Screening of Environmental Libraries

In contrast to the construction of genomic libraries from single organisms, a statistic calculation of the completeness of environmental libraries cannot be done, since the initial number of different microorganisms used for the preparation of the environmental DNA is unknown. 

Activity-based strategies employ tests that are often applied directly to colonies to identify the desired activity. Nontoxic chemical dyes and insoluble or chromo-phore-bearing derivatives of enzyme substrates, for example, can be incorporated 

DNA source Vector Number of Screening target Number of Ref. 

Clardy, J., Goodman, R. M. Chem. Biol. 1998, 5, R245 - R249. 

Edited by Susanne Brakmann and Kai Johnsson Copyright 2002 Wiley-VCH Verlag GmbH Co. KGaA ISBNs 3-527-30423-1 (Hardback) 3-527-60064-7 (Electronic) 

---

Fig. 4.1. Strategy to access and to exploit the immense genetic diversity of naturally occurring microbial assemblages by construction and screening of environmental libraries.

In the screening of genomic libraries prepared from environmental samples collected in various parts of the world, more than 200 new nitrilases were discovered that allow mild and selective hydrolysis of nitriles (150). One of them catalyzes the (J )-selective hydrolysis of 35 with an ee value of 94.5% at a substrate concentration of 100 mM (46). However, when experiments were done at a more practical concentration of 2.25 M, activity and enantioselectivity suffered (ee only 87.8%). 

Construction of Environmental Libraries for Functional Screening of Enzyme Activity... 

As a recent development, the use of these transgenic lines in which GFP expression is driven by motor neuron-specific promoters will be useful in screening the effect of libraries of environmental pollutants on the development of the neuromotor system [22, 23]. 

Another promising route was reported in patent and open hterature by both DSM and Diversa [13, 14]. This route employs a 2-deoxy-D-ribose 5-phosphate aldolase (DERA) that catalyzes a tandem aldol addition in which two equivalents of acetaldehyde (AA) are added in sequence to chloroacetaldehyde (CIAA) to produce a lactol derivative that is similar to the 3,5-dihydoxy side chain of synthetic statins (Figure 6.2e). Diversa screened environmental libraries for novel wild-type DERAs and identified an enzyme that was both tolerant to increased substrate concentrations and more active than DERA from E. coli in the target reaction [13]. 

DNA libraries and to done directly functional genes from natural samples. The knowledge of sequence information prior to cloning is not required. Another advantage is that existing environmental libraries can be employed for screening of various targets. 

---