Redesigning of Enzyme Activities through Protein Engineering.

In the June, 2000 issue of Current Opinion in Plant Biology, Dr. John Shanklin (3(3):243-48) shanklin@bnl.gov of Brookhaven National Laboratories explains the necessity of redesigning enzyme activities in view of low level expression of the gene of interest in transgenic studies. The example, he quoted, was the transfer of 16-delta 4 desaturase gene from coriander to tobacco. The accumulation of the gene product was much smaller in tobacco than in coriander. To meet such a challenging situation where the degree of expression of a gene product is at a low level, powerful methodologies have been developed to bioengineer a gene with novel specificity and/or the ability to introduce a particular functionality. Such methodologies have, in their view, accelerated the process of evolution.

The author describes the strategy of creating a gene of interest. It involves two steps: insertion of random mutations into the target gene and their subsequent reassembly. The alteration is followed by identification of improved genes through proprietary in vitro methods. The mutants are genetically recombined resulting in the generation of new genes with improved traits that investigators are looking for. Restructuring of a gene is repeated 5-7 times to produce a final version of a gene product of interest. It is of relevance here to mention that beneficial mutants that enhance the enzyme property are very rare and that for this reason, repetition of the restructuring process needs to be repeated several times.

In comparing the process of molecular breeding to conventional breeding, the author points out that the two processes are similar in many respects. For instance, backcross PCR has parallels with conventional breeding in that detrimental traits are eliminated in both the processes due to only the so-called correct fragments being amplified.

In the end, the author quotes the recent accomplishments of Miyazaki and Arnold; and Fersht et al. The former used non-natural amino acids to create a restructured enzyme improving the thermal stability of protease subtilism S41, while the latter engineered a new catalytic activity of phosphoribosylanthranilate by using indole-3-glycerol phosphate synthase

 

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