| Abstract | The mechanism by which certain oligosaccharides elicit overproduction of secondary metabolites in Bacillus licheniformis was investigated. Mannan oligosaccharides (MO) derived from locust bean gum, and oligoguluronate (OG) and oligomannuronate (OM) from alginate were used in this study.
The effect of lower (50 - 25 mg L-1) and higher (500 – 1000 mg L-1) concentrations of MO, OG and OM on bacitracin A production was investigated.
HPLC (high performance liquid chromatography) results demonstrated that the addition of lower and higher concentrations of these elicitors to cultures of B. licheniformis did not have an elicitation effect on bacitracin A production, compared to the optimal reported elicitor concentration (100 – 300 mg L-1).
HPAEC (high performance anion exchange chromatography) and TLC (thin layer chromatography) results revealed that mannobiose, mannose and mannohexaose are the main components of MO. The addition of these saccharides, separately, to B. licheniformis cultures, showed that mannobiose produced the highest and sustained increase in bacitracin A concentration whereas in mannose and mannohexaose treated cultures the effects were minimal. Similar levels of bacitracin A were obtained in MO (825 mg L-1) and mannobiose (800 mg L-1) treated cultures. Investigation of the fate of MO during the B. licheniformis shake flask and bioreactor batch growth showed that MO is degraded to mannose by the enzyme ß-mannanase.
MO (300 mg L-1) supplemented chemostat cultures of B. licheniformis did not result in a significant change in bacitracin A concentration and ß-mannanase activity compared to the control fermentation. However, when the chemostat culture was changed to batch, increases were observed both in bacitracin A concentration and ß-mannanase activity.
Incubation of B. licheniformis cells with oligosaccharide elicitors in microwell plates resulted in changes in Ca2+ influx. The highest influx was 9.2 % greater in samples incubated with MO and OG compared to the control. Addition of ionomycin (positive control) and verapamil (Ca+2 channel blocker) to the cultures of B. licheniformis resulted in 15 % increase and 74 % decrease in bacitracin A levels, respectively, as compared to the control culture.
Changes were profiled in protein expression in B. licheniformis supplemented with OG and MO elicitors in short and long term elicitation, using 2-Dimentional gel electrophoresis. Differentially expressed proteins were identified by LC-MS/MS. The proteins affected by short-term and long-term elicitation were mainly involved in energy generation, amino acid metabolism and stress responses. The addition of OG and MO to the cultures also altered the phosphorylation state of twelve proteins involved in energy generation, stress response and amino acid metabolism. Biochemical assays revealed changes in reactive oxygen species levels as well as antioxidant enzymes, superoxide dismutase and catalase in B. licheniformis cultures supplemented with elicitors. The novel findings of this work have advanced understanding of the mechanism of action of elicitors in B. licheniformis cultures. |
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