3. The soil metagenome for screening of potential polysaccharide-degrading enzymes

Objective: To study and identify polysaccharide-degrading enzymes encoding genes from constructed soil metagenomic library

Background information                                                                                                                                                         Microorganisms cultured from soil represent an important source of bioactive compounds such as antibiotics, antimicrobial agents, anticancer drugs, insecticide, herbicide, and immunosuppressant agents which are used in medical and agricultural applications. However, the rate of discovery of novel bioactive compounds has significantly decreased because the same microorganisms are recovered repeatedly by using standard cultivation techniques (Rondon et al., 1999). The progress in phylotypic analysis on community DNA and molecular microbial ecology indicate that the number of cultured microorganisms from soil represents 1% or fewer of the overall microbial population. Thus, there are 99-99.9% of microorganisms that cannot be cultured but they may have the abilities to produce useful bioactive compounds. To overcome the difficulties of culturing microorganisms and to increase the chance for discovery of bioactive compounds and enzymes from soils, cultivation-independent molecular approaches have been developed. The total microbial genome extracted directly from environmental samples can be defined as metagenome. Therefore, metagenomic is the culture-independent genomic analysis of microbial communities.  Metagenome analyses are usually initiated by the isolation of environmental DNAs and cloned into an appropriate vector to construct DNA libraries in a surrogate host system for subsequent screening and product expression.                                                                                                                                                                                      Construction of metagenomic library: DNA extraction and purification are followed by the construction of DNA libraries in appropriate cloning vectors and host strains. The classical approach includes the construction of small insert libraries (< 10 kb) by using plasmid as a vector and Escherichia coli as a host strain. However, small insert libraries do not allow detection of large gene clusters or operons. To overcome the limitation the large insert libraries are employed, such as cosmid DNA libraries with insert size ranging from 25 to 35 kb and bacterial artificial chromosome (BAC) libraries with insert size up to 200 kb. E. coli is still the preferred host for the cloning and expression of any metagenome-derived genes. Recently, other hosts such as Streptomyces lividans have been employed to identify genes involved in the biosynthesis of novel antibiotics. The development of these new host strains will increase screening efficiency due to the difference of expression system (Streit et al., 2004).           

                  Analysis of metagenomic library
: Two major strategies that have been used to identify novel biocatalysts or genes involved in the production of bioactive compounds are functional-based screening and sequence-based screening.  The former can be investigated by both physical and biochemical process.  For example, the ability to produce enzymes for digests of various substrates, the ability to produce antibacterial agent and novel bioactive compounds. The latter screening scheme bases on the use of conserved DNA sequences to design hybridization probes or PCR primers to screen for clones that contain sequences of interest.                   
                 The main aim of our research is to screen for the genes that are involved in the synthesis enzymes that exhibit polysaccharide-modifying properties such as Amylases, Cellulases, b-glucanase, and Xylanases. These enzymes are beneficial to the industry and to research in biotechnology.  Furthermore, the generation and analysis of metagenomic library will be a powerful approach to achive environmental genetic resources. It will enable us to search for other genes and biomolecules that confer novel functions and properties.

Figure 1: Metagenomics approach to assess the unculturable bacterial diversity for novel biocatalysts and                bioactive compounds. (modified from Sharma et al., 2005).

Figure 2: Extracted DNA directly from soil samples