Britta Jürgen

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations.

The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein alpha-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed on the transcriptional and the translational levels. After the induction of the tac-regulated overexpression of the recombinant model protein, a significant but transient increase of the mRNA levels of the heat shock genes lon and dnaK could be observed. The mRNA level of the gene coding for the inclusion body-associated protein IbpB showed the strongest increase and remained at a clearly higher level until the end of the fermentation. By contrast, the mRNA levels of htrA and ppiB were decreased after induction of the alpha-glucosidase overexpression. Analysis of the soluble cytoplasmic protein fraction 3 h after induction revealed increased levels of the chaperones GroEL, DnaK, and Tig and a decrease in the protein levels of the two ribosomal proteins S6 and L9, the peptidylprolyl-cis-trans-isomerase PpiB, and the sigma(38)-dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the alpha-glucosidase inclusion bodies. N-terminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous alpha-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the alpha-glucosidase protein aggregates.

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.

Abstract. Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site.

Abstract In Bacillus subtilis IS58 starved of glucose or exposed to heat shock, ethanol or salt stress, the σB-dependent general stress protein GsiB is accumulated to a higher level than other general stress proteins. This high-level accumulation of GsiB can at least partially be attributed to the remarkably long half-life (∼20 min) of the gsiB mRNA. Analysis of different gsiB-lacZ fusions revealed that this stability is not determined by sequences at the 3′ end of the transcript but rather by sequences upstream of the translational start codon. Site-directed mutagenesis established that a strong ribosome binding site was crucial for the increased stability of the gsiB mRNA. A comparison of the sequences upstream of the translational start codons of three general stress genes, gsiB, gspA and ctc, revealed a direct correlation between mRNA stability and the strength of their translational signals.

Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli

Abstract. The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator σS (σ38, rpoS gene product), is suppressed during strong overproduction of a heterologous α-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli clpP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the α-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased σS level in this mutant background.

Quality control of inclusion bodies in Escherichia coli

BackgroundBacterial inclusion bodies (IBs) are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous.ResultsIBs of α-glucosidase of Saccharomyces cerevisiae after overproduction in Escherichia coli contain a large amount of (at least 12 different) major product fragments, as revealed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass-Spectrometry (MALDI-ToF MS) identification showed that these fragments contain either the N- or the C-terminus of the protein, therefore indicate that these IBs are at least partially created by proteolytic action. Expression of α-glucosidase in single knockout mutants for the major proteases ClpP, Lon, OmpT and FtsH which are known to be involved in the heat shock like response to production of recombinant proteins or to the degradation of IB proteins, clpP, lon, ompT, and ftsH did not influence the fragment pattern or the composition of the IBs. The quality of the IBs was also not influenced by the sampling time, cultivation medium (complex and mineral salt medium), production strategy (shake flask, fed-batch fermentation process), production strength (T5-lac or T7 promoter), strain background (K-12 or BL21), or addition of different protease inhibitors during IB preparation.Conclusionsα-glucosidase is fragmented before aggregation, but neither by proteolytic action on the IBs by the common major proteases, nor during downstream IB preparation. Different fragments co-aggregate in the process of IB formation together with the full-length product. Other intracellular proteases than ClpP or Lon must be responsible for fragmentation. Reaggregation of protease-stable α-glucosidase fragments during in situ disintegration of the existing IBs does not seem to occur.

Monitoring of stress responses

New developments in the RNA analysis techniques now enable a comprehensive view on the bacterial physiology under bioprocess conditions. The DNA-chip technology allows a genome wide transcriptional profiling of bacterial cells, whose genome sequence is available. Although the analyses of microbial bioprocesses have still been somewhat limited to date, this technique has already been successfully applied in different laboratories for the investigation of stress responses of selected industrially relevant bacterial hosts. Transcriptome analyses in combination with high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for the description of general and specific stress and starvation responses of Escherichia coli and Bacillus subtilis. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. This chapter gives an overview of current RNA analysis techniques. The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised.

An acetoin-regulated expression system of Bacillus subtilis.

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.