Marilyn Wiebe - Academia.edu (original) (raw)

Papers by Marilyn Wiebe

Microbial Growth Dynamics

In 1964, Rank Hovis McDougall PLC (RHM) decided to develop a microbial fermentation to convert wh... more In 1964, Rank Hovis McDougall PLC (RHM) decided to develop a microbial fermentation to convert wheat starch (a by-product of the production of wheat protein) into high-quality protein foods for human consumption. Filamentous fungi were chosen for this process because of their nutritional and organoleptic properties, and because mycelial biomass, unlike unicellular biomass, can be formulated into a convincing facsimile of fibrous foods such as meat (Figure 1) and poultry. In 1969, Fusarium graminearum (Schwabe) was isolated from soil and this strain (A 3/5) has been used for all subsequent production of mycoprotein. The fermentation process developed for biomass production involved growing A 3/5 in continuous culture at a dilution rate of c. 0.19 h-1. Using a 1.3 m3 stirred-tank reactor (STR), RHM was able to produce c. 50 t of mycoprotein per year. In 1980, after the production of a two-million-word report describing the results of a ten-year programme of toxicology studies, RHM was...

Frontiers in Immunology, Jun 9, 2023

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced wit... more Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1.

ABSTRACT 10th European Conference on Fungal Genetics. Amsterdam, The Netherlands, 29 March - 1 Ap... more ABSTRACT 10th European Conference on Fungal Genetics. Amsterdam, The Netherlands, 29 March - 1 April 2010

Conclusion By using STRs to obtain well aerated high cell densities we successfully increased pro... more Conclusion By using STRs to obtain well aerated high cell densities we successfully increased production of scopularide A by S. brevicaulis LF580 by approx. 29-fold compared to production in stationary flasks (Yu et al. 2008). We also demonstrated that scopularide A could be produced in defined medium. • It is now possible to generate 500 mg scopularide A using less than 3 l culture.

Molecular Biotechnology, Jan 23, 2014

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothe... more The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-lm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Metabolic Engineering, Jul 1, 2012

Production of chemicals and biofuels through microbial fermentation is an economical and sustaina... more Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C 22 H 46 O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l À 1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

Text: Background: Due to its natural ability to produce riboflavin (vitamin B2), Ashbya gossypii ... more Text: Background: Due to its natural ability to produce riboflavin (vitamin B2), Ashbya gossypii is a widely used microorganism in industry. Additionally, it presents several other interesting features, such as, the smallest known eukaryotic genome, lack of extensive duplication of chromosomal segments and straight forward molecular tools for genetic manipulation. Together, these make it an interesting microorganism, not only for riboflavin production, but also for other applications such as host for recombinant protein production. The enzymatic degradation of cellulose is of great importance to the carbon cycle of the biosphere, since is the most abundant biopolymer. Bioconversion of cellulose to soluble sugars is catalyzed by a group of enzymes called cellulases. Objectives:1. Evaluate the potential of A. gossypii as a host for recombinant protein production, in particular, cellulases CBHI and EGI from Trichoderma reesei. 2. Partially characterize the heterologous cellulases secreted by A. gossypii Methods:1. Expression plasmids for production of T. reesei cellulases, namely, CBHI and EGI were used to transform A. gossypii. 2. Activity on soluble substrates was determined. 3. The secreted cellulases were partially characterized. Results: Recombinant EGI was produced by A. gossypii in complex, but not in defined medium. Production was similar at 30 and 24°C. Similar amounts of enzyme activity, were produced by A. gossypii and S. cerevisiae. CBHI activity was not detectable, but the protein could be detected by Western blotting. The cellulases secreted by A. gossypii had a different glycosylation pattern than the same proteins secreted by S. cerevisiae. Conclusions: These initial results demonstrate that CBHI and EGI are secreted by recombinant A. gossypii into the extracellular medium with similar titers to those secreted by S. cerevisiae. Further optimization of the genetic constructions and culture conditions could lead to improved secretion.

Ashbya gassypti"iS a filamentous hem1ascomycete of Industrial importance In rtbollavln (vitamin 8... more Ashbya gassypti"iS a filamentous hem1ascomycete of Industrial importance In rtbollavln (vitamin 82) production. rt has the smallest known eukaryotlc genome, ladang extenslVe duphcaoon of dvomosomal segments, and genet>c manipulation is straight-foiward. Thus A. gossypd may be useful for other apphcallODS than nboflav1n production. SUrprtsingly, the A. gossyp11 secretoly pathway and post• translational events have not been subject to extenslve study. The genome of A gossypt1 has more than 90% homology and a particular pattern of synteny with Saccharomyres Cf!f'eVIW(!, which may suggest a limited secretion ablllty. However, as a filamentous fungus A. gossypll might be expected to have efficient protein secretion at the hyphal tip, as observed In other filamentous fungi. Here we report the expression of endoglucanase I (EGJ) and cellobiohydrolase I (CSHI) from the filamentous fungus Tridlodennit reese'in A. !JOSSYPllfrom plasmids c:xintalOIQ9 the 2 moaon sequence from S. ~. under the .S: a!n!Vi5iiJe PG/(] promoter. In a>mpan50n with the yeast S. ~. A. gos:sypi secreted S11111lar arrl(Ults of EGI, but less CBHI protein. The secreted recombinant EGJ was punfied for further characterisabon. The fact that A. gossypll does not secrete large amounts of protein into the culture medium represents a clear advantage for punficaboo of recombinant protein. This was further faolltatcd by the absence of any native proteins similar to the recombinant EGI. In CX>f'ltrast, punflcation of a speoflc cetlulase from a cellulase produang host svch as T. reesei can be d11T1cult becaLISe of the CXlf'ltam1natJoo of e.g. endoglucanases with c:etloblohydrolases However, several steps of protein punflcatioo were still required to purty the recombtnant enzyme from the cult1n supernatant of ..._ !JOSSYPll.

Vaccines

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The... more SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel®‘85’-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates ...

Microbial Growth Dynamics

In 1964, Rank Hovis McDougall PLC (RHM) decided to develop a microbial fermentation to convert wh... more In 1964, Rank Hovis McDougall PLC (RHM) decided to develop a microbial fermentation to convert wheat starch (a by-product of the production of wheat protein) into high-quality protein foods for human consumption. Filamentous fungi were chosen for this process because of their nutritional and organoleptic properties, and because mycelial biomass, unlike unicellular biomass, can be formulated into a convincing facsimile of fibrous foods such as meat (Figure 1) and poultry. In 1969, Fusarium graminearum (Schwabe) was isolated from soil and this strain (A 3/5) has been used for all subsequent production of mycoprotein. The fermentation process developed for biomass production involved growing A 3/5 in continuous culture at a dilution rate of c. 0.19 h-1. Using a 1.3 m3 stirred-tank reactor (STR), RHM was able to produce c. 50 t of mycoprotein per year. In 1980, after the production of a two-million-word report describing the results of a ten-year programme of toxicology studies, RHM was...

Frontiers in Immunology, Jun 9, 2023

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced wit... more Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1.

ABSTRACT 10th European Conference on Fungal Genetics. Amsterdam, The Netherlands, 29 March - 1 Ap... more ABSTRACT 10th European Conference on Fungal Genetics. Amsterdam, The Netherlands, 29 March - 1 April 2010

Conclusion By using STRs to obtain well aerated high cell densities we successfully increased pro... more Conclusion By using STRs to obtain well aerated high cell densities we successfully increased production of scopularide A by S. brevicaulis LF580 by approx. 29-fold compared to production in stationary flasks (Yu et al. 2008). We also demonstrated that scopularide A could be produced in defined medium. • It is now possible to generate 500 mg scopularide A using less than 3 l culture.

Molecular Biotechnology, Jan 23, 2014

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothe... more The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-lm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Metabolic Engineering, Jul 1, 2012

Production of chemicals and biofuels through microbial fermentation is an economical and sustaina... more Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C 22 H 46 O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l À 1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

Text: Background: Due to its natural ability to produce riboflavin (vitamin B2), Ashbya gossypii ... more Text: Background: Due to its natural ability to produce riboflavin (vitamin B2), Ashbya gossypii is a widely used microorganism in industry. Additionally, it presents several other interesting features, such as, the smallest known eukaryotic genome, lack of extensive duplication of chromosomal segments and straight forward molecular tools for genetic manipulation. Together, these make it an interesting microorganism, not only for riboflavin production, but also for other applications such as host for recombinant protein production. The enzymatic degradation of cellulose is of great importance to the carbon cycle of the biosphere, since is the most abundant biopolymer. Bioconversion of cellulose to soluble sugars is catalyzed by a group of enzymes called cellulases. Objectives:1. Evaluate the potential of A. gossypii as a host for recombinant protein production, in particular, cellulases CBHI and EGI from Trichoderma reesei. 2. Partially characterize the heterologous cellulases secreted by A. gossypii Methods:1. Expression plasmids for production of T. reesei cellulases, namely, CBHI and EGI were used to transform A. gossypii. 2. Activity on soluble substrates was determined. 3. The secreted cellulases were partially characterized. Results: Recombinant EGI was produced by A. gossypii in complex, but not in defined medium. Production was similar at 30 and 24°C. Similar amounts of enzyme activity, were produced by A. gossypii and S. cerevisiae. CBHI activity was not detectable, but the protein could be detected by Western blotting. The cellulases secreted by A. gossypii had a different glycosylation pattern than the same proteins secreted by S. cerevisiae. Conclusions: These initial results demonstrate that CBHI and EGI are secreted by recombinant A. gossypii into the extracellular medium with similar titers to those secreted by S. cerevisiae. Further optimization of the genetic constructions and culture conditions could lead to improved secretion.

Ashbya gassypti"iS a filamentous hem1ascomycete of Industrial importance In rtbollavln (vitamin 8... more Ashbya gassypti"iS a filamentous hem1ascomycete of Industrial importance In rtbollavln (vitamin 82) production. rt has the smallest known eukaryotlc genome, ladang extenslVe duphcaoon of dvomosomal segments, and genet>c manipulation is straight-foiward. Thus A. gossypd may be useful for other apphcallODS than nboflav1n production. SUrprtsingly, the A. gossyp11 secretoly pathway and post• translational events have not been subject to extenslve study. The genome of A gossypt1 has more than 90% homology and a particular pattern of synteny with Saccharomyres Cf!f'eVIW(!, which may suggest a limited secretion ablllty. However, as a filamentous fungus A. gossypll might be expected to have efficient protein secretion at the hyphal tip, as observed In other filamentous fungi. Here we report the expression of endoglucanase I (EGJ) and cellobiohydrolase I (CSHI) from the filamentous fungus Tridlodennit reese'in A. !JOSSYPllfrom plasmids c:xintalOIQ9 the 2 moaon sequence from S. ~. under the .S: a!n!Vi5iiJe PG/(] promoter. In a>mpan50n with the yeast S. ~. A. gos:sypi secreted S11111lar arrl(Ults of EGI, but less CBHI protein. The secreted recombinant EGJ was punfied for further characterisabon. The fact that A. gossypll does not secrete large amounts of protein into the culture medium represents a clear advantage for punficaboo of recombinant protein. This was further faolltatcd by the absence of any native proteins similar to the recombinant EGI. In CX>f'ltrast, punflcation of a speoflc cetlulase from a cellulase produang host svch as T. reesei can be d11T1cult becaLISe of the CXlf'ltam1natJoo of e.g. endoglucanases with c:etloblohydrolases However, several steps of protein punflcatioo were still required to purty the recombtnant enzyme from the cult1n supernatant of ..._ !JOSSYPll.

Vaccines

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The... more SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel®‘85’-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates ...