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The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli.

ABSTRACT: While the nutrient limited fed-batch technology is the standard of the cultivation of microorganisms and production of heterologous proteins in industry, despite its advantages in view of metabolic control and high cell density growth, shaken batch cultures are still the standard for protein production and expression screening in molecular biology and biochemistry laboratories. This is due to the difficulty and expenses to apply a controlled continuous glucose feed to shaken cultures. New ready-made growth media, e.g. by biocatalytic release of glucose from a polymer, offer a simple solution for the application of the fed-batch principle in shaken plate and flask cultures. Their wider use has shown that the controlled diet not only provides a solution to obtain significantly higher cell yields, but also in many cases folding of the target protein is improved by the applied lower growth rates; i.e. final volumetric yields for the active protein can be a multiple of what is obtained in complex medium cultures. The combination of the conventional optimization approaches with new and easy applicable growth systems has revolutionized recombinant protein production in Escherichia coli in view of product yield, culture robustness as well as significantly increased cell densities. This technical development establishes the basis for successful miniaturization and parallelization which is now an important tool for synthetic biology and protein engineering approaches. This review provides an overview of the recent developments, results and applications of advanced growth systems which use a controlled glucose release as substrate supply.

SUBMITTER: Krause M

PROVIDER: S-EPMC4912726 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli.

Krause Mirja M Neubauer Antje A Neubauer Peter P

Microbial cell factories 20160617 1

While the nutrient limited fed-batch technology is the standard of the cultivation of microorganisms and production of heterologous proteins in industry, despite its advantages in view of metabolic control and high cell density growth, shaken batch cultures are still the standard for protein production and expression screening in molecular biology and biochemistry laboratories. This is due to the difficulty and expenses to apply a controlled continuous glucose feed to shaken cultures. New ready- ...[more]

PMID: 27317421

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Project description:BackgroundLack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production systems. Since microalgae are capable of consuming both nitrate and ammonium, this represents an opportunity to balance culture pH based on a nitrogen feeding strategy that does not utilize gas-phase CO₂ buffering. Stoichiometry suggests that approximately 36 weight%NH₄⁺ (balance nitrogen as NO₃⁻) would minimize the proton imbalance and permit high-density photoautotrophic growth as it does in higher plant tissue culture. However, algal media almost exclusively utilize nitrate, and ammonium is often viewed as 'toxic' to algae.ResultsThe microalgae Chlorella vulgaris and Chlamydomonas reinhardtii exclusively utilize ammonium when both ammonium and nitrate are provided during growth on excess CO₂. The resulting proton imbalance from preferential ammonium utilization causes the pH to drop too low to sustain further growth when ammonium was only 9% of the total nitrogen (0.027 gN-NH₄⁺/L). However, providing smaller amounts of ammonium sequentially in the presence of nitrate maintained the pH of a Chlorella vulgaris culture for improved growth on 0.3 gN/L to 5 gDW/L under 5% CO₂ gas-phase supplementation. Bioreactor pH dynamics are shown to be predictable based on simple nitrogen assimilation as long as there is sufficient CO₂ availability.ConclusionsThis work provides both a media formulation and a feeding strategy with a focus on nitrogen metabolism and regulation to support high-density algal culture without buffering. The instability in culture pH that is observed in microalgal cultures in the absence of buffers can be overcome through alternating utilization of ammonium and nitrate. Despite the highly regulated array of nitrogen transporters, providing a nitrogen source with a balanced degree of reduction minimizes pH fluctuations. Understanding and accommodating the behavior of nitrogen utilization in microalgae is key to avoiding 'culture crash' and reliance on gas phase CO₂ buffering, which becomes both ineffective and cost-prohibitive for commercial-scale algal culture.

Dataset Information

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli.

Publications

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli.

Similar Datasets

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