Physiological analysis of the improved ε-polylysine production induced by reactive oxygen species.

INTRODUCTION: Epsilon-poly-L-lysine (ε-PL) is produced by Streptomyces species in acidic and aerobic conditions, which inevitably induces rapid generation of reactive oxygen species (ROS). The devastating effects of ROS on biomolecules and cell vitality have been well-studied, while the positive effects of ROS are rarely reported. RESULTS: In this study, we found that a proper dose of intracellular ROS (about 3.3 µmol H2O2 /g DCW) could induce a physiological modification to promote the ε-PL production (from 1.2 to 1.5 g/L). It resulted in larger sizes of colony and mycelial pellets as well as vibrant, aggregated, and more robust mycelia, which were of high capability of ROS detoxication. Physiological studies showed that appropriate doses of ROS activated the metabolism of the pentose phosphate pathway at both transcriptional and enzymatic levels, which was beneficial for biomass accumulation. The biosynthesis of lysine was also promoted in terms of transcriptional regulatory overexpression, increased transcription and enzymatic activity of key genes, larger pools of metabolites in the TCA cycle, replenishment pathway, and diaminoheptanedioic acid pathway. In addition, energy provision was ensured by activated metabolism of the TCA cycle, a larger pool of NADH, and higher activity of the electron transport system. Increased transcription of HrdD and pls further accelerated the ε-PL biosynthesis. SIGNIFICANCE: These results indicated that ROS at proper intracellular dose could act as an inducing signal to activate the ε-PL biosynthesis, which laid a foundation for further process regulation to maintain optimal ROS dose in industrial ε-PL production and was of theoretical and practical significance. KEY POINTS: • A proper dose of intracellular ROS positively influences the ε-PL production. • Proper dose of ROS enhanced the mycelial activity and antioxidative capability. • ROS increased lysine synthesis metabolism, energy provision and pls expression.

Metabolomics analysis of salt tolerance of Zygosaccharomyces rouxii and guided exogenous fatty acid addition for improved salt tolerance.

BACKGROUND: Zygosaccharomyces rouxii plays an irreplaceable role in the manufacture of traditional fermented foods, which are produced in a high-salt environment. However, there is little research on strategies for improving salt tolerance of Z. rouxii. RESULTS: In this study, metabolomics was used to reveal the changes in intracellular metabolites under salt stress, and the results show that most of the carbohydrate contents decreased, the contents of xanthohumol and glycerol increased (fold change 4.07 and 5.35, respectively), while the contents of galactinol, xylitol and d-threitol decreased (fold change -9.43, -5.83 and -3.59, respectively). In addition, the content of four amino acids and six organic acids decreased, while that of the ten nucleotides increased. Notably, except for stearic acid (C18:0), all fatty acid contents increased. Guided by the metabolomics results, the effect of addition of seven exogenous fatty acids (C12:0, C14:0, C16:0, C18:0, C16:1, C18:1, and C18:2) on the salt tolerance of Z. rouxii was analyzed, and the results suggested that four exogenous fatty acids (C12:0, C16:0, C16:1, and C18:1) can increase the biomass yield and maximum growth rate. Physiological analyses demonstrated that exogenous fatty acids could regulate the distribution of fatty acids in the cell membrane, increase the degree of unsaturation, improve membrane fluidity, and maintain cell integrity, morphology and surface roughness. CONCLUSION: These results are applicable to revealing the metabolic mechanisms of Z. rouxii under salt stress and screening potential protective agents to improve stress resistance by adding exogenous fatty acids. © 2022 Society of Chemical Industry.

Impact of sample preparation upon intracellular metabolite measurements in 3D cell culture systems.

INTRODUCTION: Interest in cell culture metabolomics has increased greatly in recent years because of its many potential applications and advantages (e.g., in toxicology). The first critical step for exploring the cellular metabolome is sample preparation. For metabolomics studies, an ideal sample preparation would extract a maximum number of metabolites and would enable reproducible, accurate analysis of a large number of samples and replicates. In addition, it would provide consistent results across several studies over a relatively long time frame. OBJECTIVES: This study was conducted to evaluate the impact of sample preparation strategies on monitoring intracellular metabolite responses, highlighting the potential critical step(s) in order to finally improve the quality of metabolomics studies. METHODS: The sample preparation strategies were evaluated by calculating the sample preparation effect, matrix factor, and process efficiency (PE) for 16 tobacco exposition-related metabolites, including nicotine, nicotine-derived nitrosamine ketone, their major metabolites, and glutathione, using isotopically-labelled internal standards. Samples were analyzed by liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS). RESULTS: A sample drying step increased losses or variability for some selected metabolites. By avoiding evaporation, good sample preparation recovery was obtained for these compounds. For some metabolites, the cell or culture type impacted PE and matrix factor. CONCLUSION: In our sample preparation protocol, the drying-reconstitution step was identified as the main cause of metabolite losses or increased data variability during metabolomics analysis by LC-HRMS. Furthermore, PE was affected by the type of matrix. Isotopologue internal standards fully compensate losses or enhancements.

A preliminary study on the impact of exogenous A-Factor analogue 1,4-butyrolactone on stimulating bitespiramycin biosynthesis.

Bitespiramycin is composed of nine main acylated spiramycin components with isovaleryspiramycin as the major component. However, even with excellent therapeutic effects, its application and industrialization are restricted due to its low titer. In this study, the exogenous addition of A-Factor analogue 1,4-butyrolactone (1,4-BL) stimulated an improvement in bitespiramycin biological titer by 29% with a tiny influence on concentration of major component. Moreover, the mechanism of 1,4-BL stimulating effect was preliminarily explored by the analyses of three key enzyme activities, intracellular metabolite profiling and metabolic flux distribution. All results coordinately revealed that the extensive accumulation of methylmalonyl-CoA and acetyl-CoA was the direct reason for the enhanced bitespiramycin biosynthesis. This study would provide theoretical and technical basis for the application of 1,4-BL addition strategy to industrial bitespiramycin production.

Recent Progress in Lab-On-a-Chip Systems for the Monitoring of Metabolites for Mammalian and Microbial Cell Research.

Lab-on-a-chip sensing technologies have changed how cell biology research is conducted. This review summarises the progress in the lab-on-a-chip devices implemented for the detection of cellular metabolites. The review is divided into two subsections according to the methods used for the metabolite detection. Each section includes a table which summarises the relevant literature and also elaborates the advantages of, and the challenges faced with that particular method. The review continues with a section discussing the achievements attained due to using lab-on-a-chip devices within the specific context. Finally, a concluding section summarises what is to be resolved and discusses the future perspectives.

The fate of linoleic acid on Saccharomyces cerevisiae metabolism under aerobic and anaerobic conditions.

INTRODUCTION: Saccharomyces cerevisiae has been widely used for fermenting food and beverages for over thousands years. Its metabolism together with the substrate composition play an important role in determining the characteristics of the final fermented products. We previously showed that the polyunsaturated fatty acid, linoleic acid, which is present in the grape juice at trace levels, significantly affected the development of aroma compounds of the wines. However, the effect of linoleic acid on the overall cell metabolism of S. cerevisiae is still not clear. Therefore, we aimed to unlock the metabolic response of S. cerevisiae to linoleic acid using metabolomics and isotope labelling experiments. METHODS: We cultured the cells on a minimal mineral medium supplementing them with linoleic acid isomers and 13C-linoleic acid. Both intracellular and extracellular metabolite profiles were determined using gas chromatography coupled to mass spectrometry (GC-MS) to investigate which S. cerevisiae pathways were affected by linoleic acid supplementation. RESULTS: The utilisation of linoleic acid by S. cerevisiae had a significant impact on the primary carbon metabolism increasing the glucose consumption and the ethanol production under anaerobic condition. The energetic state of the cell was, therefore, affected and the glycolytic pathway, the TCA cycle and the amino acid production were up-regulated. We also observed that linoleic acid was transported into the cell and converted into other fatty acids affecting their profile even under anaerobic condition. CONCLUSION: Our data clearly shows that linoleic acid supplementation in growth medium increased glucose consumption and ethanol production by S. cerevisiae under anaerobic condition. We also suggest that S. cerevisiae might be able to perform an alternative anaerobic pathway to ß-oxidation, which has not been reported yet.

In†Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single-Cell Resolution.

Single-cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single-cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single-cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single-cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip-based live single-cell extractor (LSCE). We believe that this method will provide a valuable new tool for single-cell biology.

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea.

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible.

Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis.

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase.

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald(-) strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald(-) A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.