida. Deutsche Forschungsgemeinschaft; Alemania. Max Planck Institute of Molecular Cell Biology and Genetics; Alemania. Technische Universitat Dresden; AlemaniaFil: Tazaky, Akira. Deutsche Forschungsgemeinschaft; Alemania. Max Planck Institute of Molecular Cell Biology and Genetics; Alemania. Technische Universitat Dresden; AlemaniaFil: Rost, Fabian. Technische Universitat Dresden; AlemaniaFil: Nowoshilow, Sergej. Deutsche Forschungsgemeinschaft; Alemania. Max Planck Institute of Molecular Cell Biology and Genetics; Alemania. Technische Universitat Dresden; AlemaniaFil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Technische Universitat Dresden; AlemaniaFil: Tanaka, Elly M. Deutsche Forschungsgemeinschaft; Alemania. Max Planck Institute of Molecular Cell Biology and Genetics; Alemania. Technische Universitat Dresden; Alemani

Variation in the Structure of Bird Nests between Northern Manitoba and Southeastern Ontario

Traits that converge in appearance under similar environmental conditions among phylogenetically independent lineages are thought to represent adaptations to local environments. We tested for convergence in nest morphology and composition of birds breeding in two ecologically different locations in Canada: Churchill in northern Manitoba and Elgin in southeastern Ontario. We examined nests from four families of passerine birds (Turdidae: Turdus, Parulidae: Dendroica, Emberizidae: Passerculus and Fringillidae: Carduelis) where closely related populations or species breed in both locations. Nests of American Robins, Yellow Warblers, and Carduelis finches had heavier nest masses, and tended to have thicker nest-walls, in northern Manitoba compared with conspecifics or congenerics breeding in southeastern Ontario. Together, all species showed evidence for wider internal and external nest-cup diameters in northern Manitoba, while individual species showed varying patterns for internal nest-cup and external nest depths. American Robins, Yellow Warblers, and Carduelis finches in northern Manitoba achieved heavier nest masses in different ways. American Robins increased all materials in similar proportions, and Yellow Warblers and Common Redpolls used greater amounts of select materials. While changes in nest composition vary uniquely for each species, the pattern of larger nests in northern Manitoba compared to southeastern Ontario in three of our four phylogenetically-independent comparisons suggests that birds are adapting to similar selective pressures between locations