Abstract:
:Phosphate is one of the major constituents in growth media. It closely regulates central carbon and energy metabolism. Biochemical reactions in central carbon metabolism are known to be regulated by phosphorylation and dephosphorylation of enzymes. Phosphate scarcity can limit microbial productivity. However, microorganisms are evolved to grow in phosphate starvation environments. This study investigates the effect of phosphate-starved response (PSR) stimuli in wild-type and recombinant Escherichia coli cells cultivated in two different substrates, lactose, and glycerol. Phosphate-starved E. coli culture sustained bacterial growth despite the metabolic burden that emanated from recombinant protein expression albeit with altered dynamics of substrate utilisation. Induction of lactose in phosphate-starved culture led to a 2-fold improvement in product titre of rSymlin and a 2.3-fold improvement in product titre of rLTNF as compared with phosphate-unlimited culture. The results obtained in the study are in agreement with the literature to infer that phosphate starvation or limitation can slow down the microbial growth rate in order to produce recombinant proteins. Further, under PSR conditions, gene expression analysis demonstrated that while selected genes (gapdh, pykF, ppsA, icdA) in glycolysis and pentose phosphate pathway (zwf, gnd, talB, tktA) were up-regulated, other genes in lactose (lacY, lacA) and acetate (ackA, pta) pathway were down-regulated. We have demonstrated that cra, crp, phoB, and phoR are involved in the regulation of central carbon metabolism. We propose a novel cross-regulation between lactose metabolism and phosphate starvation. UDP-galactose, a toxic metabolite that is known to cause cell lysis, has been shown to be significantly reduced due to slow uptake of lactose under PSR conditions. Therefore, E. coli employs a decoupling strategy by limiting growth and redirecting metabolic resources to survive and produce recombinant protein under phosphate starvation conditions. KEY POINTS: • Phosphate starvation controls lactose metabolism, which results in less galactose accumulation. • Phosphate starvation modulates metabolic flow of central carbon metabolism. • Product titre improves by 2-fold due to phosphate starvation. • The approach has been successfully applied to production of two different proteins.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Pandi K,Chauhan AS,Khan WH,Rathore ASdoi
10.1007/s00253-020-10935-ysubject
Has Abstractpub_date
2020-11-01 00:00:00pages
9707-9718issue
22eissn
0175-7598issn
1432-0614pii
10.1007/s00253-020-10935-yjournal_volume
104pub_type
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