Dear all,
I am having trouble understanding the following statement as I understand both to be interchangable. I would appreciate it if someone could clarify as to what they actually mean.
"The general lack of yeast plasmids that are maintained
at very high copy number has led to the development of 2l-based vectors carrying selection markers such as LEU2-d and URA3-d (Beggs, 1978; Erhart & Hollenberg, 1983; Loison et al., 1989). The defective promoters on the markers result in an increase in copy number; hundreds of copies have been reported in selective medium, although these high copy numbers are not required for viability (Lopes et al., 1991). Such vectors are generally more useful for the overexpression of a product gene than for metabolic engineering applications. However, Ro et al. (2008) demonstrated the successful use of a plasmid carrying the LEU2-d marker (and three pathway genes) for the synthesis of artemisinic acid in nonselective, complex medium."
Any help is much appreciated, thank you! :)
For metabolic engineering applications it is desirable that your expression vectors contain a set (or at least one) working marker gene cassette. The described plasmids have inactivated marker promoter sequences. This leads to a drastic increase in copy number which is fine if you have just one gene that produces your desired protein. But if you want to construct ('engineer') a strain that contains not one but more genes necessary for product formation the applications of these plasmids are quite limited.
Cheers, Christian
Metabolic engineering does not only concern overexpression. In general engineered bacteria/yeasts had their genome (not plasmids) edited. Probably that plasmid cannot be used to integrate sequences in the genome (or remove them) for instance through double crossing over.
If your target product is just a protein, the high copy number plasmid is suitable since you need to overexpress only one gene in the plasmid. However, in many cases of metabolic engineering applications, especially your target is a metabolite, more than one gene is necessary to overexpress because many genes involved in the biosynthesis of the metabolite. Therefore, more than one plasmid is needed. If you use the high copy number plasmid in that case, the other plasmid will be segregated. (The copy number of one cell is limited.) In addition, the high copy number plasmid would lead to metabolic burden. Overall, metabolic balance is more important in the metabolic engineering applications which are many enzymes involved in biosynthesis of your target product.
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