Abstract
BackgroundEscherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.ResultsHere, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a “pre-defense” mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype.ConclusionsThe D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential “pre-defense” mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.
Highlights
Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory
It was experimentally shown that the transcripts abundance of galactitol and maltose transporter genes were drastically downregulated upon NaCl-induced osmotic stress [17], transcriptional information for other sugar transporters has not been reported
Given that the physiological functions of the two encoded proteins are both associated with transcriptional regulation, we speculated that the two mutations are likely to cause phenotypic changes in osmotolerance
Summary
Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. Since sugar molecules cannot freely travel across semi-permeable cell membranes by diffusion, the high concentrations of such external solvents lead to a strong tendency of cytoplasmic water efflux. This dehydration results in shrinkage of the cell volume and malfunction of cell membranes and embedded proteins, leading to osmotic stress [6]. To counterbalance the deleterious effect of osmotic stress, compatible solutes ( called osmoprotectants), such as potassium ions [7], glycine-betaine [8], trehalose [9], glutamate [10], and proline [11] can spontaneously accumulate in cells via de novo synthesis or transport from the medium. It was experimentally shown that the transcripts abundance of galactitol and maltose transporter genes were drastically downregulated upon NaCl-induced osmotic stress [17], transcriptional information for other sugar transporters has not been reported
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