Elucidation of the Mechanism of Elicitation in Penicillium Chrysogenum: Systematic Approach to Study the Effect of Oligosaccharides on Production of Penicillin G

The addition of an oligosaccharide elicitor results in a series of coordinated events leading to stimulation of various morphological and/or physiological responses in Penicillium chrysogenum. Whilst elicitation in P. chrysogenum has been reported and is now established, no scientific evidence has been put forward to explain the complex phenomenon behind the observations. To expand the potential of elicitation in fungi from laboratory to industrial scale it is essential to establish the potential generic nature of the elicitor’s effect and to provide concrete reasons for the changes observed. This work, serves as a starting point for the elucidation of some possible mechanisms.
Mannan oligosaccharides (MO) derived from locust bean gum, and oligomannuronate (OM) from alginate were used in these studies.
The effect of multiple addition of the same elicitor was investigated with a prospect that repeated addition would re-trigger the stimulation resulting in either maintenance of the penicillin G levels or enhancement of the production rate. However repeated addition of the same elicitor did not show any change in the production rate in comparison to single additions. Multiple additions of different elicitor types at different times and concentrations in a 5 L bioreactor showed an increase of 150% and 100% in the penicillin G production rate compared to the control and single elicitor addition cultures.
The importance of structure-activity relationship of oligosaccharides in elicitation was investigated. In this study the reducing end of MO was reduced and the modified MO (MO-R) was used to study the structure-activity relationship. Addition of MO-R resulted in a 65% decrease in the elicitor activity, but did not eliminate the ability of the oligosaccharide to enhance the production of penicillin G when compared to the untreated oligosaccharides.
The effect of single and multiple elicitor addition was also studied at the transcriptional level and showed that the transcript copy number in the elicited cultures was significantly higher (p <0.001) for the three major penicillin G biosynthetic genes (pcbAB, pcbC and penDE) in comparison to the control cultures.
The effect of elicitor addition on the cytosolic calcium level was investigated. A fluorescent method was developed that provides a dynamic and reliable technique for the analysis of cytosolic calcium changes. The addition of elicitors showed a significant increase in the cytosolic Ca2+ compared to control cultures to which no elicitor was added (p < 0.001).
The results found in this work aim to bring forward an understanding of the mechanism of action of elicitors in fungal cells.