Sodium acetate 1 13c

After 6 weeks on diet, mice were equipped with a permanent cecum catheter and allowed a recovery period of at least 5 days as described previously [27] (link). Cecal cannulas were flushed daily with phosphate buffered saline. On the day of the experiment, mice were individually housed and fasted from 6:00 to 10:00 a.m. All infusion experiments were performed in conscious, unrestrained mice. For each dietary treatment group, three different groups received solutions of phosphate buffered saline containing either sodium [1-¹³C]acetate (3 mM, 99 atom %, Sigma-Aldrich), sodium [2-¹³C]propionate (1.5 mM, 99 atom %, Sigma-Aldrich) or sodium [2,4-¹³C₂]butyrate (0.6 mM, 99 atom %, Sigma-Aldrich) via the cecum catheter at an infusion rate of 0.2 ml/h. After 6 h of infusion, animals were sacrificed by cardiac puncture under isoflurane anesthesia. Cecum content was removed quickly, frozen in liquid nitrogen, and stored at -80°C for SCFA enrichment determination.

Sodium[1-¹³C]acetate, sodium[1,2-¹³C₂]acetate and sodium[2-¹³C]acetate were purchased from Sigma Aldrich and have 99% ¹³C atom purity, whereas [¹³c₅,¹⁵n₁]-l-valine was purchased from cortecnet (98% ¹³C, 95% ¹⁵N enriched). Feeding experiments with labelled sodium[1-¹³C]acetate, sodium[1,2-¹³C₂]acetate and sodium[2-¹³C] acetate were performed in HA media (glucose 4 g·L⁻¹, yeast extract 4 g·L⁻¹, malt extract 10 g·L⁻¹, pH 7.4), whereas experiments with [¹³c₅,¹⁵n₁]-l-valine were carried out in DNPM media (Bacto Soytone 7.5 g·L⁻¹, dry yeast 5 g·L⁻¹, MOPS 21 g·L⁻¹, pH 6.8) both supplemented with appropriate antibiotics. The media was inoculated (1% v/v) with a 24 h old TSB culture of Streptomyces albus J1074::cos4. After 20 h of cultivation, determined as the starting point of rishirilide B production, the isotope labelled precursors were added aseptically into the production media. The feeding was done in 10 pulses—every six hours with a 12 h overnight break, hence 3 feedings within 24 h. The final concentration for [1-¹³C]acetate, [1,2-¹³C₂]acetate and [2-¹³C]acetate was 6.1 mM and for feeding with [¹³c₅,¹⁵n₁]-l-valine, 2 mM. Rishirilide B was isolated after 5–6 days when production reaches its maximum.

2,3-Butanediol (2,3-BD), AC, gluconic acid sodium salt, and sodium acetate-1-¹³C were purchased from Sigma (USA). N-acetylglucosamine (GlcNAc) was purchased from Aladdin (Shanghai, China). Restriction enzymes were purchased from TaKaRa Bio Inc. (China). Polymerase chain reaction (PCR) primers were provided by Sangon (Shanghai, China). FastPfu DNA polymerase and T4 DNA ligase were purchased from Transgen Biotech (China) and ThermoFisher (America), respectively. NAD⁺/NADH Quantification Colorimetric Kit was purchased from Biovision (K337-100, USA). EasyPure RNA Kit (used for RNA extraction) and TransStart Top Green qPCR SuperMix (used for qPCR) were purchased from Transgen (Beijing, China). HiScript II Q RT SuperMix for qPCR (+gDNA wiper, used for RNA reverse transcription) was purchased from Vazyme (R223-01, Nanjing, China). BacTiter-Glo™ Microbial Cell Viability Assay used for ATP detection was purchased from Promega (America). All other chemicals were of analytical grade and commercially available.