Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700 min−1. This plateau was characterized by specific power inputs between 79 and 107 W L−1 and corresponding maximal product concentrations of 6.90 μM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20 μM streptavidin in 14 days, characterized by a constant productivity of 114 nM h−1. Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development.

Streptavidin - a protein secreted by the filamentous bacterium Streptomyces avidinii - is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C. The best performing strain carrying the full-length streptavidin gene under control of PFMD was characterized in the bioreactor applying a synthetic medium and oxygen-controlled feeding of glucose. Derepression resulted in an extracellular concentration of 1.31±0.07μM of tetrameric streptavidin after 48h (27.3nMh(-1)). Feeding of glycerol improved biomass formation, but lowered the product concentration. By combining derepression and methanol induction the final extracellular streptavidin concentration increased to 11.42±0.22μM (approx. 751mgL(-1)), yielding a productivity of 52.5nMh(-1). Despite supplementing biotin the proportion of biotin-blocked binding sites in the supernatant dropped from 54.4±5.0 % after 18h to 17.2±6.5 % towards the end of glucose feeding to a final value of 1.1±3.8 %, indicating a highly bioactive product. Thus, H. polymorpha proved to be a suitable host for the production of streptavidin.

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h −1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h −1 ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog. , 32:855–864, 2016

The segregational stability of plasmids in a recombinant bioprocess is of extreme significance. Although this is commonly achieved by the selection pressure from antibiotics, their application for the production of therapeutic DNA for gene therapy or for DNA vaccines would be undesirable. Similarly, the presence of antibiotic-resistance genes in the final product would have to be avoided. In addition to the minicircle approach, a type of miniplasmid is able to fulfil the regulatory requirements. The gene tpiA is responsible for the connection of the glycerol metabolic pathway with the essential glycolytic pathway in Escherichia coli. The knockout of genomic tpiA rendered cells completely auxotrophic in minimal medium with glycerol as sole carbon source while allowing growth at a reduced rate with glucose or in complex medium. This was advantageous for optimizing antibiotic-free cloning and selection of recombinant plasmid. Complementation of the auxotrophy by plasmid-borne tpiA led to high segregational and structural plasmid stability, resulting in stable production of a model recombinant enzyme under antibiotic-free conditions in a continuous cultivation. Thus, the complementation of tpiA represents a significant alternative to antibiotics as a selection principle and is therefore of interest for the recombinant production of biotherapeutics in the form of miniplasmids.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic leaky mutant' E. coli JW1667-5 (Delta lpp-752:kan) at 30 degrees C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton (R) X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790 +/- 166 nM were reached in shake flasks at a productivity of 49.6 nM h(-1). Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608 +/- 169 nM and a productivity of 65.2 nM h(-1) in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3 +/- 4.3% indicated a highly bioactive product. (C) 2016 Elsevier B.V. All rights reserved.

Usually, the fatty acid (FA) composition of lipids from microalgae is determined using samples taken at a single time point only, often without considering the medium composition and cultivation conditions. Therefore, the results only represent the FA composition of cells in a certain growth phase. Furthermore, they may misrepresent the capability of the organism to produce certain FA or lipid mixtures. In this study, 22 FA were analysed quantitatively during the cultivation of Euglena gracilis under different cultivation modes and conditions. For cell growth, various media compositions for heterotrophic and photoheterotrophic conditions were used. Results of extensive FA analysis allowed the determination of appropriate cultivation conditions and durations for yielding lipid mixtures with optimal composition, e. g. for the production of biodiesel or functional food. Drastic differences in the ratio between n3-and n6-polyunsaturated fatty acid (PUFA) ranging from 0.04 to 1.81 were detected. This effect was strongly influenced by the cultivation mode. In addition, media with higher nitrogen concentration resulted in a higher n3/n6-PUFA-ratio as well as improved specific growth rates for all analysed combinations of glucose and nitrogen concentrations. Furthermore, it was demonstrated that the inexpensive proteose peptone medium is ideal for lipid production by E. gracilis. This work provides valuable tools to optimise yield, productivity and n3/n6-PUFA ratio concerning the cultivation of E. gracilis as well as potentially other microalgae in general.

In this study the use of Euglena gracilis biomass for ce.-tocopherol, paramylon and biogas production in a value-added chain was investigated. Therefore, we analyzed the dry cell weight and product concentrations at different growth phases during heterotrophic, photoheterotrophic and photoautotrophic cultivation in a low-cost minimal medium. Furthermore, the specific biogas yields for differently derived biomass with and without product recovery were investigated. We demonstrate that growth phase and cultivation mode not only have a significant impact on product formation, but also influence the yield of biogas obtained from anaerobic digestion of Euglena gracilis biomass. The maximum dry cell weight concentration ranged from 12.3 +/- 0.14 g L-1 for heterotrophically to 3.4 +/- 0.02 g L-1 for photoautotrophically grown Euglena gracilis cells. The heterotrophically grown biomass accumulated product concentrations of 5.3 +/- 0.12 mg L-1 of alpha-tocopherol and 9.3 +/- 0.1 g L-1 of paramylon or 805 +/- 10.9 mL of biogas g(-1) (per gram volatile solids). The results for photoautotrophically grown cells were 8.6 +/- 0.22 mg L-1 of alpha-tocopherol and 0.78 +/- 0.01 g L-1 of paramylon or 648 +/- 7.2 mL of biogas g(-1). For an energy-saving downstream procedure the extracting agent methanol does not have to be removed strictly. Samples with residual methanol showed a significantly increased biogas yield, because the solvent can be used as an additional substrate for methane production by archaebacteria. (C) 2015 Elsevier B.V. All rights reserved.

The protein streptavidin (SAV) is applied in a large variety of molecular methods due to an extraordinarily strong binding to the vitamin biotin (BIO). The protein structure is homotetrameric, characterized by one binding site for BIO per subunit. Therefore, one of the major criteria to determine the quality of SAV isolates is the proportion of BIO-blocked binding sites per tetramer. A rapid analysis of BIO-free binding sites is achieved by fluorescence quenching of biotin-4-fluorescein (B4F). However, BIO-blocked binding sites can only be determined by costly and laborious procedures such as ELISA-based methods or radioactive labeling. This study describes the systematic, model-supported development of a method for the quick and simple detection of BIO-blocked binding sites, based on a short-term heat incubation of the sample in the presence of B4F. Kinetic modeling and parameter estimation yielded dissociation constants of 1.22 +/- 0.27 x 10(-11) M for the complex SAV-BIO and 5.16 +/- 0.70 x 10(-13) M for SAV-B4F at 70 degrees C, allowing a displacement of SAV-bound BIO by B4F. This method allows the rapid monitoring of BIO-blocked binding sites in fermentation processes, independent from the chain length of SAV and the concentration of contaminating proteins, e.g. when optimizing the BIO concentration in cultivation media.

Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions. This led to the choice of a gene for knockout that only connects two essential arms of an essential metabolic pathway – the glycolysis.

Chemie Ingenieur TechnikVolume 86, Issue 9 p. 1420-1420 PosterFree Access Morphological Analysis of Streptomyces avidinii in Microplates and Shake Flasks J. M. Müller, Corresponding Author J. M. Müller jmu@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanyUniversität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, Germany===Search for more papers by this authorDr. J. M. Risse, Dr. J. M. Risse Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorProf. Dr. E. Flaschel, Prof. Dr. E. Flaschel Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this author J. M. Müller, Corresponding Author J. M. Müller jmu@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanyUniversität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, Germany===Search for more papers by this authorDr. J. M. Risse, Dr. J. M. Risse Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorProf. Dr. E. Flaschel, Prof. Dr. E. Flaschel Universität Bielefeld, Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this author First published: 28 August 2014 https://doi.org/10.1002/cite.201450638AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 J. M. Müller et al., J. Biotechnol. 2013, 163 (3), 352. 2 S. V. Sohoni et al., Microb. Cell. Fact. 2012, 11, 9. Volume86, Issue9Special Issue: ProcessNet-Jahrestagung 2014 und 31. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2014Pages 1420-1420 ReferencesRelatedInformation

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.

Chemie Ingenieur TechnikVolume 84, Issue 8 p. 1205-1206 PosterFree Access Kontinuierliche Kultivierung und Produktion extrazellulärer rekombinanter Proteine in Escherichia coli mit alternativen Plasmidselektionsmechanismen R. S. Velur Selvamani, Corresponding Author R. S. Velur Selvamani ram@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanyUniversität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorDr. techn. K. Friehs, Dr. techn. K. Friehs Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorDr. rer. nat. E. Flaschel, Dr. rer. nat. E. Flaschel Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this author R. S. Velur Selvamani, Corresponding Author R. S. Velur Selvamani ram@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanyUniversität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorDr. techn. K. Friehs, Dr. techn. K. Friehs Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this authorDr. rer. nat. E. Flaschel, Dr. rer. nat. E. Flaschel Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 Bielefeld, GermanySearch for more papers by this author First published: 25 July 2012 https://doi.org/10.1002/cite.201250085AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 U.Beshay, G.Miksch, K.Friehs, E.Flaschel, Bioprocess Biosyst. Eng. 2009, 32 (2), 149. Volume84, Issue8Special Issue: ProcessNet-Jahrestagung 2012 und 30. Jahrestagung der BiotechnologenAugust, 2012Pages 1205-1206 ReferencesRelatedInformation

Die Herstellung rekombinanter Produkte fur die pharmazeutische und industrielle Anwendung ist seit geraumer Zeit etabliert. Aufgrund der geringeren Kosten wird hierbei, sofern es moglich ist, auf Prokaryonten zuruckgegriffen. Wegen seiner einfachen Handhabung und der umfassenden Datenlage ist E. coli hier immer noch der am meisten verwendete Produktionsorganismus. Die meisten der verwendeten Expressionssysteme basieren auf der Verwendung von Plasmidvektoren. Im Rahmen dieser Prasentation sollen neue Tools vorgestellt werden, die einige der haufig auftretenden Probleme bei der Verwendung von Plasmiden speziell im industriellen Masstab verringern. Hierbei geht es vor allem um die gentechnische Optimierung und fermentationstechnische Modulierung der Gendosis zur Optimierung der Prozessproduktivitat fur schwierig herzustellende Proteine. Die etablierten Expressionsvektoren weisen einige gravierende Mangel auf, die sich unter den fur solche Proteine notwendigen Produktionsparametern negativ auf die Produktivitat auswirken. In der vorgestellten Arbeit geht es daher um die Modulierung der Plasmidkopienzahl in unterschiedlichen Prozessabschnitten, die Induktion der Proteinexpression ohne chemischen Induktor und die einfache Stabilisierung der Plasmidvektoren trotz der auf ihnen codierten wirtstoxischen Genprodukte.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.