Lovastatin, a competitive inhibitor of HMG-CoA reductase, is a powerful anti-hypercholesterolemic agent produced by Aspergillus terreus as a secondary metabolite. It is well known that highly branched filamentous morphology results in significantly hi...
Lovastatin, a competitive inhibitor of HMG-CoA reductase, is a powerful anti-hypercholesterolemic agent produced by Aspergillus terreus as a secondary metabolite. It is well known that highly branched filamentous morphology results in significantly high viscosities of culture broth in fungal cell fermentations, leading to significant reduction in mass- and oxygen-transfer capabilities. In our previous research, we also observed that cultures with compact-pelleted forms showed about 3 fold higher oxygen transfer coefficient(kLa), resulting in 1.8 fold increase in lovastatin production as compared to those with filamentous forms. It had been also demonstrated the enhanced oxygen transfer rate in the pellet-formed fermentations was due to lower viscosity of the culture medium with fungal cells of pellet morphology. In this study, for more facilitated utilization of dissolved oxygen by the high-yielding mutants, we constructed and introduced into the high producers the respective expression vector with Vitreoscilla hemoglobin(VHb) gene. Notably, the resulting transformant, SELI8-113/VHb+, harboring the wild VHb gene showed approximately 2.5 fold higher lovastatin productivity than the parallel nontransformed strains in 5L-bioreactor fermentations performed under the relatively low levels of dissolved oxygen environments. Furthermore, production stabilities of most of the strains screened from the SELI8-113/VHb+ were excellent, exhibiting sharp contrast to the results obtained from the nontransformants. Therefore, it was concluded that optimal supply of oxygen was prerequisite for the enhanced biosynthesis of lovastatin as well as production stability of the high-yielding producers.
In addition, we tested fill and draw mode of fermenter operation in order to maximally utilize the fermentation characteristics of the high-yielding transformants, as observed in the previous experiments. In the fill and draw cultures performed by exchanging 30%, 50%, and 70%, respectively, of the fermention broth with a fresh production medium, notable results were observed that the transformant(SELI8-113/VHb+) maintatined its higher lovastatin productivity as well as cell growth rate until the third round of repeated batch operations. In contrast to these results, the nontransformed strain(SELI8-113) showed significantly reduced level in terms of biosynthetic capability of lovastatin and cell proliferation in the comparative repeated batch fermentations (i.e., fill and draw culture) performed under the identical culture conditions. Also notable was the positive role of glycerol added in the production medium, since it contributed to remarkable enhancement in the lovastatin-biosynthetic capability of the transformant. The optimum concentration of glycerol supplemented to the previously utilized production medium, which did not cause catabolite repression/inhibition phenomenon to the higher producer was determined to be 12 g/L through statistical medium optimization studies. (Response surface method(RSM) based on central composite design(CCD) was adopted.). In another set of fill and draw fermentations performed with medium exchange of 70%, 80% and 90% (v/v) respectively, optimum amount of exchanged medium producing maximum amount of lovastatin was observed to be 70% (v/v), (In theses fermentations, the newly modified medium was adopted, as explained above.).