Effects of phosphoglycerate kinase overproduction in Saccheromyces cerevisiae on the physiology and plasmid stability

Abstract

In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyces cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10-15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non-selective batch cultures and continuous culture, under different growth conditions. Plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose-limited conditions. During respiro-fermentative growth, PGK-overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain. The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK-overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium

“…A limitation in the availability of essential transcription factors for both the PGK1 and ADH2 promoters may therefore influence the expression of the glycolytic enzymes and thereby affect the growth of the host organism. The disproportionally large effect of foreign gene expression may also be attributed to a competition for limiting amounts of translation factors, biosynthetic precursors, or metabolic energy (Janes et al, 1990;Shuster, 1989;Van der Aar et al, 1992).…”

“…The effect of cloned gene expression has been associated with either the energetic cost of extra protein synthesis (Bailey, 1993;Gopal et al, 1989;Snoep et al, 1995) or the competitive effect of extra protein synthesis (Gopal et al, 1989;Shuster, 1989;Snoep et al, 1995;Van der Aar et al, 1992). The "dilution" of native proteins (i.e., the reduction of the activity of native proteins by recombinant gene expression) was also proposed as a major mechanism causing a decrease in the flux through glycolysis and a decrease in the maximum specific growth rate of the host organism (Snoep et al, 1995;Van Hoek et al, 1998).…”

Super life – how and why ‘cell selection’ leads to the fastest‐growing eukaryote

“…It has been shown, however, that the control of the glycolytic enzymes on the glycolytic flux is rather small in this yeast. [15][16][17][18][19]. There is substantial, but incomplete, evidence for a high control of the glucose-uptake step on the yeast glycolysis [16,[19][20][21].…”

Studies on growth kinetics and plasmid stability of a recombinant Escherichia coli expressing a Schistosoma mansoni antigen

“…Type of promoter, if inducible, inducer used, presence of signal sequence for protein exportation/secretion, selective marker are also important aspects to be considered regarding the vector selection. The ef®cient gene expression depends also on the plasmid copy number and stability (Van Der Aar et al, 1992). pMcri-A157 is a high copy number plasmid with a strong T7 promoter inducible by IPTG, and no signal sequence for exportation/secretion.…”

“…XA, represents the number of colonies that lost the plasmid (detected onto the LB-agar without ampicillin together with the plasmid containing colonies). The rate of segregation was de®ned by the phenotype (ampR) still present after n generations and was calculated by the equation (Van Der Aar et al, 1992):…”