Browsing by Author "Kroukamp, Heinrich"

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    Improving the protein secretion capacity of Saccharomyces cerevisiae with strain engineering
    (Stellenbosch : Stellenbosch University, 2015-03) Kroukamp, Heinrich; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is frequently chosen for the production of industrial and pharmaceutical proteins, due to its rapid growth, microbial safety, eukaryotic post-translational processing and high-density fermentation capability. With the development of recombinant DNA technologies and efficient expression systems, this yeast also gained a prominent new role as a protein production host, due to its ease of genetic manipulation. Improving the production and secretion of recombinant proteins, whether for pharmaceutical, agricultural or industrial application, has the benefit of reducing the production costs and promoting accessibility to these technologies. This also holds true for the second generation biofuel production, where high levels of hydrolytic enzymes are required to break down the complex carbohydrates in lignocellulose. While high copy number expression vector systems, strong promoters and efficient secretion signals resulted in significant enhancement of protein production yields, these strategies are often limited by bottlenecks in the yeast secretion pathway. Although protein characteristics and host restrictions are likely to contribute to these bottlenecks, these factors are still poorly understood. Nonetheless, strain engineering approaches have shown great potential as a means to relieve these protein secretion bottlenecks and advancing recombinant protein production to new levels. In this study, the potential of strain engineering, with regards to enhancing cellulase secretion for second generation bioethanol production, was evaluated. Further work involved the identification of novel genetic elements enhancing protein secretion and the elucidation of possible mechanisms involved in high cellulase secretion by S. cerevisiae. When the native PSEI gene was expressed under the transcriptional control of the PGK1 promoter in S. cerevisiae, the secreted yields of recombinantly produced Neocallimastix patriciarum Cel6A, Trichoderma reesei Cel7B and Saccharomycopsis fibuligera Cel3A were increased 1.15, 1.25 and 3.70 fold, respectively. The overexpression of SOD1 did not increase any of the above mentioned cellulases. When SOD1 was overexpressed in combination with PSE1, a synergistic enhancement in secreted Cel3A was obtained. To our knowledge, this is the first reported case where SOD1 overexpression in S. cerevisiae resulted in higher heterologous protein secretion. The effect of disrupting protein N-glycosylation elongation at various steps, on the secretion of heterologously produced Neosartorya fischeri Cel12A (lacking N-glycosylation sites) and the S. fibuligera Cel3A, was investigated. The deletion of the MNN2 gene was shown to increase the extracellular Cel12A by 1.30 fold, while the deletion of MNN11 resulted in a 1.26 fold increase in extracellular Cel3A. These results implicate the cell wall as a possible barrier to protein secretion. It was also found that Cel3A with shorter N-glycosylation chains had reduced cell wall retention, compared to enzymes resembling the native glycosylation pattern. The removal of the PMR1 gene product (predicted to result in a general decrease in Golgi mannosyltranferase activities) was the only modification which enhanced the cell specific activities of both reporter cellulases, although this mutant's poor growth makes it an unlikely candidate for industrial application. The S. cerevisiae M0341 strain that secretes high levels of recombinant Talaromyces emersonii Cel7A, was mated to the Y294 control strain to produce the H3 hybrid strain. Several high secreting progeny were selected after sporulating the H3 strain. Through genome shuffling and single nucleotide polymorphism (SNP) analysis of pooled segregants, five genomic regions of the M0341 strain were identified that contain putative alleles that are beneficial to Cel7A secretion. Identifying these alleles proved problematic due to strain instability. When the T. emersonii CEL7A, N. fischeri CEL12A and S. fibuligera CEL3A were expressed in selected H3 progeny on episomal plasmids, these strains had up to ~3.5 fold increased Cel7A secretion compared to the M0341 parental strain, but no increase were observed for the other cellulase. It was also shown that cell flocculation could improve secretion. The strains constructed in this study represent a step toward efficient cellulase secreting yeasts for second generation biofuel production and presents a novel strategy to identify secretion enhancing elements for the protein production industry.