Abstract
Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds.Key points• Lysine supplementation improved the growth of E. coli cells at high temperature.• The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells.• The G474D substitution in ThrA reduced homoserine dehydrogenase activity.• E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.
Highlights
Lysine, one of the essential amino acids for humans, protects yeast cells from freezing, dehydration, and oxidative stresses (Takagi et al 1997; López-Martínez et al 2015; Olin-Sandoval et al 2019)
These results suggest that the addition of lysine to the culture medium increased intracellular lysine, leading to high-temperature stress tolerance of E. coli
Our results indicate that both Gly474Asp and Cys554Tyr substitutions in ThrA are responsible for lysine hyper-production
Summary
One of the essential amino acids for humans, protects yeast cells from freezing, dehydration, and oxidative stresses (Takagi et al 1997; López-Martínez et al 2015; Olin-Sandoval et al 2019). The resulting increase in intracellular glutathione enhanced tolerance to oxidative stress (Olin-Sandoval et al 2019). E. coli cells maintain pH homeostasis to form cadaverine by decarboxylation of lysine with intracellular protons and exchange external lysine for internal cadaverine via a cadaverine-lysine antiporter (Kanjee and Houry 2013). These findings demonstrated that lysine is closely related to adaptation and tolerance to environmental stresses; the detailed mechanism of lysine-mediated stress tolerance in microorganisms has yet to be elucidated. Little is known about the effect of the enhancement of lysine biosynthesis on stress tolerance. Lysine-mediated stress tolerance could contribute to the construction of robust host strains for microbial production of useful compounds
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