Cloning And Codon Optimization Of Chi58 From Chaetomium Cupreum And High-Level Expression In Pichia Pastoris

Chaetomium cupreum Ames is an important biocontrol fungus, which has very effective control effect to the diseases induced by various plant pathogenic fungus. Chitinase plays an important role in this biocontrol process. So it has a wide application prospect in the development of biopesticide. In this paper, we took chitinase produced by C. cupreum as research object to construct genetic engineering strain which can highly express chitinase, using bioinformatics and molecular biology technology, and improve expression level in Pichia pastoris using some methods, such as codon preference modification, screening of multi-copy, optimization of fermentation conditions. These have important value for research and development of new biopesticide.Under the condition of imitating natural environment, chitinase of C. cupreum was induced using cell wall of three plant pathogenic fungus as only carbon source. RT-PCR semi quantitative assay indicated that different carbon sourse could induce the expression of chitinase gene besides the difference of expression peak time. So it is presumed that Chi58 maybe play an important role in the biocontrol process. Consequently, the chitinase gene was cloned using inverse PCR. This should establish a foundation for the research of biocontrol mechanism of C. cupreum.The full-length sequence has been deposited in GenBank (GenBank accession number DQ 886936). Bioinformatics analysis showed that the CHI58 includes a N-terminal signal peptide sequence, a chitin binding domain (ChBD), rich cysteine joining region and glycosyl hydrolase catalytic domain. The chitinase is similar to one which is in plant in the location of chitin-binding domain and amino acid composition, so the chitinase belong to Class I with chitin-binding domain type.Two recombinant plasmids pYES2-Chi58 and pPIC9K-Chi58 were constructed and transformed into Saccharomyces cerevisiae H158 and P. pastoris GS115 respectively. The results showed that the maximal activity of chitinase from S. cerevisiae transformant was 0.33U, appearing in the 60h after the induction, and the maximal activity of chitinase from P. pastoris transformant was 39U, appearing in the 120h after induction, which was markedly enhanced about 120 times.SDS-PAGE and zymogram proved that CHI58 protein had enzyme activity in P. pastoris. It has an optimum pH of 5.8 and an optimum temperature of 45℃; it still has good heat stability at 70℃, and it still has good activity after 10 times. CHI58 protein has different degradation effect on soluble ethylene glycol, solububle colloidal chitin, chitin powder and carboxymethyl chitosan. Metal ions and some compounds have influences on chitinase CHI58 at different degree. Ba2+ and NH4+ can improve chitinase CHI58 activity with 171.2% and 80% respectively; Mg2+ and K+ can improve activity with 21.2% and 24.4% respectively; Ca2+, Mn2+ and NaN3 have weak inhibition effect on chitinase CHI58 activity; and others have inhibition on chitinase CHI58 activity at different degree.In this paper, the internal and external factors which effect Chi58 expression in P. pastoris were analyzed. The results show that modification of codon is a direct and effective way to improve exogenous gene expression. After five codons CGC in Chi58 gene were mutated to AGA, which was used in P. pastoris with highly frequency, Chi58 expression was improved about 3 times. Eventually chitinase CHI58 activity reached 246.68 U, which was 6 times before optimization.Chitin binding domain (ChBD) deletion mutant was obtained by SOE-PCR, and the mutant was constructed into P. pastoris. The results indicate that ChBD can bind and absorb insoluble chitin. Antagonism test in vitro shows that Chitin binding domain deletion chitinase CHI58 displayed lower vitro antagonistic abilities against Rhizoctonia. Solani, Septoria tritici, Pyricularia oryzae, with maximum inhiboitory rate 15%, however, chitinase CHI58 has a high inhiboitory rate of 40% against plant pathogen. Thus, Chitin binding domain (ChBD) has an important significance to biocontrol of chitinase.