Research Of Laea And Laea-like Protein Regulate The Conidia Formation And Glycoside Hydrolase

Penicillium is one of the most popular fungi in industry. Some species of Penicillium play important roles in biotechnology as well as in the medical and food industries. In addition, many Penicillium species have been reported to have the ability of producing more efficient glycoside hydrolases or high representation of extracellular proteins involved in plant cell wall degradation, that are valuable for white biotechnology. A Penicillium oxalicum （previously Penicillium decumbens） strain, and its cellulase hyper-producing mutant, has been used for industrial-scale glycoside hydrolases production for years. Interestingly, conidiation in the cellulase hyper-producing strain was severely impaired, increasing the cost of industrial production. Therefore, an in-depth understanding of cellulose synthesis and regulatory mechanism of sporogenesis will significantly promote the develpoment of bioethanol.A total of 28 gene clusters for secondary metabolism were predicted in the genome of P. oxalicum, Thus,understanding the mechanism of the regulation of second metabolism gene clusters will contribute to effectively control the production of SM. The main results of the research are as follows:1. LaeA regulate of conidiation and second metabolism in P. oxalicumLaeA can effectively coregulate of sporogenesis and second metabolism in P. oxalicum. The deletion of P. oxalicum laeA（△laeA） impaired conidiation severely. The expression of brlA and abaA was downregulated in AlaeA, showing LaeA acts upstream of them. Overexpression of brlA in △laeA could upregulate brlA, abaA, and wetA expression remarkably, but could not rescue the conidiation defect, showing the regulation of conidiation by LaeA was only partly mediated by BrlA. The double deletion of laeA and creA（△laeA△creA） blocked conidiation absolutely. Seven genes for pigmentation （albA/wA, abrB/yA, arpA, aygA, arpA-like, arpB, arpB-like）and three spore wall protein genes（rodA, rodA-like, and radβ）expression were almost silencing in △laeA△creA. Meanwhlie, differently decreasing of the spore wall protein genes expression in the three mutants resulted in the signally decreasing the toletation of CR, SDS or CFW. Of 28 secondary metabolism gene clusters in P. oxalicum, four clusters were silenced in AlaeA. Two of them were also silenced in △brlA mutant, suggesting LaeA participates in gene silencing partly mediated by BrlA. Six physically linked regions of coregulated genes were located in subtelomeric regions, showing a positional bias exists for LaeA-regulated clusters towards subtelomeric regions. The silenced clusters were derepressed in △laeAAcreA, showing that lack of CreA could remediate repression of gene clusters in △laeA background, which might be mediated by another putative LaeA-like methyltransferase PDE₀3083. In addition, the expression of PDE₀6062, which is the key gene for the production of oxalic acid was down regulated in △laeA, △creA and △laeA△creA,leading to the lower level of oxalic acid comparison with WT.Of the three mutants, △lae△AcreA displaying the duplicate effect,had the lowest level.The results showed the co-function of LaeA and CreA is required for proper asexual development and controls gene cluster expression, although there was no direct interaction between LaeA and CreA.2. Characterization of LaeA-like protein regulation of the conidiation and cellulase synthesisCellulase synthese is regulated by LaeA. Amino acid sequence analysis showed that the 23 families members including LaeA had the SAM binding domain, which are named putative methylatransferase.Recent study showed that FPase activity decreased in △laeB, contrast in △laeE and △lael, which reached to 1.5-2 times comprison to WT. The growth of △laeB under sole carton was limited with more fold. Futhermore, sporulation was quantified, confirmed that △laeB lightly lower than WT. Mircoscopic observation revealed that hyphae reduced in △laeB, sporogenesis was deleyed about 2 hours.Biomass detection of all the mutants under the liquid culture with the glucose as the sole carton showed all the strains were lower than WT, except for AlaeB, indicated that deletion of these genes affected the normal growth. Combine with the research of LaeA, LaeA interacted with the cellulase/hemicellulase transcription activator XlnR and the chromatin remodeling factor Swi6 （unpublished）. Analysis of phylogenetic tree made it clear that the 23 families members were closely related to LaeA, presuming that the mode on regulating cellulase and spores synthesis were similar to LaeA.