Bacillus megaterium: Produktion, Sekretion und Reinigung von rekombinanten Proteinen

In this thesis an alternative, competitive protein production and purification system was established using the Gram positive bacterium Bacillus megaterium. Therefore, a set of xylose-inducible gene expression vectors for the intracellular production of target proteins carrying combinations of N- and C-terminally fused His- and StrepII-affinity tags was constructed. These vectors were successfully tested using the green fluorescence protein (GFP) as model. Up to 18 mg GFP per l were produced in shaking flask cultures and were purified to apparent homogeneity by affinity chromatography. High cell density cultivation allowed upscaling of the procedure to 274 mg GFP per l. For the export of target proteins containing various combinations of N- and C-terminally fused His6- and StrepII-affinity tags into the growth medium, a second set of expression vectors was constructed. A Lactobacillus reuteri levansucrase served as model protein. The efficiency of a B. megaterium lipase (LipA), penicillin G acylase (PGA), and a computer designed leader peptide for SEC-dependent protein export were evaluated. Up to 4 mg levansucrase per l were exported into the growth medium. Coexpression of the signal peptidase gene sipM responsible for signal peptide removal increased the protein export. Deletion of the gene for the only detectable extracellular protease NprM stabilised exported proteins. Protocols for the purification of affinity-tagged exported proteins from the growth medium were developed. The alternative sucrose-inducible sacB promoter system was identified. This promoter was successfully applied for the high yield recombinant protein production and export. Finally, an expression system using two different, independently replicating plasmids was established and evaluated. The established system provides a useful alternative to the commonly used Escherichia coli systems. The majority of the developed vectors are commercialised by the MoBiTec GmbH (Göttingen, Germany).Im Rahmen dieser Arbeit ist ein Vektorsystem für die Produktion von rekombinanten Proteinen im Gram positiven Bakterium Bacillus megaterium etabliert worden, das eine Alternative zu herkömmlichen Escherichia coli Systemen bietet. Dafür wurden Expressionsvektoren entwickelt, die auf einem Xylose-induzierbaren Promotor basieren. Mit diesen Vektoren ist es nun möglich, rekombinante Proteine sowohl intra- als auch extrazellulär zu produzieren. Über diese Vektoren können Zielproteine sowohl C- als auch N-terminal mit His- oder StrepII-Affinitäts Tags fusioniert werden. Für die intrazelluläre Produktion wurde das System mit dem grün fluoreszierenden Protein (GFP) als Modell getestet. Bis zu 18 mg GFP pro l Schüttelkolben-Kultivierung konnten produziert werden. Über Affinitätschromatographie war es möglich die Fusionsproteine erfolgreich zu reinigen. Eine Hochzelldichte-Kultivierung ermöglichte die Erhöhung der Menge an intrazellulär produziertem GFP auf bis zu 274 mg pro l. Die Produktion von extrazellulären rekombinanten Proteinen wurde mit der Levansucrase aus Lactobacillus reuteri als Modell untersucht. Den Export des heterologen Proteins vermittelten die Signalpeptide der B. megaterium eigenen Lipase A (LipA) und Penicillin G Acylase (PGA) sowie ein bioinformatisch konstruiertes Peptid. Bis zu 4 mg Levansucrase pro Liter wurden so ins Medium sekretiert. Weiterhin erhöhte die Koexpression des Signalpeptidase-Gens sipM, verantwortlich für die Prozessierung des Pro-Proteins, die extrazelluläre Konzentration. Die Deletion des Gens für die extrazelluläre Protease NprM führte zur Stabilisiserung des exportierten Proteins. Für die Reinigung der Fusionsproteine aus dem Medium wurden erfolgreich Protokolle entwickelt. Weiterhin wurde ein neues Saccharose-induzierbares Promotorsystem identifiziert und erfolgreich für die heterologe Proteinproduktion genutzt. Ein Zwei-Vektor System steht nun ebenfalls für die Produktion rekombinanter Proteine in B. megaterium bereit

Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

In biocatalytic processes, the use of free enzymes is often limited due to the lack of long-term stability and reusability. To counteract this, enzymes can be crystallized and then immobilized, generating cross-linked enzyme crystals (CLECs). As mechanical stability and activity of CLECs are crucial, different penicillin G acylases (PGAs) from Gram-positive organisms have proven to be promising candidates for industrial production of new semisynthetic antibiotics, which can be crystallized and cross-linked to characterize the resulting CLECs regarding their mechanical and catalytic properties. The greatest hardness and Young’s modulus determined by indentation with an atomic force microscope were observed for CLECs of Bacillus species FJAT-PGA CLECs (26 MPa/1450 MPa), followed by BmPGA (Priestia megaterium PGA, 23 MPa/1170 MPa) and BtPGA CLECs (Bacillus thermotolerans PGA, 11 MPa/614 MPa). In addition, FJAT- and BtPGA CLECs showed up to 20-fold higher volumetric activities compared to BmPGA CLECs. Correlation to structural characteristics indicated that a high solvent content and low number of cross-linking residues might lead to reduced stability. Furthermore, activity seems to be restricted by small water channels due to severe diffusion limitations. To the best of our knowledge, we show for the first time in this study that the entire process chain for the characterization of diverse industrially relevant enzymes can be performed at the microliter scale to discover the most important relationships and limitation

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

Background: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. Results: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xyloseinducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 μL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L−1) and highest space–time-yield (8 mg L−1 h−1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). Conclusions: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space–time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains

Quantification of microaerobic growth of Geobacter sulfurreducens

Geobacter sulfurreducens was originally considered a strict anaerobe. However, this bacterium was later shown to not only tolerate exposure to oxygen but also to use it as terminal electron acceptor. Research performed has so far only revealed the general ability of G. sulfurreducens to reduce oxygen, but the oxygen uptake rate has not been quantified yet, nor has evidence been provided as to how the bacterium achieves oxygen reduction. Therefore, microaerobic growth of G. sulfurreducens was investigated here with better defined operating conditions as previously performed and a transcriptome analysis was performed to elucidate possible metabolic mechanisms important for oxygen reduction in G. sulfurreducens. The investigations revealed that cell growth with oxygen is possible to the same extent as with fumarate if the maximum specific oxygen uptake rate (sOUR) of 95 mgO2 gCDW-1 h-1 is not surpassed. Hereby, the entire amount of introduced oxygen is reduced. When oxygen concentrations are too high, cell growth is completely inhibited and there is no partial oxygen consumption. Transcriptome analysis suggests a menaquinol oxidase to be the enzyme responsible for oxygen reduction. Transcriptome analysis has further revealed three different survival strategies, depending on the oxygen concentration present. When prompted with small amounts of oxygen, G. sulfurreducens will try to escape the microaerobic area; if oxygen concentrations are higher, cells will focus on rapid and complete oxygen reduction coupled to cell growth; and ultimately cells will form protective layers if a complete reduction becomes impossible. The results presented here have important implications for understanding how G. sulfurreducens survives exposure to oxygen

The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory

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

Production of recombinant antibody fragments in Bacillus megaterium

BACKGROUND: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. RESULTS: The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His(6)-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. CONCLUSION: High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12)

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification

Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology

In this work, the structuring of iron oxide nanoparticles via spray-drying (SD) of aqueous suspensions is investigated, leading to micrometer-sized aggregates with saturation magnetization comparable to that of the individual nanoparticles. Interestingly, the superparamagnetic behavior is retained despite the multicore structure. Modification of the aggregates via the addition of silica nanoparticles to the suspension allows for control of the resulting magnetization by adjusting the iron oxide content. Moreover, the morphology of the produced aggregates is gradually shifted from irregular inflated-like shapes in case of pure iron oxide aggregates to reach spherical structures when bringing the silica content to only 20%. The aggregates with different magnetization can be effectively separated in a simple column with an attached permanent magnet. Functionalization of pure iron oxide aggregates with a previously coupled ligand holding a nitrilotriacetic acid (NTA)-like moiety and subsequent loading with Ni2+ ions leads to the ability to bind 6-histidine (His6)-tagged target proteins via chelation complexes for magnetic separation. The application of the presented system for the purification of recombinant protein A in multiple cycles is shown. The recyclability of the separation system in combination with the high degree of magnetic separation is promising for future applications in the field of preparative in situ protein purification