Supplementation of critical amino acids improves glycerol and lactose uptake and enhances recombinant protein production in Escherichia coli.

BACKGROUND: Lactose-based induction strategy in E. coli cultivation has several advantages over IPTG as it is cheap, does not impart metabolic stress to cells, and is non-toxic to cells. However, complexity of lactose as an inducer limits its application in fed-batch cultivation. A mixed glycerol-lactose based induction strategy is generally opted during fed-batch cultivation of E. coli. However, slow growth of E. coli in glycerol and lactose results in slower induction of heterologous protein. MAIN METHODS AND MAJOR RESULTS: In this study, initially we have demonstrated supplementation of critical amino acids (AAs) improves uptake rate of glycerol and lactose in wildtype E. coli BL21(DE3) in defined medium. A feeding strategy of mixed glycerol-lactose feed along with supplement of critical AAs enhances recombinant production of pramlintide multimer (rPramlintide). High cell density cultivation of E. coli using mixed glycerol-lactose feed and critical AAs supplement resulted in final cell density of 52.2 ± 0.90 g L-1  and rPramlintide titer of 7.8 g L-1 . RT-qPCR analysis of genes involved in glycerol and lactose metabolism of recombinant culture showed upregulation with AAs supplementation. CONCLUSIONS AND IMPLICATIONS: We hypothesize that supplementation of critical AAs serves dual purpose: (i) faster assimilation of carbon sources, and (ii) combating metabolic stress arises due to AAs starvation. The substrate uptake and gene expression profiles demonstrate that AAs addition enhances glycerol and lactose assimilation due to overall improvement in their metabolism governed by global regulators of carbon metabolism.

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

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.