Project description:In this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only 2 mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical application, deleting gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.
Project description:At selected timepoints during the S erythrea fermentation process, samples of fermentation broth were collected. Proteins in the samples were separated by 2D-PAGE and differentially expressed proteins were identified by LC-MS/MS.
Project description:In this study, an in vitro antifungal growth experiment showed that the inhibitory rate of the MCF broth on pathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, Trichothecium roseum, and Colletotrichum gloeosporioides) was less than that of B. amyloliquefaciens culture fermentation (BCF). Moreover, the content and gene expression of lipopeptide antibiotics was also lower than that in the BCF group. However, the pot experiments based on irrigation with appropriately diluted fermentation broth showed that the biocontrol effect of MCF on tomato Fusarium wilt was significantly higher than that of TCF (T. longibrachiatum culture fermentation) and BCF, and was approximately 15.79% higher than that of the BTF group which made by mixing equivalent amounts of BCF and TCF. In MCF broth, two microorganisms antagonized and coexisted, and the growth of T. longibrachiatum was inhibited. Using transcriptomic methods, we speculated that MCF can up-regulate the expression of genes related to carbon and nitrogen metabolism, oxidation–reduction activity, sporulation, environmental information response and chemotaxis, and biosynthesis of secondary metabolites of B. amyloliquefaciens, which might enhance the nutrient substances metabolism and competitiveness, survival ability, colonisation, and adaptability to the environment to increase its biocontrol potential.
Project description:S. bombicola was fermented under two different nitrogen sources (yeast extract or ammonium sulfate). The fermentation broth after 7 days’ cultivation was taken for extracellular proteins analysis.
Project description:Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient thera-peutic agents for various diseases, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drug. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb can be identified at the intact protein level. The molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Simultaneously, absolute quantification of mAb content in fermentation broth yielded concentrations of 67 and 2578 ng.mL-1 at the beginning and end of fermentation, respectively. The whole workflow features excellent robustness as well as retention time and peak area stability of 0.3% and 4.9% RSD. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production as well as targeted biosimilar development.
