Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry.
The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the 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-bearing 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 encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.
Lú Chau
T
T
Department of Chemical Engineering. Institute of Technology, Alfonso X el sabio. 27002-Lugo, Spain.
Guillán
A
A
Roca
E
E
Núñez
M J
MJ
Lema
J M
JM
D019149:Bioreactors; D048429:Cell Size / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D005285:Fermentation; D005434:Flow Cytometry / Q000592:standards*; D017930:Genes, Reporter; D043326:Glucan 1,3-beta-Glucosidase; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase