Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes andinvolve the interplay of a multitude of proteins. A unique collection of thermosensitivesecretory mutants allowed scientists to demonstrate that the secretory pathway of theyeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. Allproteins commence their journey in the endoplasmic reticulum, where they undergoamino-linked core glycosyl modification. After passage through the Golgi apparatus,where the remodelling of the glycosyl chains is completed, proteins are transported totheir final destinations, which are either the cell surface, periplasmic space or thevacuole.Proteins destined for secretion are usually synthesised with a transientamino-terminal secretion leader of varying length and hydrophobicity, which plays acrucial role in the targeting and translocation of their protein cargo. Considerableeffort has been made to elucidate the molecular mechanisms involved in theseprocesses, especially due to their relevance in a rapidly expanding biotech industry.The advantages of S. cerevisiae as a host for the expression of recombinantproteins are well documented. Unfortunately, S. cerevisiae is also subject to anumber of drawbacks, with a relative low product yield being one of the majordisadvantages.Bearing this in mind, different secretion leaders were compared with the aim ofimproving the secretion of the LKA 1 and LKA2 a-amylase enzymes from theS. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known forits ability to degrade raw starch and an improved secretion of its amylase enzymesfrom S. cerevisiae paves the way for a potential one-step starch utilisation process.Three sets of constructs were prepared containing the LKA 1 and LKA2 genesseparately under secretory direction of either their native secretion leader, theS. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). Theinclusion of a spacer peptide in the latter set of constructs ensured improved Kex2pproteolytic processing of the leader/protein fusion. Strains expressing the amylasegenes under their native secretion leaders resulted in the highest saccharolyticactivity in the culture medium. In contrast to this, strains utilising the syntheticsecretion leader produced the highest fermentation yield, but had a lower thanexpected extracellular activity. We hypothesise that the native amylase leaders mayfunction as intramolecular chaperones in the folding and processing of theirpassenger proteins, thereby increasing processing efficiency and concomitantenzyme activity.

[发布机构] Stellenbosch University