Mining And Functional Identification Of Cellulase Genes From Animal Gastrointestinal Microorganisms

Cellulose,the main component of crop straws,is the key factor limiting the utilization of straws in animal feeding.Mining efficient cellulases are effective method to improve straw utilization rate.The gastrointestinal microorganisms of plant-eating animals are regarded as important screening sources of cellulase genes.However,most of the studies mainly focus on the ruminal microorganisms of ruminants with a lack of investigations on the intestinal microorganisms of other animals with strong cellulose-degrading ability.Additionally,most of cellulase genes of microorganisms cannot be obtained by traditional microbial culture methods.Therefore,this study avoided the traditional microbial cultivation methods and used the direct metagenomic sequencing technology to(i)explore the differences of community structures of cellulose-degrading microorganisms,(ii)investigate metabolic functions and cellulase gene distribution among three plant-eating animals,and(iii)mine novel and efficient cellulase genes by metagenomic fosmid libraries.Furthermore,the feasibility of screening novel and highly efficient cellulase genes by fosmid libraries was evaluated by the sequence analysis,heterologous expression of the selected genes,and the degradation effects of cellulases on straws.The main research results in this study are as follows:1.Field emission scanning electron microscope(FE-SEM),X-ray diffraction(XRD),and Fourier transform infrared spectroscopy(FTIR)analyses showed that the cellulosic structures of the woods were seriously damaged after being eaten by Reticulitermes Labralis(R.Labralis)and Sipalus gigas(S.gigas),which indicated that these two insects have strong degradation effects on cellulosic biomass.2.The rumens of five Tibetan goats,the hindguts of 1,800 R.Labralis and the guts of 24S.gigas were analyzed using direct metagenomic sequencing technology.The results revealed that the abundances of Prevotella sp.FD3004 and Ruminococcus flavefaciens in the rumen of Tibetan goats were significantly higher than those in the guts of the investigated two insects(P<0.05).The abundances of Treponema primitia,Treponema azotonutricium,Treponema endosymbiont of Eucomonympha sp.and Treponema brennaborense in the hindguts of R.Labralis were significantly higher than those in the gastrointestinal tracts of the Tibetan goats and S.gigas(P<0.05).The abundances of Lactococcus lactis,Dysgonomonas capnocytophagoides and Lactobacillus fabifermentans in the guts of S.gigas were significantly higher than those in the rumens of Tibetan goats and in the hindguts of R.Labralis(P<0.05).These different microbial species have a function of cellulosic degradation.3.?-glucosidase activities in the rumens of Tibetan goats were significantly lower than those in the guts of the other two insects by comparing the(hemi)cellulase activities of the gastrointestinal contents(P<0.05).Additionally,the cellulase activities in the guts of S.gigas were higher than those of the Tibetan goats and R.Labralis(P<0.05).4.The functional annotation of genes predicted by direct metagenomic sequencing showed that the abundances of ko00520,ko00500,and ko00052 in the rumens of Tibetan goats were significantly higher than those in the other two insects(P<0.05);ko00620 was the highest in the hindguts of R.Labralis(P<0.05),and ko00640 was the the most abundant in the guts of S.gigas(P<0.05).The abundances of GH5,GH45,GH124,GH3,and GH10 in the rumens of Tibetan goats were significantly higher than those in the other two insects(P<0.05).The abundances of GH51 and GH116 in the hindguts of R.Labralis were significantly higher than those in the Tibetan goats and S.gigas(P<0.05).The abundances of GH8,GH9,and GH1 in the S.gigas were significantly higher than those in the Tibetan goats and R.Labralis(P<0.05).Furthermore,the abundances of EC3.2.1.40,3.2.1.21,and 3.2.1.80 in the rumens of Tibetan goats were significantly higher than those in the other two insects(P<0.05).The above results indicated that the gastrointestinal microbiomes of these animals can be used as important sources for screening cellulase genes.5.The novel genes of cellulase cannot be obtained by directly metagenomic sequencing.In this study,three metagenomic fosmid libraries were successfully constructed,including in the rumens of Tibetan goats(1,700 clones;with a coverage rate of 68 Mb),the hindguts of R.Labralis(1,900 clones;76 Mb),and the whole guts of S.gigas(2,112 clones;84.48 Mb).A total of 98,244 and 153 positive clones were identified and screened from the fosmid libraries of the rumens of Tibetan goats,the hindguts of R.Labralis and guts of S.gigas,respectively.Three positive clones(R-A2,R-E1,and T-B4)were screened by comparing the sizes of different functional hydrolytic circles,which could simultaneously produce endoglucanase and?-glucosidase,and used to perform the following gene screening experiments.6.Bifunctional genes expressing endoglucanase and?-glucosidase were screened by constructing the subclone libraries from the fosmid plasmids R-A2,R-E1,and T-B4.The screened genes were named A2-Y12-2,Rbs A,and Y14,respectively.Amino acid sequences alignment analysis showed that A2-Y12-2 and Rbs A were similar to two known proteins in National Center for Biotechnology Information(NCBI)database(>99%);however,they had lower similarities with the reported amino acid sequences of cellulase.In addition,Y14showed 53%similarity with other proteins in the Gen Bank database,suggesting that Y14 as a novel cellulase gene.According to the predicted tertiary structure of Y14,the catalytic activity sites were predicted at Ser385 and Asn388.7.The bifunctional genes,A2-Y12-2,Rbs A,and Y14,were seamlessly cloned into p ET-28a(+)vector,respectively,and they were finally transferred to BL21(DE3)competent Escherichia coli(E.coli),inducing a successful expression.Endoglucanase expressed by Y14showed the optimal activity at 50?C and p H 5.5(specific activity 11.79 U/mg),while?-glucosidase expressed by Y14 showed the optimal activity at 37?C and p H 8.5(specific activity0.41 U/mg).They were higher than the specific activity of endoglucanase produced by A2-Y12-2(50?C,p H 5.5)and Rbs A(37?C,p H 5.5)under optimum reaction conditions,and higher than the specific activity of?-glucosidase produced by A2-Y12-2(50?C,p H 8.5)and Rbs A(50?C,p H 5.5)under optimum reaction conditions.8.The crude fiber(CF)contents decreased when wheat and rice straw were degraded by bifunctional cellulases Rbs A,A2-Y12-2 or Y14 at 50?C from the 0^th to the 7^th day.This indicated that all of the three different cellulases had thermal stability at 50?C.Bifunctional cellulase,Y14,had the best degradation effects on rice straw and the degradation rate of CF in rice straws was 18.16%on the 7^th day.Bifunctional cellulase,A2-Y12-2,had the best degradation effect on wheat straw,and the degradation rate of CF in wheat straw was 17.53%on the 7^th day.The results of FE-SEM,XRD,and FTIR also showed that the bifunctional cellulases could degrade the celluloses in the examined straws.Among them,bifunctional enzymes,A2-Y12-2 and Y14,had better degradation effects on the cellulose of wheat and rice straw than Rbs A.In summary,the gastrointestinal metagenomes in the rumens of Tibetan goats,the hindguts of R.Labralis,and the whole guts of S.gigas can be used as important sources for screening cellulase genes.The bifunctional genes screened from these fosmid libraries enrich the resources of existing cellulase degradation-associated genes.This provides a reference for improving the crop straw utilization efficiency for feeding programs of livestock and poultry.