Amino acid supplementation for enhancing recombinant protein production in E. coli

Abstract

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent‐like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth‐promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

Machine learning modeling of the effects of media formulated with various yeast extracts on heterologous protein production in Escherichia coli

“…Kumar et al also reported that 20 mixed amino acids with chemically defined media increased recombinant peptide production by 40% in E . coli BL21 (DE3) (Kumar et al, 2020). Generally, the addition of amino acids to the growth medium can influence E .…”

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

“…In spite of the advantages, some problems are also associated with the production of recombinant proteins in E. coli : the formation of inclusion bodies, the inefficient protein translocation, or the metabolic burden ( Mairhofer et al, 2013 ; Baig et al, 2014 ; Marschall et al, 2017 ). Some current strategies developed to solve these drawbacks are focused on supplementation of essential precursors or use of co-cultures ( Gurramkonda et al, 2018 ; Slouka et al, 2019 ; Chiang et al, 2020 ; Kumar et al, 2020 ; Wang et al, 2020 ). Metabolic burden relates to imbalance suffered by a host cell when a heterologous gene is expressed.…”

Modeling the effects of media formulated with various yeast extracts on heterologous protein production by Escherichia coli using machine learning

“…Kurmar et al also reported that 20 mixed amino acids with chemically defined media increased recombinant peptide production by 40% in E. coli BL21 (DE3). [25] Generally, the addition of amino acids to growth medium can influence E. coli protein expression. In rich medium, E. coli cells grow faster, and expression of the majority of the translation apparatus genes is significantly elevated.…”