진화와 대사공학을 통한 슈도모나스 푸티다(Pseudomonas putida)의 포도당과 자일로스로부터 뮤콘산 생산

For shake flask and growth curve experiments, seed cultures were inoculated from glycerol stocks into 14-mL round bottom Falcon® tubes containing 5 mL of LB Miller medium and incubated overnight at 30 °C and 225 rpm. Overnight cultures were then inoculated into a 125-mL baffled shake flask containing 10 mL LB medium to an initial OD600 of 0.2. These second seed cultures were cultivated at 30 °C at 225 rpm for 4 h to reach an OD600 of ~2. The second seed cultures were washed twice with M9 salts (6.78 g L−1 Na2HPO4, 3 g L−1 KH2PO4, 0.5 g L−1 NaCl, 1 g L−1 NH4Cl) and then inoculated into a 125-mL baffled shake flask containing 25 mL modified M9 minimal medium (6.78 g L−1 Na2HPO4, 3 g L−1 KH2PO4, 0.5 g L−1 NaCl, 1 g L−1 NH4Cl, 2 mM MgSO4, 100 µM CaCl2, 18 µM FeSO4) supplemented with either 30 mM xylose, 30 mM glucose, or mixture of 30 mM glucose and 15 mM xylose, to an initial OD600 of 0.1. The molar ratio of glucose and xylose in mixed substrates is 2:1, which is the ratio typical of corn stover hydrolysates10% v/v) during fermentation without hydrolysate purification or concentration. Energy Environ. Sci. 9, 1237–1245 (2016)." href="/articles/s41467-022-32296-y#ref-CR56" id="ref-link-section-d4408857e3149"56. All growth curves were characterized on Bioscreen C Pro analyzers (Growth Curves US) using 300-µL cultures inoculated as described for the shake flasks above. Shake flasks and plate reader data were plotted and analyzed using GraphPad Prism version 8.4.2. Absolute growth rate (μA) and growth lag (λ) were calculated based on Gompertz equation using default settings of the FittR tool deposited in GitHub (https://github.com/scott-saunders/growth_curve_fitting)57. In some cases, death phase was removed from the growth curve to fit the model./p>

Cells were inoculated in 0.5 L bioreactors (BioStat-Q Plus, Sartorius Stedim Biotech) at an initial OD600 of 0.2. The batch phase consisted of growth on 300 mL of modified M9 with 10.6 g L−1 glucose and 4.4 g L−1 xylose, which mimics the sugar ratio in sugar hydrolysates from corn stover10% v/v) during fermentation without hydrolysate purification or concentration. Energy Environ. Sci. 9, 1237–1245 (2016)." href="/articles/s41467-022-32296-y#ref-CR56" id="ref-link-section-d4408857e3452"56. The fed-batch phase was initiated when the sugar concentration was approximately 7 g L−1, at which point sugars were fed to maintain sugar concentrations between ~2–10 g L−1 by manually modifying feeding rates. The feeding solution contained 353 g L−1 glucose and 147 g L−1 of xylose, and its pH was adjusted to pH 7 with NaOH. The bioreactors were controlled at pH 7 by the addition of 4 N NH4OH, at 30 °C, and air was sparged at 1 vvm. The initial agitation speed in the batch phase was 350 rpm. When the dissolved oxygen (DO) reached a value of 30%, it was automatically controlled at that level by automatic agitation adjustments. Samples were taken periodically to evaluate bacterial growth and to analyze sugar concentrations and muconate./p>10% v/v) during fermentation without hydrolysate purification or concentration. Energy Environ. Sci. 9, 1237–1245 (2016)./p>