Isolation And Identification Of Soil Cladosporium And Functional Characterization Of Extremophilic Xylanases

Hemicellulose is one of the major components of plant cell wall,which can be degraded by xylanolytic enzymes.It is of great importance to obtain highly efficient hemicellulose-degrading fungal strains and develop new xylanases with great application potentials.In this study,17 Cladosporium strains were isolated from the plateau soil of Xinjiang,Qinghai and Yunnan.By combining the genetic(ITS,ACT and TEF1)and phenotypic analysis,six new species Cladosporium neopsychrotolerans SL-16~T(= CGMCC 3.18031~T),C.paralimoniforme XJ4974~T(= CGMCC3.18104~T),C.prolongatum XJ5436~T(= CGMCC 3.18036~T),C.sinuatum QH1-5~T(= CGMCC 3.18096~T),C.tianshanense SL-14~T(－CGMCC 3.18033~T)and C.verruculosum QH1-6~T(= CGMCC 3.18099~T)were proposed,which enrich the Cladosporium resource.It’s the first time to reveal soil as an important niche of Cladosporium.The psychrophilic C.tianshanense SL-14 and psychrotolerant C.neopsychrotolerans SL-16 isolated from the rhizosphere soil of Tianshan snow lotus had great lignocellulose-degrading capabilities.To explore the genetic resources of xylanolytic enzymes,strains SL-14 and SL-16 were then sequenced to generate two genomes of 33.8 Mb and 35.9 Mb that contain 9,826 and 13,456 predicted genes,respectively.Functional annotation on predicted genes suggested that these fungi have 8,027 and 11,684 functional genes and 408 and 571 carbohydrate active enzyme(CAZyme)genes.In comparison to psychrophilic SL-14,psychrotolerant SL-16 contains more putative transmembrane proteins(967 vs.518)and CAZymes(571 vs.408),and more CAZymes involved in the degradation of plant cell wall(235 vs.215)and xylan(46 vs.19).These advantages make C.neopsychrotolerans more adaptable to various niches and valuable in mining novel xylanases.A xylanolytic system of seven glycoside hydrolases(4 GH10,1 GH11,and 2 GH43)was identified in C.neopsychrotolerans SL-16 based on the genome analysis.Based on the quantative PCR analysis of gene expression levels,three representative genes of each family(xyn10A,xyn11A and xyl43A)were selected for gene cloning and heterologous expression in Escherichia coli BL21(DE3).The purified recombinant enzymes showed the typical traits of cold-active proteins including optimal temperature at 35-40℃ and being thermolabile(<40℃).The enzymes had broad substrate specificity and showed synergistic action on xylan degradation by utilizing different cleavage modes.The enzymes in simultaneous and sequential combinations released xylose as the final product with a synergy degree of up to 15.32.Under neutral ambient conditions(pH 6.5 and 30℃),the combination of commercial cellulase Neutral Max and sequential enzyme system Xyn11A→Xyn10A→Xyl43A enhanced the sacchrification efficiency of steam-exploded corn stover,releasing greater amounts of glucose and xylose(1.47-fold and 11.81-fold,respectively).By removing the blocking xylan from lignocellulosic materials and converting it into fermentable xylose,this low-temperature-active xylanolytic system may represent great prospect in the production of biofuels in an energy-saving,environment-friendly.and highly-efficient way.To obtain xylanases having prebiotic xylooligosaccharide-producing capabilities,four GH11 xylanase-encoding genes(MtxynllA,Mtxyn11At,Mtxyn11B and Mtxynll)were cloned from the compost fungus Mycothermus thermophilus CGMCC3.18119,and successfully overexpressed in Pichia pastoris GS115.The recombinant enzymes had neutral(maximal activities at pH 6.0-6.5)and thermophilic(65 0C)properties as well as strong xylan-degrading capabilities(95.0-96.6%).To verify the prebiotic effect of xylooligosaccharides on probiotics,the xylan hydrolysates of different xylanases were used as the sole carbon source to cultivate Lactobacillus strains L.brevis 1.2028,L.rhamnosus GG,L.casei BL23,and L.plantarum WCSFI.All Lactobacillus strains showed capacity to utilize the xylooligosaccharides efficiently(83.8-98.2%).In comparison to other enzymes,MtXyn11A is superior in high yield,high specific activity,high catalytic efficiency,high xylan conversion rate and significant promoting effect on the growth of probiotic bacteria,and thus shows great potentials in the production of xylooligosaccharides.In summary,this study firstly reported on six new Cladosporium species,generated and compared two genomes of cold-active Cladosporium strains,and functionally verified the synergistic mechanism of a low-temperature-active xylanolytic enzyme system in the degradation of xylan and lignocellulose and the promoting effect of xylooligosaccharides produced by four thermophilic xylanases on Lactobacillus growth.This study not only provides valuable cold-active and thermophilic enzymes to undermine the thermal adaptation mechanism,but also is of great importance in the basic and applied studies of industrial enzymes.