Kaisa Ukkonen, Antti Vasala, Heikki Ojamo and Peter Neubauer

Abstract:

This report describes the combined use of an enzyme-based glucose release system (EnBase(R)) and high-aeration shake flask (Ultra Yield FlaskTM). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

Microbial Cell Factories 2011, 10:107

Julia Glazyrina, Mirja Krause, Stefan Junne, Florian Glauche, Dirk Strom, Peter Neubauer

Abstract:

The enzyme controlled substrate delivery cultivation technology EnBase(®) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3L bioreactor and 150L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40gL(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.

The ePublication can be found at N Biotechnol. 9 Nov. 2011

Reference publications using EnBase Technology

updated January 2012

BioSilta.Publications.1.2012.pdf

Stefan Junne, Arne Klingner, Johannes Kabisch, Thomas Schweder and Peter Neubauer

Abstract:

This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.

The publication can be read in the Biotechnology Journal - Special Issue: Bioprocess Scale-up/down

Biotechnology Journal. 2011. 6: 1009–1017

Maciej Pilarek, Julia Glazyrina and Peter Neubauer

Abstract

Background: Liquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase.

Results: In EnBase(R) cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 uM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40 % compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase.

Conclusions: PFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

Abstract and provisional publication available at Microbial Cell Factories.

Microb. Cell. Fact. 2011, 10:50

Juozas Šiurkus and Peter Neubauer

Abstract

Background: The eukaryotic RNase ribonuclease/angiogenin inhibitors (RI) are a protein group distinguished by a unique structure – they are composed of hydrophobic leucine-rich repeat motifs (LRR) and contain a high amount of reduced cysteine residues. The members of this group are difficult to produce in E. coli and other recombinant hosts due to their high aggregation tendency.

Results: In this work dithiothreitol (DTT) was successfully applied for improving the yield of correctly folded ribonuclease/angiogenin inhibitor in E.coli K12 periplasmic and cytoplasmic compartments. The feasibility of the in vivo folding concepts for cytoplasmic and periplasmic production were demonstrated at batch and fed-batch cultivation modes in shake flasks and at the bioreactor scale. Firstly, the best secretion conditions of RI in the periplasmic space were evaluated by using a high throughput multifactorial screening approach of a vector library, directly with the Enbase fed-batch production mode in 96-well plates. Secondly, the effect of the redox environment was evaluated in isogenic dsbA+ and dsbA- strains at the various cultivation conditions with reducing agents in the cultivation medium. Despite the fusion to the signal peptide, highest activities were found in the cytoplasmic fraction. Thus by removing the signal peptide the positive effect of the reducing agent DTT was clearly proven also for the cytoplasmic compartment. Finally, optimal periplasmic and cytoplasmic RI fed-batch production processes involving externally added DTT were developed in shake flasks and scaled up to the bioreactor scale.

Conclusions: DTT highly improved both, periplasmic and cytoplasmic accumulation and activity of RI at low synthesis rate, i.e. in constructs harbouring weak recombinant synthesis rate stipulating genetic elements together with cultivation at low temperature. In a stirred bioreactor environment RI folding was strongly improved by repeated pulse addition of DTT at low aeration conditions.

Free download of this publication from Microbial Cell Factories

Microb. Cell. Fact. 2011, 10:31.

Hanna Tegel, Louise Yderland, Tove Boström, Cecilia Eriksson, Kaisa Ukkonen, Antti Vasala, Peter Neubauer, Jenny Ottosson and Sophia Hober

Abstract

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

The publication is available online here.

Tatu J. K. Haataja, M. Kristian Koski, J. Kalervo Hiltunen and Tuomo Glumoff

Abstract

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics. All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2. 

Link to the publication abstract 

Biochem. J. 435(3),771-81.

Sanna Taskila, Ekaterina Osmekhina, Mika Tuomola, Jari Ruuska and Peter Neubauer

Abstract

Rapid detection of Salmonella in foods is often limited by the high demand for the sensitivity of detection, poor physiological conditions of the target cells, and high concentration of background flora. In this study, the conditions of nonselective enrichment cultivation were modified in order to improve the quantitative detection of heat-injured Salmonella in minced meat. The effect of the modifications on the recovery was observed by means of RNA-based sandwich hybridization, which was adjusted for the quantification of Salmonella enterica 23S rRNA in crude cell extracts. The supplementation of buffered peptone water with the enzyme-controlled substrate delivery system EnBase-Flo and ferrioxamine E was shown to improve the recovery of cells in both single strain cultures and in the presence of minced meat. The presented results can be used for the development of more efficient enrichment cultivation media for faster detection of food borne Salmonella.

Link to publication

J. Food Sci. 2011:76,M157-M162

Van Dat Nguyen, Feras Hatahet, Kirsi EH Salo, Eveliina Enlund, Chi Zhang and Lloyd W Ruddock

Abstract

Background: Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.

Results: Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible

Conclusions: Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.

Free download of this publication from Microbial Cell Factories

Microb. Cell. Fact. 2011,10:1

Ralf Hortsch and Dirk Weuster-Botz

Abstract

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.

Link to the publication abstract

Alexander Ehrmann, Klaus Richter, Florian Busch, Julia Reimann, Sonja-Verena Albers and Reinhard Sterner

Abstract

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate L-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for L-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of L-serine.

Link to Publication

Biochemistry 2010, 49, 10842–10853

Ekaterina Osmekhina, Antje Neubauer, Katharina Klinzing, Johanna Myllyharju, and Peter Neubauer

Abstract

Background: We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.

Results: We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.

Conclusions: Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Free download of this publication from Microbial Cell Factories

Julia Glazyrina, Eva-Maria Materne, Thomas Dreher, Dirk Storm, Stefan Junne, Thorsten Adams, Gerhard Greller and Peter Neubauer

Abstract

Background: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.

Results: Cultivation of E. coli in the BIOSTAT CultiBag RM system in a conventional batch mode without control yielded an opti cal density (OD(600)) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD(600) of 60 (20 g L(-1) cell dry weight). By the use of an internal controlled glucose delivery system, EnBase Flo, OD(600) of 30 (10 g L(-1) cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.

Conclusions: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Free download of this publication from Microbial Cell Factories

Juozas Šiurkus, Johanna Panula-Perälä, Uwe Horn, Mario Kraft, Renata Rimšeliene and Peter Neubauer

Abstract

Background: Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.

Results: Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.

Conclusions: Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditions from the first screening phase not only shortens the developmental line by granting that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.

Free download of this publication at Microbial Cell Factories

Mirja Krause, Kaisa Ukkonen, Tatu Haataja, Maria Ruottinen, Tuomo Glumoff, Antje Neubauer, Peter Neubauer and Antti Vasala

Abstract

Background: Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.

Results: The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 ~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l-1 cell dry weight could be achieved with the developed technique. In comp arison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.

Conclusions: We have demonstrated that by applying the novel EnBase® Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.

Publication can be download for free at Microbial Cell Factories

Sanna Taskila, Peter Neubauer, Mika Tuomola, Antje Breitenstein, Jukka Kronlöf and Tomi Hillukkala

Abstract

Lactic acid bacteria (LAB) are known as predominant beer spoilers. They cause turbidity, acidity, gas formation and offflavors in beer by formation of side metabolites. Beer spoiling LAB have a substantial financial impact in the brewing industry making their rapid detection and identification essential. Despite the developed rapid diagnostic methods, the bottleneck in detection remains the lengthy enrichment cultivation step. This paper describes the applicability of a novel glucose auto delivery system, EnBase, for the improved enrichment cultivation of beer spoiling LAB in MRS medium. By means of the applied system, glucose is slowly released into the culture during growth, which results in faster enrichment. Growth of Lactobacillus brevis DSM 20054T and several beer spoiling LAB was accelerated resulting in up to a 300% increase in the cell density after 48 h of cultivation compared to the commonly used MRS medium. A test of naturally contaminated beer samples indicated that the addition of glucose by means of EnBase allows faster detection of LAB in breweries.

Free download of this publication at The Journal of the Institute of Brewing and Distilling

Johanna Panula-Perälä, Juozas Šiurkus, Antti Vasala, Robert Wilmanowski, Marco G Casteleijn and Peter Neubauer

Abstract

Background: Here we describe a novel cultivation method, called EnBase™, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.

Results: High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel. Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.

Conclusion: The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

Free download of this publication from Microbial Cell Factories