Dataset Information

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.

ABSTRACT:

Background

In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in biotechnology has led to the development of many different strains, and selecting an ideal host to produce a specific protein of interest is an important step in developing a production process. E. coli B and K-12 strains are frequently employed in large-scale production processes, and therefore are of particular interest. We previously evaluated the individual cultivation characteristics of E. coli BL21 and the K-12 hosts RV308 and HMS174. To our knowledge, there has not yet been a detailed comparison of the individual performances of these production strains in terms of recombinant protein production and system stability. The present study directly compared the T7-based expression hosts E. coli BL21(DE3), RV308(DE3), and HMS174(DE3), focusing on evaluating the specific attributes of these strains in relation to high-level protein production of the model protein recombinant human superoxide dismutase (SOD). The experimental setup was an exponential carbon-limited fed-batch cultivation with minimal media and single-pulse induction.

Results

The host strain BL21(DE3) produced the highest amounts of specific protein, followed by HMS174(DE3) and RV308(DE3). The expression system HMS174(DE3) exhibited system stability by retaining the expression vector over the entire process time; however, it entirely stopped growing shortly after induction. In contrast, BL21(DE3) and RV308(DE3) encountered plasmid loss but maintained growth. RV308(DE3) exhibited the lowest ppGpp concentration, which is correlated with the metabolic stress level and lowest degradation of soluble protein fraction compared to both other strains.

Conclusions

Overall, this study provides novel data regarding the individual strain properties and production capabilities, which will enable targeted strain selection for producing a specific protein of interest. This information can be used to accelerate future process design and implementation.

SUBMITTER: Marisch K

PROVIDER: S-EPMC3698069 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.

Marisch Karoline K Bayer Karl K Cserjan-Puschmann Monika M Luchner Markus M Striedner Gerald G

Microbial cell factories 20130611

<h4>Background</h4>In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in biotechnology has led to the development of many different strains, and selecting an ideal host to produce a specific protein of interest is an important step in developing ...[more]

PMID: 23758670

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Project description:Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan(TM) DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.

Project description:BackgroundSmall-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved.ResultsCustomization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well.ConclusionsThe optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

Dataset Information

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.

Background

Results

Conclusions

Publications

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.

Similar Datasets

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