Isolation And Identification Of Carbendazim Tolerant Strinas YF-2~T,YF-3~T And Expression Of The MheI Gene

Carbendazim is a broad spectrum,systemic benzimidazole fungicide,which has been widely used in China and abroad.Chemical character of carbendazim is stable and its half-life period is long.As the usage amount of carbendazim has increased year by year,itsnegative influence on environment becomes more obvious and serious.Carbendazim is degradaed mainly by microorganisms in the environment;so far strainscapable of degrading carbendazimwere Gram-staining positive.Two carbendazim tolerant strains YF-2~T(1000mg·L-1)andYF-3~T(1200mg·L-1)were isolated from the soilsubjected to the long-term application of carbendazim.Polyphasic classification was carried out on these two strains.Cells of YF-2~T are Gram-staining negative,non-motile,non-spore-forming,rod-shaped,approximately 0.4-0.6 μm width,2.3-2.6 μm in length.Colonies on R2A agar inoculated 8h are circular with a shiny surface and entire edges,light-yellow-pigmented(flexirubin-type),translucent and mucoid.Growth occurs at between 25-37℃,at pH 5.0-8.0,and with 0-5%(w/v)NaCl.The strain contains menaquinone-6 as the major respiratory quinone.Polar lipids consist of phosphatidylethanolamine,seven unidentified lipids and three unidentified aminolipids.The predominant fatty acids(≥5%)are summed feature 3(comprising C16:1 ω7c and/or C16:1 ω6c),iso-C15:0,iso-C17:0 3-OH,C16:0 and anteiso-C15:0.The G+C contents of strain YF-2~T is 35.2%.Analysis based on sequence of the 16S rRNA gene showed that strain YF-2~T was closely related to strains Flavobacterium akiainvivens IK-1T and Flavobacterium hauense BX12~T with 95.99%and 95.92%sequence similarity,respectively.In conclusion,strain YF-2~T shouldrepresent a novel species of the genus Flavobacterium,for which the name Flavobacterium shanxiense sp.nov.is proposed.Cells of YF-3~T are Gram-stainingnegative,non-motile,non-spore-forming,rod-shaped,approximately 0.5-0.7 μm in width,2.1-2.3 μm in length.Colonies grown 12h on TSA are circular with a shiny surface and entire edges,yellow-orange-pigmented(flexirubin-type,non-diffusible),translucent and mucoid.Growth conditions are 25-37℃,at pH 5.0-8.0,with 0-5%(w/v)NaCl.Menaquinone-6 is the main respiratory quinone.The predominant fatty acids(≥50%)are iso-C15:0,iso-C17:0 3-OH,summed feature 9(comprising iso-C17:1ω9cand/or C16:0 10-methyl)and summed feature 3(comprising C16:1ω7c and/or C16:1 ω6c).Polar lipids consist of phosphatidylethanolamine,five unidentified lipids and two unidentified aminolipids.Sym-homospermidine is the predominant polyamine but minor amounts of spermidine arealso present.The DNA G+C content of strain YF-3~T is 37.0mol%.The 16S rRNA gene sequence of strain YF-3~T showed similarity withChryseobacterium hispalense AG13~T and Chryseobacterium taiwanense BCRC 17412~T are 98.71%and 96.93%respectively.DNA-DNA relatedness to reference strains Chryseobacterium hispalense AG 13~T and Chryseobacterium taiwanense BCRC 17412~T are 31.7±2.1(33.5±3.6)and 28.4±5.4(25.7±4.4)respectively,which are well below the threshold of 70%recommended for the delineation of bacterial species.In conclusion,strain YF-3~T shouldrepresent a novel species of the genus Chryseobacterium,for which the name Chryseobacterium shandongense sp.nov.is proposed.The key step of carbendazim degradation is the hydrolysis of carbendazimto2-Aminobenzimidazole,which is catalyzed by MheI.The codons of the mhel gene from the carbendazim-degrading strain mbc-1 was optimized and synthesized in order to be suitable for the its expression in Bacillus subtilis WB800,and then the optimized gene(mhel)was cloned intoshuttle expression vector pP43NMK,which has strong promoter P43 and nprB signal peptide.Then the constructed plasmidpP43NMK-mheIis transformedinto Bacillus subtilis WB800.Gene(mhel)is expressed successfully and efficiently,which provides the foundationfor the production of enzymeMheI.