Molecular analysis of pigment production in Fusarium

In this doctoral thesis, the role of the aurofusarin biosynthesis gene cluster in Fusarium graminearum and F. culmorum on mycelium pigmentation and the effect of the extract containing aurofusarin and its precursor's on antibacterial and biofilm formation were investigated at the molecular level. In this context, 3F, 7F and Fg174 (F. graminearum) and 9F, 11F, F1 and F14 (F. culmorum) were investigated by morphological and thin layer chromatography in terms of stable pigments production in 47 F. graminearum, 24 F. culmorum isolates. The presence of Pks12, Gip6, Gip7, Gip4, which are involved in the biosynthetic pathway, and the StuAp gene, which encodes the general transcription factor, in the genomic DNA of these isolates with references of F. graminearum 88-1 and H-11 were amplified via polymerase chain reaction (PCR). Sanger sequencing showed that there were transition and transversion type single nucleotide changes in the amplified Gip6 and StuAp partial gene regions, and single, double and triple in-del regions with these single nucleotide changes in Pks12, Gip7 and Gip4 genes. The effect of different environmental conditions on the expression level alterations of five genes on mycelium pigmentation was investigated by real-time PCR and high performance liquid chromatography in terms of temperature of 15 °C and 25 °C and pH variables of 5.6 and 7.0. The highest expression levels for all genes were determined in pH 5.6 medium at 15 °C. Spearman correlation analysis identified a significant correlation between aurofusarin MOLECULAR ANALYSIS OF PIGMENT PRODUCTION IN Fusarium biosynthesis with Pks12 expression at pH 5.6 and 25 °C; with Pks12, Gip7 and StuAp expressions at pH 5.6 and 15 °C; with Gip4 expression at pH 7.0 and 15 °C. Multiple regression and principal component analysis revealed that pH and temperature parameters alone did not affect mycelium coloration. Mycelium coloration did not occur at acidic pH (3.0). The extract of Fusarium isolate (9F) containing 0.72 ng/µl aurofusarin decreased the growth of P. aeruginosa PAO1 (ATCC 15692) by 38.89%; the extract containing 0.36 ng/µl aurofusarin reduced the biofilm layer by approximately 116%. Correlation analysis showed that 0.36 ng/µl aurofusarin inhibited biofilm formation by causing a decrease in the metabolic activity of cells and the expression levels of LasI, LasB and LasR associated with quorum-sensing (QS). Findings from the study not only provided information about the molecular mechanism of aurofusarin biosynthesis and mycelium pigmentation, but also provided important data on the effects of environmental conditions on secondary metabolite biosynthesis in Fusarium. In this study, it has been shown that aurofusarin has an antibacterial effect as well as an inhibitory effect on biofilm formation. The doctoral thesis has great importance in terms of laying the groundwork for the high-scale production of aurofusarin under laboratory conditions without any manipulation of the genome.