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.

A model‐based framework for parallel scale‐down fed‐batch cultivations in mini‐bioreactors for accelerated phenotyping

“…An initial challenge of these systems was the difficulty in implementation of controlled feeding strategies, as in laboratory bioreactors. However, various solutions have been developed recently to enable fed-batch-like conditions, such as the gradual supply of glucose to the culture through enzyme-based glucose release systems (Krause, Neubauer, & Neubauer, 2016), the application of micropumps for continuous feed supply (Gebhardt, Hortsch, Kaufmann, Arnold, & Weuster-Botz, 2011), model-based intermittent feeding (Sawatzki et al, 2018), and membrane-based substrate release systems (Philip et al, 2018). These improvements bring the screening system closer to actual cultivation conditions, such that a better performing strain can be selected for scale-up.…”

Accelerated Bioprocess Development of Endopolygalacturonase-Production with<em> Saccharomyces cerevisiae</em> Using Multivariate Prediction in a 48 Mini-Bioreactor Automated Platform

“…stress. An improvement of the cultivation conditions can be achieved by enzymatic glucose release mimicking continuous fed-batch conditions [53].…”

Comparative Characteristics of Biotechnological Approaches to Obtaining Recombinant Human Cytokines in Bacterial Expressing Systems

“…Необмежена доступність поживних речовин і експоненціальний ріст у системах періодичного типу призводять до утворення побічних метаболітів і підкислення культур. Дезамінування амінокислотних субстратів, яке відбувається при використанні пептонів або дріжджового екстракту, може викликати підвищення рН унаслідок виділення в середовище аміаку [30]. Використання мінімальних середовищ дає змогу вирішити ці проблеми і досягти вищого рівня синтезу [19].…”