Browsing by Author "Jacob, Odwa"

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    Comparison of multi-gene integration strategies in CRISPR-based transformation of Saccharomyces cerevisiae
    (University of Western Cape, 2021) Jacob, Odwa; den Haan, Riaan
    Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.
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    Comparison of multi-gene integration strategies in CRISPR-based transformation of Saccharomyces cerevisiae
    (University of the Western Cape, 2021) Jacob, Odwa; Den Haan, R.
    Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method. We first utilized the CRISPR systems for targeting the T.r.eg2 gene to single locus chromosomal sites for single copy integration. Subsequently, we then targeted the same gene to repeated sequences in the genome, namely the delta sites, for multi-copy integration. The procedure was repeated with a different gene, T.e.cbh1, integrated into the same sites to ascertain reporter gene specific effects. High integration efficiency was achieved, since all the strains successfully integrated the genes. However, we discovered significant differences in enzyme activities between the two genes when targeted to different loci, as well as varying copy numbers as determined by qPCR. The T.e.cbh1 gene was highly expressed by yeast transformants targeted at the repeated delta sequences used for multi-copy integration, reaching maximum levels of 248 mU/gDCW. The T.r.eg2 gene was highly expressed in yeast transformants targeted to the single locus site on chromosome 12, reaching a maximum of 160U/gDCW, though it was shown that off-target integration likely occurred. We then used the information from these observations to construct a CBP yeast strain containing three cellulase genes: T.r.eg2, T.e.cbh1, and S.f.BGL1. Significant differences in enzyme activities were observed between the three genes, and it was shown that the S.f.BGL1 gene was poorly expressed by the CBP yeast strain, whereas the T.r.eg2 gene was highly expressed. Notably, due to the fact that marker containing plasmids could be cured from these strains, many additional genetic changes can still be made. Overall, our two CRISPR-Cas9 systems were efficient at engineering strains that produce recombinant proteins and can be used in future studies for a variety of applications, including metabolic engineering in S. cerevisiae
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    Crispr-based multi-gene integration strategies to create saccharomyces cerevisiae strains for consolidated bioprocessing
    (MDPI, 2022) Jacob, Odwa; van Lill, Gert Rutger; den Haan, Riaan
    Significant engineering of Saccharomyces cerevisiae is required to enable consolidated bioprocessing (CBP) of lignocellulose to ethanol. Genome modification in S. cerevisiae has been successful partly due to its efficient homology-directed DNA repair machinery, and CRISPR technology has made multi-gene editing even more accessible. Here, we tested the integration of cellulase encoding genes to various sites on the yeast genome to inform the best strategy for creating cellulolytic strains for CBP. We targeted endoglucanase (EG) or cellobiohydrolase (CBH) encoding genes to discreet chromosomal sites for single-copy integration or to the repeated delta sites for multi-copy integration. CBH1 activity was significantly higher when the gene was targeted to the delta sequences compared to single gene integration loci. EG production was comparable, though lower when the gene was targeted to a chromosome 10 site. We subsequently used the information to construct a strain containing three cellulase encoding genes. While individual cellulase activities could be assayed and cellulose conversion demonstrated, it was shown that targeting specific genes to specific loci had dramatic effects on strain efficiency. Since marker-containing plasmids could be cured from these strains, additional genetic changes can subsequently be made to optimize strains for CBP conversion of lignocellulose.