in 1898 , a microbiologist , heinrich winterberg , first described that there was a discrepancy in the number of microorganisms between culturable bacteria on nutrient media and the total bacteria in nature counted by microscopy . since then , microbial unculturability , the so - called ' great plate count anomaly , ' has long been recognized in microbiology . while microbial unculturability is not fully understood so far , microbial diversity has been analyzed extensively in various environments . the development of culture - independent analysis adopted amplification and dna sequencing of microbial signature rrna sequences from nature without cultivation of entire microbial species . therefore , analysis of the microbial phylogenetic marker genes , such as the 16s rrna gene , revealed that microorganisms are the true dominant organism in nature . the number of prokaryotic cells was estimated to be 4 - 6  10 cells on earth , and the prokaryotes represent the largest pool of carbon , nitrogen , and phosphorus among living organisms . microbial diversity has also been recognized from various soils , and only a small fraction of total microbial species in soil ( i.e. , less than 1% ) has been characterized by cultivation - based methods [ 5 , 6 ] . since the majority of unculturable bacteria in soil has not been cultured , it has not been functionally characterized . . culture - independent analysis of microbial diversity in soil revealed that most bacterial members abundant in soils are members of the phyla proteobacteria , acidobacteria , actinobacterium , firmicutes , and some verrucomicrobia [ 8 , 9 ] , showing that a large number of the 16s rrna gene clones originated from uncultured bacterial species . for example , the forest soils retained higher members of the phylum acidobacteria but fewer -proteobacterial members . on the other hand , agricultural soils harbor higher numbers of members in -proteobacteria but less acidobacteria . the interesting result recently reported by mendes et al .   indicated that the relative abundance of bacterial phyla , not their profiles , could be important for microbial community function in a specific soil , such as disease - suppressive soil . the first proposal of the term ' metagenome ' was by handelsman et al . , while the first approach using environmental dna was reported in 1995 to search for genes encoding cellulases . the soil metagenome can be isolated directly from various types of soil either directly or indirectly without bacterial cultivation [ 14 - 16 ] . while the microbial diversities of various microbial habitats are being actively investigated by taking advantage of next - generation sequencing technology to analyze a large number of 16s rrna gene amplicon sequences derived from a variety of soils , the advances in bioprospecting metagenomics , the purified soil metagenomic dna would be cloned into several different plasmid vectors . in general , high - molecular - weight dnas cloned into fosmid , cosmid , or bacterial artificial chromosome ( bac ) vectors are introduced into a surrogate host bacterium , such as escherichia coli . especially , the fosmid - based preparation of a metagenomic library is the most frequently used strategy , due to their high cloning efficiency , the improved stability in e. coli , and the feasibility to construct a medium - sized ( 40 kb)-insert dna library . considering the low frequency of finding genes for novel bioactivities from a soil metagenomic library , the cloning efficiency is a crucial factor to construct a large clone member library . one of the most critical steps for successful library construction is the isolation of pure high - molecular - weight dna , which is suitable for cloning into the vectors [ 8 , 20 ] . however , high contents of soil - derived humic acids are co - purified during the high - molecular - weight dna isolation process , and humic acids interfere with the cloning process of high - molecular - weight dna into a vector . various methods to remove humic acids during soil metagenomic dna purification usually end up with the recovery of relatively low - molecular - weight dna , which is not suitable to clone into a fosmid or bac . several commercial kits would be plausible to purify soil metagenomic dna for the amplification purpose of 16s rrna genes , which will be subsequently used for microbial diversity analysis . however , the sizes of dna fragments isolated by most of the commercial kits are rather small to clone into vectors , such as fosmids and bacs . therefore , optimal methods for the direct isolation of high - molecular - weight dna from soil have been developed and used for bioprospecting metagenomics [ 14 , 16 , 20 ] . although obtaining a large number of metagenomic clones from soil is a prerequisite to search for novel microbial genes from soil , the technologies for metagenomic library construction were generalized using fosmids and cosmids . various novel microbial resources could be expected from soil metagenomic libraries , because soils bear the highest microbial diversity compared to any other microbial community . since functional metagenomics ( or bioprospecting metagenomics ) was attempted from a soil ecosystem , a number of microbial genes encoding novel enzymes or bioactive compounds have been identified and characterized [ 21 , 22 ] . here , we summarize several technical points to be considered for bioprospecting metagenomics and the major results of novel enzymes and bioactivities from the soil metagenome . metagenomic libraries with either large - insert dna or small - insert dna were used to search for novel microbial genes . the library with the small - insert dna could be feasible to identify novel enzymes , while that with the large - insert dna is also frequently used . screening of both types of metagenomic libraries has been conducted and successfully identified many novel enzymes and bioactivities . two different approaches for screening the metagenomic libraries are frequently used , such as function - based ( expression - dependent ) screening and sequence - based ( homology - dependent ) screening . function - based screening is dependent on the expression of metagenomic genes in a surrogate host , such as e. coli , and subsequently detects metabolic activities in the heterologous host . therefore , the proper expression of metagenomic genes is essential to detect the phenotypic characteristics of the desired activity by the function - based screening approach . it is arguable that the majority of genes from uncultured soil microorganisms would not be expressed properly in a host bacterium . there has been no extensive analysis to assess the expression ratio of metagenomic genes in defined culturable bacteria . in spite of the expression barrier of cloned metagenomic genes in e. coli , the function - driven approach has proven to be feasible to search for novel genes and gene products . recently , function - based screening approaches have been advanced and modified to search for novel microbial genes compared with the original approach - i.e . , the direct detection of metabolic function . host strains or mutants were used to identify genes for functional complementation in trans by metagenomic clones , and another advance in function - based screening was combined with hts to detect the induced gene expression in a host bacterium with substrates , autoinducers , and metabolic products provided exogenously [ 25 - 27 ] . one of the approaches to overcome the host expression barrier is to develop a broad - host range vector to express a metagenomic library in a variety of host bacteria . this approach , in fact , is promising and nourishing the function - based screening of metagenomics to obtain diverse microbial resources for enzymes and biomolecules . the sequence - based approach is to screen for marker genes using dna probe or pcr primers designed from dna sequences of already known genes . since this approach would detect gene variants with the conserved motif from the metagenome , it is arguable if this approach would really screen true novel genes from microbial diversity nevertheless , there are a number of examples with some successful identification of novel microbial enzymes [ 22 , 29 ] . a recent approach with sequence - based screening is taking advantage of the advances in next - generation sequencing . direct sequencing of metagenomic dna by next - generation sequencing is now used increasingly to study gene inventories in the metagenomes of specific microbial communities . direct sequencing of a metagenome generates a large dataset , which should be analyzed by support of the appropriate informatics . analysis of the soil metagenome by the sequencing - based approach is still a huge challenge , since the soil microbial community is the most complicated one with the highest microbial diversity compared with any other microbial community . a combination of appropriate bioinformatics , such as comparative sequence analysis , with the large set of soil metagenome sequences will be a new opportunity to search for microbial enzymes and novel bioactivities in the future . if both function - based and sequence - based screening is combined with hts , novel microbial enzymes and bioactivities would be detected at higher frequency compared with conventional screening . this is mainly because the large numbers of metagenomic clones in libraries should be searched for novel enzymes and biomolecules to tap the great soil microbial diversity . some of the recently adopted hts methods for drug discovery may enable the discovery of novel enzymes and biomolecules from the soil metagenome . multidisciplinary efforts , combined with bioinformatics , analytical chemistry , and high - throughput technology , is required to search for novel enzymes and bioactivities from soil metagenomes . many novel enzymes were identified from various metagenomic studies , and we summarized the examples of enzyme recovery from soil metagenome studies in table 1 [ 10 , 32 - 58 ] . one of the most prevailing novel enzymes found from the soil metagenome is esterase / lipase . lipolytic enzymes , such as esterases and lipases , are important biocatalysts for biotechnological applications . the interesting features of lipolytic enzymes include no requirement for cofactors , remarkable stability in organic solvents , broad substrate specificity , stereoselectivity , and positional selectivity . the features of lipolytic enzymes are especially attractive for organic synthesis if the enzymes with specific chiral resolution could be retrieved from the soil metagenome . therefore , the discovery of novel lipolytic enzymes from the soil metagenome has received much attention compared to other enzymes . considering that lipolytic enzymes are the most well characterized and highly studied enzymes among biocatalysts , the finding of several novel families of esterase / lipase enzymes is quite surprising . one of our previous results with estd2 revealed that estd2 was not similar to any previously described enzymes , while the enzyme clearly displayed esterase activity . a similar protein found in the genbank database also exhibited the same activity , while it was initially annotated as a hypothetical protein . therefore , function - driven selection of estd2 constituted a novel family of esterases and re - annotated many proteins as esterase / lipases that were previously known as hypothetical proteins . similar studies with enzymes found in the soil metagenome suggested that soil microbial diversity truly bears the great extent of novel microbial enzymes . cellulolytic enzymes and cell wall - degrading enzymes ( cwdes ) of plant cells are also enzymes with biotechnological interest , mostly due to biomass degradation , with the purpose of bioenergy production . in contrast to the enzymatic interest , the identification of novel cellulolytic enzymes or other cwdes is not well documented . the reason for the rare recovery of cellulases and cwdes is poor secretion of these enzymes in e. coli , the major host bacteria , and incompatible detection methods of enzymatic activity for a large member of metagenomic libraries from the soil metagenome . for the successful detection of cellulases and cwdes , technical elaboration with hts would be necessary to enhance the detection of proper enzyme activity . bioinformatics , such as comparative sequence analysis of the metagenome , could be another choice to identify cellulolytic enzymes and cwdes from massive datasets derived from direct sequencing of the soil metagenome . a number of enzymes for other carbohydrate metabolism and a few lactonases were also detected from the soil metagenome , and they are listed in table 1 . however , many enzymes are not feasible to detection by simply screening for the bacterial phenotypic changes , while the lipolytic enzymes were simply identified by tributyrin hydrolysis on culture medium . in addition to the novel enzymes , soil metagenomes are rich sources of a variety of small molecules with bioactivities , such as antibiotics and other pharmaceutically applicable activities . the search for novel bioactivities is primarily based on the phenotypic detection of bacterial traits of the host bacteria bearing metagenomic libraries . antagonistic activity and colony color changes of the host bacterium are typical examples to detect metagenomic clones , potentially associated with novel secondary metabolite production . however , the conventional approach exhibits a rarer chance to identify novel gene clusters and bioactivities . therefore , it is necessary to develop new detection strategies for novel bioactivities from the soil metagenome . nonetheless , the simple detection of antibiosis or color changes of the host bacterial colonies with metagenomic clones brought some interesting results , suggesting the great potential of novel bioactivities from the soil metagenome . we summarize the identified bioactivities and their gene clusters from the soil metagenome in table 2 . these bioactivities include antibacterial turbomycins , glycopeptides , cyanobactins , type ii polyketides , trans - acyltransferase polyketides , and the anticancer agent et-743 . some of the bioactivities from the soil metagenome may also be discovered by homology - based screening . whole metagenome sequences from soil , which will be accomplished in the future , would be a rich source to be probed to identify gene clusters for bioactive compound production . since functional metagenomics ( or bioprospecting metagenomics ) was attempted from a soil ecosystem , a number of microbial genes encoding novel enzymes or bioactive compounds have been identified and characterized [ 21 , 22 ] . here , we summarize several technical points to be considered for bioprospecting metagenomics and the major results of novel enzymes and bioactivities from the soil metagenome . metagenomic libraries with either large - insert dna or small - insert dna were used to search for novel microbial genes . the library with the small - insert dna could be feasible to identify novel enzymes , while that with the large - insert dna is also frequently used . screening of both types of metagenomic libraries has been conducted and successfully identified many novel enzymes and bioactivities . two different approaches for screening the metagenomic libraries are frequently used , such as function - based ( expression - dependent ) screening and sequence - based ( homology - dependent ) screening . function - based screening is dependent on the expression of metagenomic genes in a surrogate host , such as e. coli , and subsequently detects metabolic activities in the heterologous host . therefore , the proper expression of metagenomic genes is essential to detect the phenotypic characteristics of the desired activity by the function - based screening approach . it is arguable that the majority of genes from uncultured soil microorganisms would not be expressed properly in a host bacterium . there has been no extensive analysis to assess the expression ratio of metagenomic genes in defined culturable bacteria . in spite of the expression barrier of cloned metagenomic genes in e. coli , the function - driven approach has proven to be feasible to search for novel genes and gene products . recently , function - based screening approaches have been advanced and modified to search for novel microbial genes compared with the original approach - i.e . , the direct detection of metabolic function . host strains or mutants were used to identify genes for functional complementation in trans by metagenomic clones , and another advance in function - based screening was combined with hts to detect the induced gene expression in a host bacterium with substrates , autoinducers , and metabolic products provided exogenously [ 25 - 27 ] . one of the approaches to overcome the host expression barrier is to develop a broad - host range vector to express a metagenomic library in a variety of host bacteria . this approach , in fact , is promising and nourishing the function - based screening of metagenomics to obtain diverse microbial resources for enzymes and biomolecules . the sequence - based approach is to screen for marker genes using dna probe or pcr primers designed from dna sequences of already known genes . since this approach would detect gene variants with the conserved motif from the metagenome , it is arguable if this approach would really screen true novel genes from microbial diversity nevertheless , there are a number of examples with some successful identification of novel microbial enzymes [ 22 , 29 ] . a recent approach with sequence - based screening is taking advantage of the advances in next - generation sequencing . direct sequencing of metagenomic dna by next - generation sequencing is now used increasingly to study gene inventories in the metagenomes of specific microbial communities . direct sequencing of a metagenome generates a large dataset , which should be analyzed by support of the appropriate informatics . analysis of the soil metagenome by the sequencing - based approach is still a huge challenge , since the soil microbial community is the most complicated one with the highest microbial diversity compared with any other microbial community . a combination of appropriate bioinformatics , such as comparative sequence analysis , with the large set of soil metagenome sequences will be a new opportunity to search for microbial enzymes and novel bioactivities in the future . if both function - based and sequence - based screening is combined with hts , novel microbial enzymes and bioactivities would be detected at higher frequency compared with conventional screening . this is mainly because the large numbers of metagenomic clones in libraries should be searched for novel enzymes and biomolecules to tap the great soil microbial diversity . some of the recently adopted hts methods for drug discovery may enable the discovery of novel enzymes and biomolecules from the soil metagenome . multidisciplinary efforts , combined with bioinformatics , analytical chemistry , and high - throughput technology , is required to search for novel enzymes and bioactivities from soil metagenomes . many novel enzymes were identified from various metagenomic studies , and we summarized the examples of enzyme recovery from soil metagenome studies in table 1 [ 10 , 32 - 58 ] . one of the most prevailing novel enzymes found from the soil metagenome is esterase / lipase . lipolytic enzymes , such as esterases and lipases , are important biocatalysts for biotechnological applications . the interesting features of lipolytic enzymes include no requirement for cofactors , remarkable stability in organic solvents , broad substrate specificity , stereoselectivity , and positional selectivity . the features of lipolytic enzymes are especially attractive for organic synthesis if the enzymes with specific chiral resolution could be retrieved from the soil metagenome . therefore , the discovery of novel lipolytic enzymes from the soil metagenome has received much attention compared to other enzymes . considering that lipolytic enzymes are the most well characterized and highly studied enzymes among biocatalysts , the finding of several novel families of esterase / lipase enzymes is quite surprising . one of our previous results with estd2 revealed that estd2 was not similar to any previously described enzymes , while the enzyme clearly displayed esterase activity . a similar protein found in the genbank database also exhibited the same activity , while it was initially annotated as a hypothetical protein . therefore , function - driven selection of estd2 constituted a novel family of esterases and re - annotated many proteins as esterase / lipases that were previously known as hypothetical proteins . similar studies with enzymes found in the soil metagenome suggested that soil microbial diversity truly bears the great extent of novel microbial enzymes . cellulolytic enzymes and cell wall - degrading enzymes ( cwdes ) of plant cells are also enzymes with biotechnological interest , mostly due to biomass degradation , with the purpose of bioenergy production . in contrast to the enzymatic interest , the identification of novel cellulolytic enzymes or other cwdes is not well documented . the reason for the rare recovery of cellulases and cwdes is poor secretion of these enzymes in e. coli , the major host bacteria , and incompatible detection methods of enzymatic activity for a large member of metagenomic libraries from the soil metagenome . for the successful detection of cellulases and cwdes , technical elaboration with hts would be necessary to enhance the detection of proper enzyme activity . bioinformatics , such as comparative sequence analysis of the metagenome , could be another choice to identify cellulolytic enzymes and cwdes from massive datasets derived from direct sequencing of the soil metagenome . a number of enzymes for other carbohydrate metabolism and a few lactonases were also detected from the soil metagenome , and they are listed in table 1 . however , many enzymes are not feasible to detection by simply screening for the bacterial phenotypic changes , while the lipolytic enzymes were simply identified by tributyrin hydrolysis on culture medium . in addition to the novel enzymes , soil metagenomes are rich sources of a variety of small molecules with bioactivities , such as antibiotics and other pharmaceutically applicable activities . the search for novel bioactivities is primarily based on the phenotypic detection of bacterial traits of the host bacteria bearing metagenomic libraries . antagonistic activity and colony color changes of the host bacterium are typical examples to detect metagenomic clones , potentially associated with novel secondary metabolite production . however , the conventional approach exhibits a rarer chance to identify novel gene clusters and bioactivities . therefore , it is necessary to develop new detection strategies for novel bioactivities from the soil metagenome . nonetheless , the simple detection of antibiosis or color changes of the host bacterial colonies with metagenomic clones brought some interesting results , suggesting the great potential of novel bioactivities from the soil metagenome . we summarize the identified bioactivities and their gene clusters from the soil metagenome in table 2 . these bioactivities include antibacterial turbomycins , glycopeptides , cyanobactins , type ii polyketides , trans - acyltransferase polyketides , and the anticancer agent et-743 . some of the bioactivities from the soil metagenome may also be discovered by homology - based screening . whole metagenome sequences from soil , which will be accomplished in the future , would be a rich source to be probed to identify gene clusters for bioactive compound production . the soil metagenome , a rich microbial source , is still unexplored , since major microbial species have not been cultured and characterized from various soils . both function - driven metagenomics and homology - dependent metagenomics have started out in soils and brought some successful discovery of novel microbial enzymes and secondary metabolites . further analysis of soil microbial communities and the appropriate enrichment of target resources from soils will increase the chance to discover novel microbial enzymes and bioactivities from the soil metagenome . in addition , the adoption of hts , the advances in sequencing technology , and the proper bioinformatics would be necessary to improve the efficacy of bioprospecting metagenomics of soils for the discovery of novel enzymes and bioactivities .
the microbial diversity in soil ecosystems is higher than in any other microbial ecosystem . 
 the majority of soil microorganisms has not been characterized , because the dominant members have not been readily culturable on standard cultivation media ; therefore , the soil ecosystem is a great reservoir for the discovery of novel microbial enzymes and bioactivities . 
 the soil metagenome , the collective microbial genome , could be cloned and sequenced directly from soils to search for novel microbial resources . 
 this review summarizes the microbial diversity in soils and the efforts to search for microbial resources from the soil metagenome , with more emphasis on the potential of bioprospecting metagenomics and recent discoveries .