Phosphate starvation controls lactose metabolism to produce recombinant protein in Escherichia coli.

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.

Microaerobic fermentation alters lactose metabolism in Escherichia coli.

Microaerobic fermentation has been shown to improve lactose transport and recombinant protein production in Escherichia coli. Mechanistic correlation between lactose and dissolved oxygen has been studied and it has been demonstrated that E. coli can switch its genetic machinery upon fluctuations in dissolved oxygen levels and thereby impact lactose transport, resulting in product formation. Continuous induction of lactose in microaerobic fermentation led to a 3.3-fold improvement in product titre of rLTNF oligomer and a 1.8-fold improvement in product titre of rSymlin oligomer as compared with traditional aerobic fermentation. Transcriptome profiling indicated that ribosome synthesis, lactose transport and amino acid synthesis genes were upregulated during microaerobic fermentation. Besides, novel lactose transporter setB was examined and it was observed that lactose uptake rate was 1.4-fold higher in microaerobic fermentation. The results indicate that microaerobic fermentation can offer a superior alternative for industrial production of recombinant therapeutics, industrial enzymes and metabolites in E. coli. KEY POINTS: • Microaerobic fermentation results in significantly improved protein production • Lactose transport, ribosome synthesis and amino acid synthesis are enhanced • Product titre improves by 1.8-3.3-fold.

Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production.

Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression.

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster®/B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.