Engineering and Fermenter Optimization of Fungi GLA Productions in Pichia Pastoris GS115 Using Oil Waste

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in both levels in human and animal feed. GLA is used in many nutritional and medicinal applications such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the main dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, which is one of the important enzymes in the GLA production pathway was isolated from Mucor rouxii DSM1194 and expressed in Pichia pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in fermenter by oil waste along 5 days and results detected 6.3 g / l lipid and 103 mg/g GLA was produced in 72 hours. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.

Molecular evolution and functional characterisation of tunicate xenobiotic receptors

<p>Marine microorganisms generate a wide range of ’bioactive’ compounds that can have far-reaching effects on biological and ecological processes. Metazoans have developed specialised biochemical pathways that metabolise and eliminate potentially toxic chemicals (xenobiotics) from their bodies. The vertebrate xenobiotic receptor, pregnane X receptor (PXR), is a ligand-activated nuclear receptor transcription factor regulating expression of multiple detoxification genes. Ligand-binding domains (LBDs) of vertebrate PXR orthologues may have adaptively evolved to bind toxins typically encountered by these organisms. Marine invertebrate filter-feeders are exposed to relatively high concentrations of xenobiotics associated with their diet. Tunicates (phylum: Chordata) are of particular interest as they form the sister clade to the Vertebrata. Genomes of the solitary tunicate Ciona intestinalis and the colonial tunicate Botryllus schlosseri both encode at least two xenobiotic receptors that are orthologues to both the vertebrate vitamin D receptor (VDR) and PXR. Pursuing the idea that tunicate xenobiotic receptors (VDR/PXR) may adaptively evolve to bind toxic chemicals commonly present in an organism’s environment, this thesis aims to identify if: (i) adaptive evolution is acting on putative tunicate VDR/PXR orthologues to enhance binding of dietary xenobiotics; (ii) these receptors are activated by dietary xenobiotics (e.g. microalgal biotoxins) and; (iii) tunicate VDR/PXR LBDs can be used as sensor elements in yeast bioassays for the detection of both natural and synthetic bioactive compounds. To identify genetic variation and to search for evidence of positive selection, next-generation sequencing was performed on three tunicate VDR/PXR orthologues genes. Recombinant yeast (Saccharomyces cerevisiae) cell lines were developed for the functional characterisation of tunicate VDR/PXR LBDs. These tunicate VDR/PXR LBD-based yeast bioassays were utilised to detect known microalgal biotoxins, natural bioactive compounds, and environmental contaminants. Next-generation sequencing revealed both an unusually high genetic diversity and strong purifying selection in VDR/PXR orthologues from C. intestinalis and B. schlosseri. Single-base-deletion allelic variants were found in C. intestinalis VDR/PXR orthologues resulting in predicted proteins having a DNA-binding domain but lacking a LBD. The persistence of these variants may reflect constitutive expression of detoxification genes as a selective advantage in the marine environment. To assess the functional characteristics of tunicate VDR/PXR orthologues, recombinant yeast cell lines were developed that express VDR/PXRα LBDs from C. intestinalis and B. schlosseri. These chimeric proteins mediate liganddependent expression of a lacZ reporter gene which encodes an easily assayed enzyme (β-galactosidase). These yeast bioassays were highly sensitive towards both synthetic and natural toxins (coefficients of variance, CV <25%). Microalgal biotoxins (okadaic acid and portimine) were two orders of magnitude more potent than synthetic chemicals, which was consistent with the hypothesis that tunicate xenobiotic receptors can bind marine bioactive compounds frequently present in a filter-feeder’s diet. Following these functional studies, the yeast bioassays were tested in a more applied context by screening the following compounds: (i) natural bioactive compounds that represent promising compounds for drug development and; (ii) synthetic chemicals that are common environmental pollutants. Of the 34 compounds tested, 30 were active in the tunicate yeast bioassays. The yeast bioassays were particularly sensitive towards a small number (n = 11) of marine and terrestrial bioactive compounds (CJ-13-014, CJ-13-104, thysanone and naringin) and emerging contaminants such as pharmaceuticals (ketoconazole), antifungals (radicicol), preservatives (butylated hydroxtoluene) and surfactants (oil dispersants), generating CV values <25%. Activities of the remaining 19 compounds were highly variable and appeared to depend on several factors, such as solvent used, duration of exposure and type of recombinant protein expressed (e.g. C. intestinalis versus B. schlosseri VDR/PXRα). In conclusion, the yeast bioassay developed in this thesis, with further development, may provide a template for novel bioassays that may find application in routine microalgal biotoxin testing and environmental monitoring. These bioassays may also assist in the identification of marine bioactive compounds as drug lead compounds.</p>

Molecular evolution and functional characterisation of tunicate xenobiotic receptors

<p>Marine microorganisms generate a wide range of ’bioactive’ compounds that can have far-reaching effects on biological and ecological processes. Metazoans have developed specialised biochemical pathways that metabolise and eliminate potentially toxic chemicals (xenobiotics) from their bodies. The vertebrate xenobiotic receptor, pregnane X receptor (PXR), is a ligand-activated nuclear receptor transcription factor regulating expression of multiple detoxification genes. Ligand-binding domains (LBDs) of vertebrate PXR orthologues may have adaptively evolved to bind toxins typically encountered by these organisms. Marine invertebrate filter-feeders are exposed to relatively high concentrations of xenobiotics associated with their diet. Tunicates (phylum: Chordata) are of particular interest as they form the sister clade to the Vertebrata. Genomes of the solitary tunicate Ciona intestinalis and the colonial tunicate Botryllus schlosseri both encode at least two xenobiotic receptors that are orthologues to both the vertebrate vitamin D receptor (VDR) and PXR. Pursuing the idea that tunicate xenobiotic receptors (VDR/PXR) may adaptively evolve to bind toxic chemicals commonly present in an organism’s environment, this thesis aims to identify if: (i) adaptive evolution is acting on putative tunicate VDR/PXR orthologues to enhance binding of dietary xenobiotics; (ii) these receptors are activated by dietary xenobiotics (e.g. microalgal biotoxins) and; (iii) tunicate VDR/PXR LBDs can be used as sensor elements in yeast bioassays for the detection of both natural and synthetic bioactive compounds. To identify genetic variation and to search for evidence of positive selection, next-generation sequencing was performed on three tunicate VDR/PXR orthologues genes. Recombinant yeast (Saccharomyces cerevisiae) cell lines were developed for the functional characterisation of tunicate VDR/PXR LBDs. These tunicate VDR/PXR LBD-based yeast bioassays were utilised to detect known microalgal biotoxins, natural bioactive compounds, and environmental contaminants. Next-generation sequencing revealed both an unusually high genetic diversity and strong purifying selection in VDR/PXR orthologues from C. intestinalis and B. schlosseri. Single-base-deletion allelic variants were found in C. intestinalis VDR/PXR orthologues resulting in predicted proteins having a DNA-binding domain but lacking a LBD. The persistence of these variants may reflect constitutive expression of detoxification genes as a selective advantage in the marine environment. To assess the functional characteristics of tunicate VDR/PXR orthologues, recombinant yeast cell lines were developed that express VDR/PXRα LBDs from C. intestinalis and B. schlosseri. These chimeric proteins mediate liganddependent expression of a lacZ reporter gene which encodes an easily assayed enzyme (β-galactosidase). These yeast bioassays were highly sensitive towards both synthetic and natural toxins (coefficients of variance, CV <25%). Microalgal biotoxins (okadaic acid and portimine) were two orders of magnitude more potent than synthetic chemicals, which was consistent with the hypothesis that tunicate xenobiotic receptors can bind marine bioactive compounds frequently present in a filter-feeder’s diet. Following these functional studies, the yeast bioassays were tested in a more applied context by screening the following compounds: (i) natural bioactive compounds that represent promising compounds for drug development and; (ii) synthetic chemicals that are common environmental pollutants. Of the 34 compounds tested, 30 were active in the tunicate yeast bioassays. The yeast bioassays were particularly sensitive towards a small number (n = 11) of marine and terrestrial bioactive compounds (CJ-13-014, CJ-13-104, thysanone and naringin) and emerging contaminants such as pharmaceuticals (ketoconazole), antifungals (radicicol), preservatives (butylated hydroxtoluene) and surfactants (oil dispersants), generating CV values <25%. Activities of the remaining 19 compounds were highly variable and appeared to depend on several factors, such as solvent used, duration of exposure and type of recombinant protein expressed (e.g. C. intestinalis versus B. schlosseri VDR/PXRα). In conclusion, the yeast bioassay developed in this thesis, with further development, may provide a template for novel bioassays that may find application in routine microalgal biotoxin testing and environmental monitoring. These bioassays may also assist in the identification of marine bioactive compounds as drug lead compounds.</p>

Humoral and cellular immunity in mice immunized with whole recombinant yeast expressing complex NS2B/NS3 protein of dengue serotype 3

A nonpathogenic edible yeast, Saccharomyces cerevisiae, has been identified as a vehicle to express many foreign antigens which elicit the immune response in mice. The complex NS2B/NS3 is a protease that represents a prime target for rational drug design for dengue infection. During infection, the NS3 protein is the main target for CD4+ and CD8+ T cell responses, which may be protective. However, no studies have been undertaken evaluating the use of recombinant yeast Saccharomyces cerevisiae INVSc1 expressing complex NSB/NS3 protease as a protective antigen against dengue infection. In the present study, we evaluated the humoral and cellular immune response elicited by recombinant yeast compared to wild-type yeast in the mouse model. Intraperitoneal (i.p.) administration of recombinant and wild-type yeast at 1 and 25 yeast units into BALB/c mice was used. These studies demonstrated that administration at a low concentration of recombinant yeast at 1 yeast units (YU) significantly elicits antibodies against DENV NS3 antigen. Furthermore, real-time PCR analysis revealed that NS2B/NS3-specific cytocines (TNF-a, IFN-©, IL-2) increased with moderate mode compared to wild-type yeast. The results in this study show the potential of recombinant yeast as an edible vaccine platform against dengue infection.

An Integrated Approach to Produce a Recombinant Yeast Pichia Pastoris For GLA Production

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) used in many nutritional and medicinal applications such as the treatment of cancer, inflammatory disorders, and diabetes. However, GLA level of the total fatty acids in plant seeds and nuts as prominent sources of GLA is not enough to utilize on an industrial scale. The study aimed to improve the expression of delta-6 desaturase, which is one of the important enzymes in GLA production pathway. The expression vector pPICZC was selected for clone M.rouxii delta-6 desaturase. The engineered vector was first cloned into E. coli DH5α and after plasmid extraction and confirmation of sequencing was transformed by electroporation into Pichia pastoris GS115. The results indicated that the recombinant yeast strain expressed the gene in the presence of methanol 0.5%. The lipids and essential fatty acids especially GLA were extracted to confirm the expression. The results of studies of lipid and fatty acid production by Sudan black and Nile red staining, GC, and flow cytometry revealed that recombinant strain can produce GLA levels up to 19.2% of total fatty acids. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.