Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry

The plasmid instability of genetically modified microorganisms during prolo nged bioreactor operations is one of the major problems to be overcome in t he production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearin g cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encode s the enzyme exo-beta -glucanase, were used to determine the P+ fraction. O ther parameters such as viability, cellular protein, cell size and structur e were also monitored using flow cytometry. This technique has two main adv antages over the conventional method of determining the P+ fraction (platin g in selective and nonselective solid media): (a) it takes a very short per iod of time to obtain a measurement that provides multiple parametric infor mation; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous o peration (432 h) with the recombinant strain in a CSTR was carried out to t est the application of this technique. Measurements of cellular exo-beta -g lucanase activity and cellular protein content closely correlates to the me asured fraction of plasmid-containing cells in the population. Moreover, th e standard deviation of the fraction of P+ cells determined using this tech nique was very low (about 2%). Recombinant protein production also increase d the size of the yeast cells, whereas the recombinant cells also had a mor e complex internal structure than the non-recombinant host strain.