Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

Khoomrung, Sakda; Martínez, José Luis; Tippmann, Stefan; Jansa-Ard, Suwanee; Buffing, Marieke F.; Nicastro, Raffaele; Nielsen, Jens

doi:10.1016/j.ancr.2015.10.001

Cited by 15 publications

(11 citation statements)

References 32 publications

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“…For rapid sampling and quenching, 10 mL of fermentation broth was quenched quickly by adding 40 mL of chilled (−80 °C) 80% (v/v) methanol and the pellet was collected by centrifugation (8000 rpm for 5 min at 4 °C). − For metabolite extraction, 5 mL of hot (95 °C) ethanol was added, vortex-mixed for 30 s, incubated at 95 °C for 3 min followed by freezing at −40 °C for 15 min. The supernatant was collected by centrifugation (5000 rpm for 5 min at 4 °C) and freeze-dried at −80 °C under reduced pressure.…”

Section: Methodsmentioning

confidence: 99%

Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces

et al. 2017

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With the advent of the genomics era, heterologous gene expression has been used extensively as a means of accessing natural products (NPs) from environmental DNA samples. However, the heterologous production of NPs often has very low efficiency or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in native producers, especially in the cases of genome mining, it is also difficult to rationally and systematically engineer synthetic pathways to improved NPs biosynthetic efficiency. In this study, various strategies ranging from heterologous production of a NP to subsequent application of omics-guided synthetic modules optimization for efficient biosynthesis of NPs with complex structure have been developed. Heterologous production of spinosyn in Streptomyces spp. has been demonstrated as an example of the application of these approaches. Combined with the targeted omics approach, several rate-limiting steps of spinosyn heterologous production in Streptomyces spp. have been revealed. Subsequent engineering work overcame three of selected rate-limiting steps, and the production of spinosad was increased step by step and finally reached 1460 μg/L, which is about 1000-fold higher than the original strain S. albus J1074 (C4I6-M). These results indicated that the omics platform developed in this work was a powerful tool for guiding the rational refactoring of heterologous biosynthetic pathway in Streptomyces host. Additionally, this work lays the foundation for further studies aimed at the more efficient production of spinosyn in a heterologous host. And the strategy developed in this study is expected to become readily adaptable to highly efficient heterologous production of other NPs with complex structure.

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Section: Methodsmentioning

confidence: 99%

Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces

et al. 2017

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“…Intracellular yeast metabolites were prepared by the procedure described previously, with modifications [ 21 ]. Treatment cultures were prepared in YES broth medium with an initial yeast cells OD 600 of 0.05 and supplemented with 11αOH-KA (45 µg mL −1 ), then incubated until the mid-log phase with constant shaking (120 rpm) at 30 °C.…”

Section: Methodsmentioning

confidence: 99%

The Antiaging Effect of Active Fractions and Ent-11α-Hydroxy-15-Oxo-Kaur-16-En-19-Oic Acid Isolated from Adenostemma lavenia (L.) O. Kuntze at the Cellular Level

et al. 2020

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Background: The extract of Adenostemma lavenia (L.) O. Kuntze leaves has anti-inflammatory activities and is used as a folk medicine to treat patients with hepatitis and pneumonia in China and Taiwan. The diterpenoid ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA) is the major ingredient in the extract and has wide-spectrum biological activities, such as antitumor and antimelanogenic activities, as well as anti-inflammatory activity. However, the physical and biological properties of this compound as an antioxidant or antiaging agent have not been reported yet. Methods: In addition to in vitro assays, we monitored antioxidative and antiaging signals in Schizosaccharomyces pombe (yeast) and mouse melanoma B16F10 cells. Results: A. lavenia water and chloroform fractions showed antioxidant properties in vitro. The A. lavenia extracts and 11αOH-KA conferred resistance to H2O2 to S. pombe and B16F10 cells and extended the yeast lifespan in a concentration-dependent manner. These materials maintained the yeast mitochondrial activity, even in a high-glucose medium, and induced an antioxidant gene program, the transcriptional factor pap1+ and its downstream ctt1+. Accordingly, 11αOH-KA activated the antioxidative transcription factor NF-E2-related factor 2, NRF2, the mammalian ortholog of pap1+, in B16F10 cells, which was accompanied by enhanced hemeoxygenase expression levels. These results suggest that 11αOH-KA and A. lavenia extracts may protect yeast and mammalian cells from oxidative stress and aging. Finally, we hope that these materials could be helpful in treating COVID-19 patients, because A. lavenia extracts and NRF2 activators have been reported to alleviate the symptoms of pneumonia in model animals.

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“…Furthermore, most analytical methods used for targeted metabolite analysis are designed to quantify a particular class of metabolites, such as FAs, 38 lipids, 39 or amino acids. 40 By applying this method as a screening strategy, we could determine that lipids were the metabolites most affected by Alum administration. The majority of these lipids were TAGs, as well as several unidentified species.…”

Section: ■ Conclusionmentioning

confidence: 99%

Metabolic Profiling and Compound-Class Identification Reveal Alterations in Serum Triglyceride Levels in Mice Immunized with Human Vaccine Adjuvant Alum

et al. 2019

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Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (LCMS)-based metabolic and lipid profiling to monitor the effects of Alum adjuvant on mouse serum at 6, 24, 72 and 168 h post-vaccination. We propose a new strategy termed Subclass Identification and Annotation for Metabolomics (SIAM) for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long chain unsaturated fatty acids were decreased at 24 h, and TAGs with short chain fatty acids were decreased at 168 h. To *

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Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

Cited by 15 publications

References 32 publications

Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces

Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces

The Antiaging Effect of Active Fractions and Ent-11α-Hydroxy-15-Oxo-Kaur-16-En-19-Oic Acid Isolated from Adenostemma lavenia (L.) O. Kuntze at the Cellular Level

Metabolic Profiling and Compound-Class Identification Reveal Alterations in Serum Triglyceride Levels in Mice Immunized with Human Vaccine Adjuvant Alum

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