Chen, Yaping; (1994) Improving the productivity of yeast as a host for synthesis of secreted products. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The low secretory capacity of the yeast Saccharomyces cerevisiae limits its productivity for synthesis of secreted products. Mutagenesis is widely adopted to increase the secretory efficiency of this host and to provide "supersecretor" strains. This study was based upon the chance observation (made by Zeneca Pharmaceuticals) that S. cerevisiae cells engineered to secrete elafin also produced extracellular ubiquitin. Since there was no ubiquitin secretion by cells prior to the introduction of the elafin gene, there was an apparent association between this ubiquitin secretion and elafin production. Congenic and isogenic S. cerevisiae strains were therefore employed in order to investigate if cellular ubiquitin levels might influence elafin secretion. Loss of the stress-inducible polyubiquitin gene (UBI4) reduced elafin secretion 3 to 4-fold. Conversely, overexpression of ubiquitin by galactose induction of an integrated UBI4 gene under GAL1 promoter control, enhanced elafin secretion 7 to 10-fold and caused appreciable association of free ubiquitin with the yeast periplasm. However production of a-factor and certain other natural yeast proteins is unaffected in ubiquitin overexpressing strains, indicating that ubiquitin overexpression can only be used to enhance the secretion of certain proteins. This is the first study to show that the secretion of a heterologous protein can be influenced by ubiquitin. In parallel studies, the physiological effects of ubiquitin overexpression were examined. Ubiquitin has been shown to have a chaotropic action on proteins, which may be why it is not normally in excess in cells. However overexpressing ubiquitin did not have detrimental effects on growth and even increased certain stress tolerances (e.g. tolerance of aminoacid analogues). Besides the elafin expression studies, work was also conducted on intracellular expression of the native alcohol dehydrogenase of yeast. Plasmids for moderate and high overexpression of this tetrameric enzyme were constructed and inserted into yeast in an attempt to identify any problems that may be associated with very high expression (to 40–50% total cell protein) of a multimeric, NAD-requiring dehydrogenase.

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