Microtiter Plate Cultivation Research Articles

High-throughput G protein-coupled receptor-based autocrine screening for secondary metabolite production in yeast.

Biosensors are valuable tools in accelerating the test phase of the design-build-test-learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein-coupled receptor (GPCR)-based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR-based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment. Here, we explore the integration of production and sensing modalities into a single Saccharomyces cerevisiae strain and compartmentalization using three different methods: (1) cultivation in microtiter plates, (2) spatial separation on agar plates, and (3) encapsulation in water-in-oil-in-water double emulsion droplets, combined with analysis and sorting via a fluorescence-activated cell sorting machine. Employing tryptamine and serotonin as proof-of-concept target molecules, we optimize biosensing conditions and demonstrate the ability of the autocrine screening method to enrich for high producers, showing the enrichment of a serotonin-producing strain over a nonproducing strain. These findings illustrate a workflow that can be adapted to screening for a wide range of complex chemistry at high throughput using commercially available microfluidic systems.

DoE-based medium optimization for improved biosurfactant production with Aureobasidium pullulans.

Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus Aureobasidium pullulans are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of A. pullulans have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48gL-1, and the space-time yield from 0.13 to 0.20gL-1h-1 in microtiter plate cultivations. This was followed by a successful transfer to a 1L bioreactor, reaching a polyol lipid concentration of 41gL-1. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.

Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates

BackgroundOne critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms.ResultsSince most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter.ConclusionIn this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Microbioreactor‐assisted cultivation workflows for time‐efficient phenotyping of protein producing Aspergillus niger in batch and fed‐batch mode

In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.

High-throughput screening of lycopene-overproducing mutants of Blakeslea trispora by combining ARTP mutation with microtiter plate cultivation and transcriptional changes revealed by RNA-seq

Random mutagenesis is still the main strategy for increasing the lycopene yield from Blakeslea trispora because of its complex, unclear genetic regulation. In this study, an effective mutation breeding method was established to obtain lycopene-overproducing mutants, and the transcriptional changes between the mutant and parent strains were then revealed by RNA-seq. After ARTP mutation and preliminary screening on solid plates, 50 color mutants were simultaneously inoculated into a microtiter plate-based system (MTPS) and traditional flasks to estimate the dependability of the high-throughput screening method. As expected, a significant linear correlation (R2 = 0.924) was observed between the absorbance of pigment in the MTPs and lycopene production in flask fermentation. A mutant (WY-239) showing a maximum lycopene concentration of 21.80 ± 1.58 mg/g was obtained, which represented a 56.27 % increment compared with the parent strain. Transcriptomic analysis showed that the carbon metabolism flowed more intensively to acetyl-CoA and then to lycopene in the WY-239 mutant compared with the parent strain. Some primary metabolic and sexual activity may be correlated with lycopene biosynthesis. The mutation breeding method could also be used as a reference for breeding other filamentous fungi, and the findings of RNA-seq can serve as a basis for metabolic engineering.

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture.

Cocultures bear great potential in the conversion of complex substrates and process intensification, as well as, in the formation of unique components only available due to inter-species interactions. Dynamic data of coculture composition is necessary for understanding and optimizing coculture systems. However, most standard online determined parameters measure the sum of all species in the reactor system. The kinetic behavior of the individual species remains unknown. Up to now, different offline methods are available to determine the culture composition, as well as the online measurement of fluorescence of genetically modified organisms. To avoid any genetic modification, a noninvasive online monitoring tool based on the scattered light spectrum was developed for microtiter plate cultivations. To demonstrate the potential, a coculture consisting of the bacterium Lactococcus lactis and the yeast Kluyveromyces marxianus was cultivated. Via partial least squares regression of scattered light spectra, the online determination of the individual biomass concentrations without further sampling and analyses is possible. The results were successfully validated by a Coulter counter-analysis, taking advantage of the different cell sizes of both organisms. The findings prove the applicability of the new method to follow in detail the dynamics of a coculture.

FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into d-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.

Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium

BackgroundCulture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097.ResultsBacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth.ConclusionsThe introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.

Microbioreactor Systems for Accelerated Bioprocess Development.

In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

Differential transcriptomic analysis reveals hidden light response in Streptomyces lividans.

Recently, a comprehensive screening workflow for the filamentous bacterium Streptomyces lividans, a highly performant source for pharmaceutically active agents was introduced. This framework used parallelized cultivation in microtiter plates to efficiently accelerate early upstream process development. Focusing on growth performance, cultivation was successfully scaled-up to 1 L stirred tank reactors. However, metabolic adaptation was observed on the transcriptomic level as among others, several genes incorporated in light response were upregulated during bioreactor cultivation. Despite it was assumed that this was attributed to the fact that reactor cultivations were performed in glass vessels exposed to daylight and artificial room light, this setup did not allow distinguishing exclusively between light and other effects. Upon that, the present study directly investigates the influence of light by defined illumination of microtiter plate cultures. Almost identical growth performance was observed for cultures grown in the dark or with illumination. Transcriptomics revealed the upregulation of seven genes of which 6 have previously been described to be relevant for carotenoid synthesis and its regulation. These pigments are effective quenchers of reactive oxygen species. The seventh transcript coded for a photo-lyase incorporated in UV-damage repair of DNA further confirming induced light response. However, this was fully compensated by metabolic adaptation on the transcriptomic level and overall process performance was maintained. Consequently, environmental conditions need extremely careful control and evaluation during in-depth omics analysis of bioprocesses. Otherwise metabolic adaptation induced by such issues can easily be misinterpreted, especially during studies addressing cultivation system comparisons. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:287-292, 2018.

Microtiter plate cultivation of oleaginous fungi and monitoring of lipogenesis by high-throughput FTIR spectroscopy

BackgroundOleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi.ResultsThe microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R2 = 0.86–0.96) and satisfactory residual predictive deviation of cross-validation (RPDCV = 2.6–5.1) values demonstrated the goodness of these models.ConclusionsWe have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.

Fast and reliable strain characterization of Streptomyces lividans through micro-scale cultivation.

Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms. To address this challenge, we developed an integrated cultivation workflow for Streptomyces lividans based on a parallelized shaken 48-well microtiter-plate (MTP) cultivation device. In a first step, a feasible pre-culture method was identified and validated, revealing high comparability in subsequent main cultivations (coefficient of variation of 1.1% for in-plate replicates and 3.2% between different pre-cultures). When validating the growth performance in 1 mL MTP cultivation against an established 1,000 mL lab-scale cultivation system, highly comparable cultivation patterns were found for online (pH, dissolved oxygen), as well as for offline derived parameters (glucose uptake, cell-dry-weight, and pellet size). Additionally, the two cultivation regimes were compared with respect to transcriptional and protein secretion activity of Streptomyces, showing overall good comparability with minor, but well explainable discrepancies, most probably caused by different energy dissipation (shaking vs. stirring) and adaption effects due to different illumination conditions. Embedded within the presented cultivation workflow, the 1 mL MTP-based parallelized cultivation system seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. This can contribute to widen the field of application for these organisms and facilitate screening and early-stage bioprocess development. Biotechnol. Bioeng. 2017;114: 2011-2022. © 2017 Wiley Periodicals, Inc.

Screening of an Escherichia coli promoter library for a phenylalanine biosensor.

In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

Fed-batch like cultivation in a micro-bioreactor: screening conditions relevant for Escherichia coli based production processes.

ObjectivesRecombinant protein production processes in Escherichia coli are usually operated in fed-batch mode; therefore, the elaboration of a fed-batch cultivation protocol in microtiter plates that allows for screening under production like conditions is particularly appealing.ResultsA highly reproducible fed-batch like microtiter plate cultivation protocol for E. coli in a micro-bioreactor system with advanced online monitoring capabilities was developed. A synthetic enzymatic glucose release medium was employed to provide carbon limited growth conditions without external substrate feed and the required buffer capacity to keep the pH value within 7 ± 1. Accurate process design allowed for cultivation up to cell densities of 10 g biomass l−1 without any limitations in oxygen supply [dissolved oxygen (DO) level above 30 %]. In the micro-bioreactor system (BioLector) online monitoring of cell growth, DO and pH was performed. Furthermore, the influence of the cultivation temperature, the applicability for different host strains as well as the transferability of results to lab-scale bioreactor cultivations was evaluated.ConclusionThis robust microtiter plate cultivation protocol allows for screening of E. coli systems under conditions comparable to lab-scale bioreactor cultivations.

(13)C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H.

Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically 13C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from 13C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with 13C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments.

Bioprocess automation on a Mini Pilot Plant enables fast quantitative microbial phenotyping.

BackgroundThe throughput of cultivation experiments in bioprocess development has drastically increased in recent years due to the availability of sophisticated microliter scale cultivation devices. However, as these devices still require time-consuming manual work, the bottleneck was merely shifted to media preparation, inoculation and finally the analyses of cultivation samples. A first step towards solving these issues was undertaken in our former study by embedding a BioLector in a robotic workstation. This workstation already allowed for the optimization of heterologous protein production processes, but remained limited when aiming for the characterization of small molecule producer strains. In this work, we extended our workstation to a versatile Mini Pilot Plant (MPP) by integrating further robotic workflows and microtiter plate assays that now enable a fast and accurate phenotyping of a broad range of microbial production hosts.ResultsA fully automated harvest procedure was established, which repeatedly samples up to 48 wells from BioLector cultivations in response to individually defined trigger conditions. The samples are automatically clarified by centrifugation and finally frozen for subsequent analyses. Sensitive metabolite assays in 384-well plate scale were integrated on the MPP for the direct determination of substrate uptake (specifically D-glucose and D-xylose) and product formation (specifically amino acids). In a first application, we characterized a set of Corynebacterium glutamicum L-lysine producer strains and could rapidly identify a unique strain showing increased L-lysine titers, which was subsequently confirmed in lab-scale bioreactor experiments. In a second study, we analyzed the substrate uptake kinetics of a previously constructed D-xylose-converting C. glutamicum strain during cultivation on mixed carbon sources in a fully automated experiment.ConclusionsThe presented MPP is designed to face the challenges typically encountered during early-stage bioprocess development. Especially the bottleneck of sample analyses from fast and parallelized microtiter plate cultivations can be solved using cutting-edge robotic automation. As robotic workstations become increasingly attractive for biotechnological research, we expect our setup to become a template for future bioprocess development.

Scale-down of vinegar production into microtiter plates using a custom-made lid

As an important food preservative and condiment, vinegar is widely produced in industry by submerged acetic acid bacteria cultures. Although vinegar production is established on the large scale, up to now suitable microscale cultivation methods, e.g. using microtiter plates, are missing to enable high-throughput cultivation and to optimize fermentation conditions. In order to minimize evaporation losses of ethanol and acetic acid in a 48-well microtiter plate during vinegar production a new custom-made lid was developed. A diffusion model was used to calculate the dimensions of a hole in the lid to guarantee a suitable oxygen supply and level of ventilation. Reference fermentation was conducted in a 9-L bioreactor to enable the calculation of the proper cultivation conditions in the microtiter plate. The minimum dissolved oxygen tensions in the microtiter plate were between 7.5% and 23% of air saturation and in the same range as in the 9-L bioreactor. Evaporation losses of ethanol and acetic acid were less than 5% after 47 h and considerably reduced compared to those of microtiter plate fermentations with a conventional gas-permeable seal. Furthermore, cultivation times in the microtiter plate were with about 40 h as long as in the 9-L bioreactor. In conclusion, microtiter plate cultivations with the new custom-made lid provide a platform for high-throughput studies on vinegar production. Results are comparable to those in the 9-L bioreactor.

Automation Highlights from the Literature

Automation Highlights from the Literature

Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

BackgroundConventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP).ResultsAs an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct.ConclusionsOur low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.