High cell density cultivation of E. coli in shake flasks for the production of recombinant proteins

Ganjave, Snehal D.; Dodia, Hardik; Sunder, Avinash Vellore; Madhu, Swati; Wangikar, Pramod P.

doi:10.1016/j.btre.2021.e00694

Cited by 18 publications

(18 citation statements)

References 41 publications

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“…This intein self-cleavage system also requires no auxiliary enzymes or chemicals to remove the carrier protein. Additionally, by using bioreactors, volumetric protein production ( E. coli cell density) could be improved up to 10–34 fold using a fed-batch strategy compared to batch cultivation [ 57 ]. Therefore, this intein self-cleavage system presents a potential method to produce AMPs in E. coli and is beneficial to reducing the cost of production for commercial scale production.…”

Section: Resultsmentioning

confidence: 99%

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

Chih

Hsieh

et al. 2022

Biomedicines

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Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

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Section: Resultsmentioning

confidence: 99%

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

Chih

Hsieh

et al. 2022

Biomedicines

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“…During the 24 h period, the bacteria are in rapid accumulation and sugar is used primarily for cell growth. Rapid bacterial growth leads to large specific growth rates, allowing for greater synthesis of the by-product acetate, so a lower agitation speed and feeding of CCH (total sugar concentration was 223 g/L as determined in the first section) was chosen to maintain normal bacterial growth (Ganjave et al 2022). However, after this, the organism began to enter a formal 2,3-BDO production period, requiring higher agitation rates and the provision of sufficient sugar (500 g/L glucose) for rapid production and accumulation of 2,3-BDO.…”

Section: Fed-batch Fermentation Using Cch As Carbon Source For 23-bdomentioning

confidence: 99%

Utilization of corncob hydrolysate enables 2,3-butanediol production in Enterobacter cholerae

Wang

Zhi

et al. 2022

BioRes

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2,3-Butanediol (2,3-BDO) is an important industrial diol that could function in various fields. Currently, there are many substrates used for 2,3-BDO biosynthesis, but studies using green carbon sources such as corncob hydrolysate as a substrate are lacking. As a widely distributed waste lignocellulose-derived substrate, corncob hydrolysate is nutrient-rich and cost-effective. The present study evaluated 2,3-BDO production via an Enterobacter cholerae strain using corncob hydrolysate as carbon source. Chemical component analysis showed that concentrated corncob hydrolysate contained 233 g/L total sugar and showed no inhibitory effect, but it was beneficial for 2,3-BDO synthesis. Optimization experiments for fermentation resulted in a titer of 47.23 g/L 2,3-BDO with a yield of 0.30 g/g and a productivity rate of 0.66 g/L•h. This study is expected to provide insights for large-scale bioproduction of bulk chemicals utilizing corncob hydrolysates.

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“…Microfluidic devices have been implemented to perform fed-batch schemes in microtiter plates [ 12 ]. Other devices have been proposed to feed glucose solutions to shake flasks [ 13 , 14 ]. The specific growth rate can be efficiently controlled in mini bioreactors using mathematical models coupled to feeding strategies [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains

et al. 2022

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Background Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. Results The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h−1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h−1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h−1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. Conclusion The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

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High cell density cultivation of E. coli in shake flasks for the production of recombinant proteins

Cited by 18 publications

References 41 publications

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

Utilization of corncob hydrolysate enables 2,3-butanediol production in Enterobacter cholerae

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains

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