Fed‐batch production of recombinant β‐galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations

Prytz, Ingela; Sandén, Anna Maria; Nyström, Thomas; Farewell, Anne; Wahlström, Åsa; Förberg, Cecilia; Prágai, Zoltán; Barer, Michael R.; Harwood, Colin R.; Larsson, Gen

doi:10.1002/bit.10716

Cited by 14 publications

(5 citation statements)

References 20 publications

(18 reference statements)

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“…The rpoS and csiE exhibits a very low basal level of GFP expression, whereas this basal level is high in the case of the uspA strain. This quite high basal level has been previously observed with an equivalent lacZ transcriptional reporter strain cultivated in fed-batch mode (Prytz, 2003). A significant difference has been noticed at the level of the induction profile between classical and scale-down bioreactor for the rpoS and csiE strains.…”

Section: Fig 10 Evolution Of the Gfp-related Fluorescence For The Rsupporting

confidence: 67%

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations - Mathematical Modeling and Related Experimental Tools

Delvigne

Brognaux²,

Gorret³

et al. 2011

Biosensors - Emerging Materials and Applications

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Section: Fig 10 Evolution Of the Gfp-related Fluorescence For The Rsupporting

confidence: 67%

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations - Mathematical Modeling and Related Experimental Tools

Delvigne

Brognaux²,

Gorret³

et al. 2011

Biosensors - Emerging Materials and Applications

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“…This effect is not seen in the fed-batch experiment presented in this work, nor has the effect been observed during fed-batch experiments for extended time periods in the same 10-l reactor as was used for the batch cultivation (unpublished data). This behaviour has previously been attributed to differential substrate uptake rates of the cells in a population due to different expression levels of the sugar permeases, leading to different production rates in different cells [20]. …”

Section: Discussionmentioning

confidence: 99%

Optimisation of surface expression using the AIDA autotransporter

2011

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BackgroundBacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.ResultsThe use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.ConclusionsA number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

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“…The expression of padA, padR and usp1 was low and relatively constant during the growth. The expression of Usp proteins from E. coli is often induced by entrance into the stationary phase of growth [Nyström and Neidhardt, 1996;Prytz et al, 2003]. This is not the case for Usp1 from L. plantarum.…”

Section: Resultsmentioning

confidence: 99%

Kinetics and Intensity of the Expression of Genes Involved in the Stress Response Tightly Induced by Phenolic Acids in Lactobacillus plantarum

et al. 2007

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In Lactobacillus plantarum, PadR, the negative transcriptional regulator of padA encoding the phenolic acid decarboxylase, is divergently oriented from padA. Moreover, it forms an operonic structure with usp1, a genewhose products display homology with proteins belonging to the UspA family of universal stress proteins. PadR is inactivated by the addition of p-coumaric, ferulic or caffeic acid to the culture medium. In order to better characterize the stress response of this bacterium to phenolic acids, we report here the kinetics and quantitative expression by qRT-PCR of the 3 genes from the padA locus. The expression of the 3 genes is very low in the non-induced condition, while the addition of 1.2 mMp-coumaric acid induces an increase in the expression of padA, padR and usp1 by factors of 8,000, 37 and 13, respectively. These maximum relative transcript levels are obtained after 5 min of induction at the end of the exponential growth phase, while phenolic acid decarboxylase activity, not detectable before induction, is increased by a factor of 8,000 in 10 min. The apparent half-life of padA mRNA is about 1.4 min. The padA-padR system displays dynamic characteristics that are valuable to the development of tools for gene expression in this bacterium.

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Fed‐batch production of recombinant β‐galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations

Cited by 14 publications

References 20 publications

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations - Mathematical Modeling and Related Experimental Tools

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations - Mathematical Modeling and Related Experimental Tools

Optimisation of surface expression using the AIDA autotransporter

Kinetics and Intensity of the Expression of Genes Involved in the Stress Response Tightly Induced by Phenolic Acids in <i>Lactobacillus plantarum</i>

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