Metagenome

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

Figure 3.9 Cloning and DNA-isolation strategies commonly used in metagenomics.

The rapidly increasing number of novel metagenome-derived genes also led to the identiflcation of major bottlenecks. These are mainly linked to the eflhciency of heterologous gene expression and the amount of protein produced, in particular. 

Functional screening of a soil metagenomic library for cellulases revealed a total of eight cellulolytic clones, one of which was purified and characterized [58]. Despite the fact that this library had been generated from a soil sample collected from a... 

Metagenomics has been applied to the search for novel genes encoding the synthesis of vitamins such as biotin and vitamin C [47,76]. Seven cosmids were detected in metagenomic libraries obtained after avidin enrichment of environmental samples. The highest levels of biotin production in this study were detected in a cosmid... 

Table 3.1 Lipolytic genes identified in metagenome screens.

Nitrilases are quite rare in bacterial genomes and less than 20 were reported prior to the application of metagenomics for their detection in environmental DNA [81]. Two studies targeting environmental genomes report the detection of more than 337 novel nitrilases. This has dramatically increased the amount of information about nitrilases, and the newly discovered diversity can be applied for the enantioselective production of hydroxy carboxylic add derivatives [81]. 

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

Proteases are hydrolytic enzymes with important application in industries, in particular, in detergent and in the food industry. A metagenomic study in which 100 000 plasmid clones were screened for proteolytic activity found one positive done, which was determined to be novel by sequencing analysis [84]. 

In industrial processes, 1,3-propanediol is used for the production of polyester fibers, polyurethanes and cydic compounds [85]. 1,3-Propanediol can be produced from glucose with the limiting step catalyzed by glycerol dehydratase. A metagenomic survey for glycerol hydratases from the environment resulted in seven positive clones, one of which displayed a level of catalytic efficiency and stability making it ideal for application in the produdion of 1,3-propanediol from glucose. 

An effective and generic method of screening of BVMO activity is indispensable for screening metagenome libraries as these libraries are often in the range of 100000-1000000 clones. Therefore, it is a challenge to develop a novel way to efficiently detect BVMO activity in gene libraries. This would facilitate... 

The recently developed methodology for enzyme discovery that is based on random DNA isolation and subsequent screening (metagenome mining) is... 

Illustrative examples of their biotechnological applications using metagenomes include the following ... 

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