Alimjan Idiris

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well‐characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high‐level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein‐expressing host systems are still required for the production of heterologous proteins involved in post‐translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission‐yeast strains.

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe.

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host‐specific proteases. As an initial solution to this problem, we constructed a protease‐deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine‐ and/or cysteine‐type proteases in the culture medium. These findings also demonstrated that construction of a protease‐deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required. Copyright

The gap‐filling sequence on the left arm of chromosome 2 in fission yeast Schizosaccharomyces pombe

We report a gap‐filling sequence between SPBPB21E7.09 (in contig c1348) and SPBPB10D8.01 (in contig pB10D8) on the left arm of chromosome 2 in the fission yeast, Schizosaccharomyces pombe. The sequence was determined from a BAC clone overlapping SPBPB21E7.01c (eno102) (in contig c1348) and SPBC1683.07 (mal1) (in contig pB10D8). The gap‐filling sequence is 17 881 bp in length and contains five putative open reading frames, which were systematically named as SPBC460.01c, SPBC460.02c, SPBC460.03, SPBC460.04c and SPBC460.05. Their deduced amino acid sequences respectively include protein motifs corresponding to amino acid permease, glutathione S‐transferase C‐terminal domain, taurine catabolism dioxygenase TauD TfdA family and major facilitator superfamily, whereas their functions are unknown. The sequence has been submitted to the international DNA database (DDBJ/EMBL/GenBank) under Accession No. AB325691. Copyright

Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells

Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications.

Minimum Genome Factories in Schizosaccharomyces pombe

This chapter gives an overview of the “minimum genome factory” (MGF) of the fission yeast Schizosaccharomyces pombe (S. pombe). The S. pombe genome is one of the smallest found in free-living eukaryotes. We engineered a reduction in the number of S. pombe genes using a large-scale gene deletion method called the LATOUR method. This method enabled us to identify the minimum gene set required for growth under laboratory conditions. The genome-reduced strain has four deleted regions: 168.4 kb of the left arm of chromosome I; 155.4 kb of the right arm of chromosome I; 211.7 kb of the left arm of chromosome II; and 121.6 kb of the right arm of chromosome II. These changes represent a loss of 223 genes of an estimated 5,100. The 657.3-kb deletion strain was less efficient at taking up glucose and some amino acids from the growth media than the parental strain. This strain also showed increased gene expression of the mating pheromone M-factor precursor and NADP-specific glutamate dehydrogenase. There was also a 2.7-fold increase in the concentration of cellular ATP, whereas levels of heterologously produced proteins, such as the green fluorescent protein and the secreted human growth hormone, increased by 1.7 fold and 1.8 fold, respectively.