Screening Antibacterial Substance From Entophytic Fungi And Construction Of ECB Deacylase Genetically Engineered Strain

Methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus, MRSA) has become a major pathogen of nosocomial infection all over the world.It is a difficult problem in the treatment of clinical anti-infection. Inclduing vancomycin, teicoplanin, rifampin and etc., these medicines were proved to be this resistented by MRSA in a certain extent. It has not yet developed the new effective antibiotics to treat MRSA.It is a serious problem to find new drugs for development of precursors. Endophytic fungus was commonly found in healthy plant tissues, which has the bacteriostasis function among varied active functions. The laboratory tries to find active strains which has antiMRSA function among entophytic fungi and researchs the optimized method in fermentation conditions,and has laid a working basis for the future development of the drug treatment of MRSA.We used 14 d solid state fermentation, and extracte fermentation product by ethyl acetate. We used Bacillus subtilis, Candida albicans, Staphylococcus aureus, Sahroeter lutea, Escherichia coli as the identification of pathogenic bacteria strains, screening 529 endophytic fungi (mainly plant Bupleurum, wheat, tomato, rice, angelica, Cep, forsythia, ingot grass cinnabar root, Eucommia, pheasant tail, peony, Agastache rugosa, etc.) by disc agar diffusion method. It showed fermented products extracts for 50 against Escherichia coli,66 strains against Candida albicans and 262 strains against Staphylococcus aureus,124 strains against Sarcina lutea,159 strains against Bacillus subtilis. Also used MRSA as the identification of strains, screening selected 55 strains which had a broad antibacterial spectrum of endophytic bacteria. It showed that 19 strains had anti-MRSA activity. Then we selected a broad spectrum antimicrobial activity of endophytic fungi SIPI 3.0070 to identify strains. Through morphological and cultural characteristics observation (3 d,7 d colony morphology and microscopic morphology), we had the initial identification of the strain is Aspergillus, followed by molecular biology methods, the 18S rDNA strain SIPI 3.0070 sequencing were submitted to GenBank, the sequence of registration number FJ864683, sequence homology is also on Aspergillus fumigatus (homology 100%), the final identification of SIPI 3.0070 is Aspergillus fumigatus. Also we optimized bacteria culture and soaking conditions with inhibition zone size as the evaluation criteria. It showed that millet as a solid medium, the fermentation period 14 d, culture soaked with ethyl acetate extract had higher activity to anti-gram Gram positive bacteria. If used methanol extracting fermentation products, extract antifungal effect is good.Aspergillus nidulans produces echinocandin B0(ECB), a neutral lipopeptide. A deacylase from Actinoplanes utahensis catalyzes cleavage of the linoleoyl group from echinocandin B, a key step in generating a potential antifungal agent. Access to literature, the current basic traditional Chinese Aspergillus nidulans fermentation to obtain the ECB amide hydrolase, there is no associated bacteria through genetic engineering obtained ECB amide hydrolase. Since the use of sophisticated genetic engineering methods, and the relative traditional fermentation method, a more rational and orientation, the establishment of genetically engineered bacteria to produce acid amide hydrolase ECB is particularly important. The laboratory used two types of genetically engineered bacteria build strategy. A fragment containing the ECB deacylase encoding region and its flank genes was amplified from Actinoplanes utahensis SIPI-A.2001, EcoRâ… and Xbaâ… digestion of the gene and the pSP72 plasmid, connection, construct pCQ-02-44-1 plasmid, gene sequencing constracting the line corresponding amide hydrolase ECB was 99.8% homology. and linked into the overexpression vector pTGV2, named pCQ-03-11-1. Then the plasmid was transformed into Streptomyces lividans TK24. This genetically engineered strain can transform ECB into ECB nucleus. HPLC analysis showed that the genetically engineered strain can effectively cleave ECB side chain, and the conversion rate was as high as 53.2%. Aspergillus nidulans actinomycetes conversion rate is 11.74%,as high as nearly 5 times. Another strategy cloned ECB amide hydrolase (approximately 2.8 kb) gene, online sequence alignment homology was 99.8%, and then cloned erythromycin strong promoter (ermEp) gene, sequence homology of 100% on by restriction to connect to construct containing the promoter and the ECB erythromycin amide hydrolase cloned plasmid into plasmid pTGV2. This work using genetically engineered bacteria for the next ECB substrate conversion basis, and explore the use of genetically engineered bacteria to high conversion efficiency of the possibility of transforming ECB.