Cloning And Analysis Of Genes Related To Cell Wall Construction In Shear-sensitive Marine Fungus Aspergillus Glaucus HB1-19

Marine-derived filamentous fungus Aspergillus glaucus HB1-19, producing aspergiolide A which is a novel anti-tumor compound, is considerable sensitive to mechanical shear stress, which is a barrier for fermentation scale-up in bioreactor. Generally, researchers increased production by optimizing fermentor structure and rotation speed for the shear-sensitive strains. In this study, we aim to clone genes related to cell wall biosynthesis of A. glaucus HB1-19 and analyze their functions, which would provide reference to furture studies on construction of shear resistent strains for bioreactor fermentation.Degenerate PCR and genome walking methods were used to obtain the gene sequences of AgugeA, AgugmA and AgugtA followed by construction of pSUhwl, pSUhw2 and pSUhw3 by homologous integration of double crossover with the Zeocin resistance gene Sh ble as a marker. After identifying numerous transformants, we gained none of the three desired mutants. Phenylglyoxal hydrate (2 mmol/L), a specific inhibitor to AgugeA in Aspergillus genus, severely inhibits the growth of A. glaucus HB1-19 comparing to A. nidulans. We assume that AgugeA is essential for growth in A. glaucus HB1-19.Depending on the assumption, we construct several plasmids assembled with promoters PgpdA, PtrpC and terminator TtrpC using GFP as a marker, silencing and quelling AgugeA respectively for identifying its role in growth. By gene silening, no transformants grew but the control grew well, which further proved the above assumption.For further research of the function of AgugeA, we then constructed the vector containing the inducible promoter PalcA by integrating part of AgugeA into the recipient chromosome by a single crossover event, which would be helpful to furture studies on inducible expression of cell wall synthesis related genes to investigate its changes of shear-sensitivity, growth, development and metabolism.