Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Schematic map of the shuttle vector pOriT-4CM (A), the reporter vector (B) and the expression vector pEV (C).
repA, replication-related gene in Pseudoalteromonas sp. SM20429; oriT, origin of transfer; AmpR and CAT, selective marker for ampicillin resistance and chloramphenicol resistance, respectively; Phage f1 region, replication-related genes in E. coli; cel308, catalytic domain with its signal peptide of the cold-adapted cellulose gene; Pxyl, promoter of the xylanase gene from Pseudoalteromonas sp. SM20429.
Fig 2.
Cellulase activity of Pseudoalteromonas sp. SM20429 harboring promoter reporter vectors p4xc or p4cc at different temperatures.
The activity of the cellulase expressed by p4xc at 15°C for 72 h was taken as 100%.
Fig 3.
Pseudoalterin production after induction under different conditions.
(A) Pseudoalteromonas sp. SM20429 harboring pEV-Psn was induced by different concentrations of oat spelt xylan at 15°C. (B) Pseudoalteromonas sp. SM20429 harboring pEV-Psn was induced by 2% oat spelt xylan at different temperatures.
Fig 4.
Pseudoalterin expressed by SM20429 and by E. coli.
(A) SDS-PAGE analysis of the purified pseudoalterin from SM20429. M, marker; lane 1, the purified pseudoalterin. (B) SDS-PAGE analysis of pseudoalterin expressed by E. coli BL21 (DE3) after cloning the pseudoalterin gene into the vector pGEX-4T-1 and induced by IPTG at 15°C. M, marker; lane 1, soluble recombinant pseudoalterin fused with a GST tag.
Table 3.
Comparison of the substrate specificity between recombinant pseudoalterin and native pseudoalterin.
Fig 5.
UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase expressed by SM20429 and E. coli.
(A) SDS-PAGE analysis of the purified proteins expressed by E. coli BL21 (DE3) M, marker; lane 1, UDP-ManNAc dehydrogenase; lane 2, UDP-GlcNAc 2-epimerase. (B) SDS-PAGE analysis of the purified proteins expressed by SM20429. M, marker; lane 1, UDP-GlcNAc 2-epimerase; lane 2, UDP-ManNAc dehydrogenase.