Classical recombinant technique

In the cases described below we will generally focus on metabolic pathway engineering rather than on classical strain improvement, for obvious reasons. One should be aware, however, that the classical approach has strengths that can make it a powerful partner of the rational approach. Thus, regulation problems have been addressed by the development of a feedback-resistant enzyme, using selective pressure and random mutagenesis as described above, in a research species, followed by introduction of the altered gene in the production species via recombinant techniques. 

Since a growing number of newly discovered peptidases are specifically expressed in single tissues, especially, at low expression levels or often only at certain development stages, it is very complicated to isolate the enzymes in sufficient quantities using classical biochemical procedures. Therefore, the only alternative is the cloning and expression of these peptidases. In addition, recombinant techniques allow directed structural alterations in order to program mechanistic or functional features. Peptidases can be expressed in most of the developed expression systems (yeast, viral, bacterial, insect cells and mammalian). It is not usually easy to predict which expression system is the method of choice. For functional expression of recombinant peptidases various examples have been presented[371. 

This screening for the best substrate is probably the easiest method for a chemist of improving the bio transformation. Nevertheless, if expertise in the handling of microorganisms or if a microbiological facility is available, then the best strain for a certain reaction should be found by screening many different microorganisms or, alternatively, a strain can be improved by classical means, such as mutation, or by more modern recombination techniques. 

The production hosts are often further improved by either classical mutation or recombinant techniques to allow ease of production. The primary target for strain improvement is the removal of unwanted enzyme activities, such as extra cellular protease and amylase to improve product purity and stability. 

A recently introduced technique that also targets the phenotype is whole genome shuffling. This latter approach involves the amplification of the genetic diversity within a population through genetic recombination [19, 20]. Next, similar to the traditional procedure, the newly created library is screened for mutants with the desired properties. Its main advantage is that improvement is much faster than is possible with classical mutation and selection as described above. 

The application of recombinant DNA techniques to the study of neuropeptide biosynthesis is essential because these techniques facilitate structural analyses and evolutionary studies, clarify biosynthetic pathways, provide the necessary background and probes for analysis of synthesis regulation at the levels of mRNA and gene transcription and assist in classical genetic experimentation. Examples of these benefits are cited and problems encountered in insect systems are discussed. 

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