Supplementation of critical amino acids improves glycerol and lactose uptake and enhances recombinant protein production in Escherichia coli.
Biotechnol J
; 16(8): e2100143, 2021 Aug.
Article
en En
| MEDLINE
| ID: mdl-34047499
ABSTRACT
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 MAJORRESULTS:
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 dualpurpose:
(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.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Escherichia coli
/
Lactosa
Idioma:
En
Revista:
Biotechnol J
Asunto de la revista:
BIOTECNOLOGIA
Año:
2021
Tipo del documento:
Article
País de afiliación:
India