A phenotypic study of a unique respiratory strain of Saccharomyces cerevisiae and its application in protein production
During aerobic growth at high glucose concentrations, the yeast Saccharomyces cerevisiae prefers to catabolise glucose through fermentation rather than through respiration. Previously fully respiratory growth could only be achieved when a low external glucose concentration was maintained, in fed-batch and continuous cultures, but since the development of the respiratory strain, TM6*, in our laboratory this can now be achieved even at high external glucose concentration.
In this thesis the transferability of the respiratory phenotype has been confirmed by integration of the chimeric TM6* construct into a hxt1-hxt7 wine strain. The genomic profile of the resulting V5.TM6*P strain was investigated
by miniarray and the results showed that genes involved in the TCA cycle, glyoxylate cycle, gluconeogenesis pathway and the respiratory chain were upregulated compared to the parental strain. The transcriptional responses in the
respiratory strain were not altered when the external glucose concentration changed in contrast to the wild type strain where a change in transcription (both up and down regulated) was observed when glucose was changed from high to low concentration in a batch culture. It was further observed that the respiratory strain had a lower concentration of fructose-1, 6-bisphosphate compared to its parent strain. Modelling of regulation of glycolysis showed that there are differences in the kinetics of the phosphofructokinase between the TM6* strain and the wild type; suggesting a key role of this enzyme in the shift
between a respiro-fermentative and a respiratory metabolism.
As a result of its respiratory metabolism, the TM6* strain is able to produce more biomass, making the TM6* strain an excellent candidate as a protein production host. In this thesis a two-fold yield improvement, in overproduction
of a membrane protein, Fps1, is demonstrated, compared to two wild type strains.