pEV-UDP-D

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	The constructed vector pEV-Psn was transferred into SM20429 by conjugation and the conditions for pseudoalterin expression were optimized. SM20429 harboring pEV-Psn was cultured to logarithmic phase (OD600≈0.6) at 20°C in 50 ml marine LB broth containing 50 μg/ml ampicillin and 12.5 μg/ml chloramphenicol. Different concentrations of oat spelt xylan (1%, 2% and 3%) were added in the culture prior to incubation at 15°C with shaking at 100 rpm.

Protocol tips

The constructed vector pEV-Psn was transferred into SM20429 by conjugation and the conditions for pseudoalterin expression were optimized. SM20429 harboring pEV-Psn was cultured to logarithmic phase (OD600≈0.6) at 20°C in 50 ml marine LB broth containing 50 μg/ml ampicillin and 12.5 μg/ml chloramphenicol. Different concentrations of oat spelt xylan (1%, 2% and 3%) were added in the culture prior to incubation at 15°C with shaking at 100 rpm.

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"The constructed vector pEV-Psn was transferred into SM20429 by conjugation and the conditions for pseudoalterin expression were optimized. SM20429 harboring pEV-Psn was cultured to logarithmic phase (OD600≈0.6) at 20°C in 50 ml marine LB broth containing 50 μg/ml ampicillin and 12.5 μg/ml chloramphenicol. Different concentrations of oat spelt xylan (1%, 2% and 3%) were added in the culture prior to incubation at 15°C with shaking at 100 rpm. Samples were collected at different culture times and the extracellular elastolytic activity was measured as previously described [11] to determine the optimal xylan concentration for pseudoalterin expression. To determine the optimal culture temperature, cultures with 2% oat xylan were grown at 5°C, 10°C, 15°C or 20°C, and the extracellular elastolytic activity was measured at different culture times. The experiments were repeated three times. Three parallels were set in each assay, and errors were calculated. UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase were expressed in SM20429 under the same optimal conditions as for pseudoalterin expression. SM20429 harboring pEV-UDP-E or pEV-UDP-D was cultured to logarithmic phase (OD600≈0.6) at 20°C and then induced by 2% oat spelt xylan at 15°C for 48 h.

To express these proteins in E. coli, the three constructed expression vectors were transformed into E. coli BL21 (DE3) separately. E. coli BL21 (DE3) harboring an expression vector was cultured to logarithmic phase (OD600≈0.6–0.8) at 37°C and then induced by IPTG at 15°C."

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