Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as ...an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T m of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive γ-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.
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
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Lysine supplementation improved the growth of E. coli cells at high temperature.
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The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells.
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The G474D substitution in ThrA reduced homoserine dehydrogenase activity.
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E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.
Draws on archive of material, a first in English to take an in-depth look at Kyoto's modern transformation - its reinvention after 'collapse' (Meiji Restoration) and relocation of the imperial court ...to Tokyo. Includes: introduction, chapters on notable historical elements that sustain Kyoto as a quintessentially modern 'ancient capital' today.
Hypophysitis, which is often accompanied by pituitary dysfunction, is classified into several subtypes based on the cause, histology, and the location of inflammation in the pituitary gland. A ...definitive diagnosis requires pituitary biopsy, which is invasive, and the process is limited to specialized clinical settings. In this opinion paper, we review the literature associated with hypophysitis, and provide the guidelines of the Japan Endocrine Society for the diagnosis and treatment of autoimmune and IgG4-related hypophysitis.
Purine catabolism is regarded as a housekeeping function that remobilizes nitrogen for plant growth and development. However, emerging evidence suggests that certain purine metabolites might ...contribute to stress protection of plants. Here, we show that in Arabidopsis, the intermediary metabolite allantoin plays a role in abiotic stress tolerance via activation of abscisic acid (ABA) metabolism. The aln loss‐of‐function of ALN, encoding allantoinase, results in increased allantoin accumulation, genome‐wide up‐regulation of stress‐related genes and enhanced tolerance to drought‐shock and osmotic stress in aln mutant seedlings. This phenotype is not caused by a general response to purine catabolism inhibition, but rather results from a specific effect of allantoin. Allantoin activates ABA production both through increased transcription of NCED3, encoding a key enzyme in ABA biosynthesis, and through post‐translational activation via high‐molecular‐weight complex formation of BG1, a β‐glucosidase hydrolysing glucose‐conjugated ABA. Exogenous application of allantoin to wild‐type plants also activates the two ABA‐producing pathways that lead to ABA accumulation and stress‐responsive gene expression, but this effect is abrogated in ABA‐deficient and BG1‐knockout mutants. We propose that purine catabolism functions not only in nitrogen metabolism, but also in stress tolerance by influencing ABA production, which is mediated by the possible regulatory action of allantoin.
Nitric oxide (NO) is an important signaling molecule involved in various biological phenomena in many organisms. The physiological functions and metabolism of NO in yeast, a unicellular ...microorganism, are still unknown, mainly because it is difficult to analyze the intracellular NO levels accurately. Here, we developed a new method of more accurately measuring NO content in yeast cells with the detection limit of 6 nM, by treating the cells with an NO-specific fluorescence probe followed by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). This approach successfully detected and quantified the NO content inside yeast cells treated with an NO donor. Moreover, the HPLC/FLD analysis indicates that the fluorescence induced under some environmental stress conditions, such as ethanol, vanillin, and heat-shock, was not derived from NO. The HPLC/FLD method developed in this study provides a new strategy for measuring the intracellular NO concentration with higher accuracy.
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•We developed an NO measurement system using HPLC and a fluorescence NO probe.•Our measurement system successfully quantified the intracellular NO level in yeast.•The previous method using flowcytometry produced false positives for NO detection.
A cell population characterized by the release of glucose repression and known as
emerges spontaneously in the yeast
. This study revealed that the
variants exhibit retarded alcoholic ...fermentation when glucose is the sole carbon source. To identify the key to the altered glucose response, the gene expression profile of
cells was examined. Based on RNA-seq data, the
status was linked to impaired function of the Cyc8p-Tup1p complex. Loss of Cyc8p led to a decrease in the initial rate of alcoholic fermentation under glucose-rich conditions via the inactivation of pyruvate decarboxylase, an enzyme unique to alcoholic fermentation. These results suggest that Cyc8p can become inactive to attenuate alcoholic fermentation. These findings may contribute to the elucidation of the mechanism of non-genetic heterogeneity in yeast alcoholic fermentation.
Glutamate decarboxylase (GAD) catalyses the decarboxylation of L‐glutamate to gamma‐aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low‐cost synthesis of ...GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence‐based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full‐consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half‐life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l‐glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one‐pot synthesis.
Sequence‐based protein design has the potential to generate highly functional enzymes. A full consensus glutamate decarboxylase, FcGAD, has high thermal stability and catalytic rate, which can be used in the synthesis of gamma‐aminobutyric acid (GABA). Utilizing E. coli which express FcGAD as biocatalyst, we achieved the synthesis of GABA at gram scale in a one‐pot reaction.
In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the ...formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.
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