PDF(Pubmed)
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.
摘要：
赖氨酸,一种营养上重要的氨基酸，参与各种生物对环境压力的适应和耐受性。以前的研究报告说，赖氨酸的积累是对胁迫的反应，补充赖氨酸可以增强胁迫耐受性；然而，赖氨酸生物合成增强对胁迫耐受性的影响尚未阐明。在这项研究中,我们证实，在高温（42.5°C）下，向培养基中补充赖氨酸会增加细胞内赖氨酸含量并改善大肠杆菌的细胞生长。然后通过常规诱变从赖氨酸类似物S-腺苷甲硫氨酸抗性突变体中分离出过量生产赖氨酸的菌株，并表现出比野生型菌株更高的对高温胁迫的耐受性。我们在ThrA上鉴定了新的氨基酸取代Gly474Asp和Cys554Tyr，一种双功能天冬氨酸激酶/高丝氨酸脱氢酶（AK/HSDH），在赖氨酸过量生产的突变体中。有趣的是,ThrA的Gly474Asp和Cys554Tyr变体诱导赖氨酸积累并赋予大肠杆菌细胞高温胁迫耐受性。酶分析显示，ThrA中的Gly474Asp取代降低了HSDH活性，表明细胞内天冬氨酸半醛的水平，它是HSDH的底物和赖氨酸生物合成的中间体，由于HSDH活性的丧失而升高并在大肠杆菌中转化为赖氨酸。本研究表明，补充赖氨酸和增强赖氨酸生物合成都能提高大肠杆菌细胞的高温胁迫耐受性。我们的发现表明，过量生产赖氨酸的菌株具有作为耐受胁迫的微生物的潜力，并且可以应用于强大的宿主细胞，用于微生物生产有用的化合物。关键点:•赖氨酸补充改善了高温下大肠杆菌细胞的生长。•G474D和C554Y变体ThrA增加了大肠杆菌细胞中的赖氨酸生产率。•ThrA中的G474D取代降低了高丝氨酸脱氢酶活性。•过量产生赖氨酸的大肠杆菌细胞表现出高温胁迫耐受性。
