5.1.2. Molecular techniques

Recent molecular technological advances in mutagenesis and gene transfer methods expanded considerably the range of microorganisms into which DNA from unrelated organisms can be introduced. The genus barrier and the kingdom barrier are no longer insurmountable obstacles.

Current methods used in bacteria allow the inserted gene recombinant constructs to be integrated at specific sites into the chromosome or the plasmids. Nevertheless, the procedures for gene modification should be subjected to safety considerations. The following important features are a matter of discussion: peculiarities of the host microorganism in respect to inserted gene(s), characteristics of the vector and construct; DNA transfer methods.

When bacteria are used as host microorganisms a procedure for safety assessment requires a history for safe consumption of this microorganism either as a food or as a food component. If this evidence lacks, the safety of the host must be established. For the eukaryiotic host the same safety considerations are applied.

The inserted gene(s) could be taken from the same microbial species or from evolutionary more distant organism. The inserted gene products should have a history of safety use in food or its safety should be substantiated. The shorter the inserted DNA fragment, the more reduced/facilitated the procedure for evaluation of food safety would be.

As regards the characteristics of the vector and the construct - if the vector used is a part of the genome of GMM the whole DNA sequence should be characterized including; replicons, promoters, selective markers, linkers as well as any other parts of DNA. The vector must contain nucleotide sequence from microorganisms with history of safe use in food. The selective markers must be selected very strictly and based on safe use and the antimicrobial resistance marker must be avoided. In case of use, an application of specific methods for removal from GMM genome should be applied (e. g. sequence specific recombination). For the eukaryotes specific cloning vectors like centromeric plasmids, yeast artificial chromosome, plasmids based on killer factor determinants etc. have been designed.

Speaking about DNA transfer methods - recommendation exists for use of methods of DNA transfer of physical, chemical and biological nature, which minimize major genetic rearrangements in host genome. In case of use of integrative vectors, the nucleotide sequence of the flanking regions at the integration site of the chromosome should be characterized. This information is necessary to predict the risk of the methods used. In eukaryotes there are dependable methods for directed integration of in vitro modified or composed gene constructs into specific chromosomal sites and for deletion of genes applicable in certain species. On the basis of these methods, transgenic constructs have been made and they are highly stable during vegetative growth of cells. A possibility of recombination by mating with related strains of indigenous microflora exists. When the genetic properties of the used strains are not well known, the insufficient information about possible recombinant events makes it impossible to forecast the mechanism and integration site of the foreign genes. Thus, it is possible the methodology used in genetic manipulation of yeasts and filamentous fungi to enable integration at variable sites, which could cause appearance of different biotechnological properties and genetic stability of GMMs.