scholarly journals Harnessing the yeast Saccharomyces cerevisiae for the production of fungal secondary metabolites

2021 ◽  
Author(s):  
Guokun Wang ◽  
Douglas B. Kell ◽  
Irina Borodina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secondary Metabolites ◽  
High Throughput Screening ◽  
Large Scale ◽  
Strain Improvement ◽  
Cell Factory ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Factories ◽  
Genetically Engineer ◽  
Fungal Secondary Metabolites

Abstract Fungal secondary metabolites (FSMs) represent a remarkable array of bioactive compounds, with potential applications as pharmaceuticals, nutraceuticals, and agrochemicals. However, these molecules are typically produced only in limited amounts by their native hosts. The native organisms may also be difficult to cultivate and genetically engineer, and some can produce undesirable toxic side-products. Alternatively, recombinant production of fungal bioactives can be engineered into industrial cell factories, such as aspergilli or yeasts, which are well amenable for large-scale manufacturing in submerged fermentations. In this review, we summarize the development of baker’s yeast Saccharomyces cerevisiae to produce compounds derived from filamentous fungi and mushrooms. These compounds mainly include polyketides, terpenoids, and amino acid derivatives. We also describe how native biosynthetic pathways can be combined or expanded to produce novel derivatives and new-to-nature compounds. We describe some new approaches for cell factory engineering, such as genome-scale engineering, biosensor-based high-throughput screening, and machine learning, and how these tools have been applied for S. cerevisiae strain improvement. Finally, we prospect the challenges and solutions in further development of yeast cell factories to more efficiently produce FSMs.

scholarly journals The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

2019 ◽  
Vol 104 (2) ◽  
pp. 675-686 ◽  
Author(s):  
Saowalak Changko ◽  
Priscilla D. Rajakumar ◽  
Rosanna E. B. Young ◽  
Saul Purton
Keyword(s):  
Oral Delivery ◽  
Large Scale ◽  
Technical Problem ◽  
Crop Protection ◽  
Strain Improvement ◽  
Viral Pathogen ◽  
Algal Biotechnology ◽  
Bacterial Gene ◽  
Cell Factories ◽  
Phosphite Oxidoreductase

AbstractEdible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during set-up and implementation of ‘crop-protection’ strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bio-available form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the ‘retro-fitting’ of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

scholarly journals Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae

2019 ◽  
Vol 116 (39) ◽  
pp. 19415-19420 ◽  
Author(s):  
Behrooz Darbani ◽  
Vratislav Stovicek ◽  
Steven Axel van der Hoek ◽  
Irina Borodina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dicarboxylic Acid ◽  
Acid Production ◽  
Dicarboxylic Acids ◽  
Scale Production ◽  
Protein Motif ◽  
Yeast Saccharomyces Cerevisiae ◽  
Organic Acid Production ◽  
Cell Factories ◽  
Voltage Dependent

Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion–sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.

scholarly journals Leveraging budding yeast Saccharomyces cerevisiae for discovering aging modulation substances for functional food

2019 ◽  
Vol 9 (5) ◽  
pp. 297
Author(s):  
Shaoyu Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Bioactive Compounds ◽  
Functional Food ◽  
Budding Yeast ◽  
Model Organism ◽  
Cellular Systems ◽  
Cell Factory ◽  
Compression Of Morbidity ◽  
Bioactive Substances ◽  
Yeast Saccharomyces Cerevisiae

Background: Discovery of bioactive substances contained in functional food and the mechanism of their aging modulation are imperative steps in developing better, potent and safer functional food for promoting health and compression of morbidity in the aging population.  Budding yeast (Saccharomyces cerevisiae) is invaluable model organism for aging modulation and bioactive compounds discovery. In this paper we have conceptualised a framework for achieving such aim. This framework consists of four components: discovering targets for aging modulation, discovering and validating caloric restriction mimetics, acting as cellular systems for screening natural products or compounds for aging modulation and being a biological factory for producing bioactive compounds according to the roles the yeast systems play. It have been argued that the component of being a biological factory for producing bioactive compounds has much underexplored which also present an opportunity for new active substance discovery and validation for health promotion in functional food industry.Keywords: Aging modulation, budding yeast, functional food, bioactive substances, cell factory

scholarly journals Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

2021 ◽  
Author(s):  
Eline Postma ◽  
Else-Jasmijn Hassing ◽  
Venda Mangkusaputra ◽  
Jordi Geelhoed ◽  
Pilar de la Torre ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Metabolic Networks ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Data-driven rational biosynthesis design: from molecules to cell factories

2019 ◽  
Vol 21 (4) ◽  
pp. 1238-1248
Author(s):  
Fu Chen ◽  
Le Yuan ◽  
Shaozhen Ding ◽  
Yu Tian ◽  
Qian-Nan Hu
Keyword(s):  
Large Scale ◽  
Protein Domain ◽  
Data Driven ◽  
Cell Factory ◽  
Dna Assembly ◽  
Metabolic Reaction ◽  
Cell Factories ◽  
Metabolic Systems ◽  
One Stop ◽  
Target Molecules

Abstract A proliferation of chemical, reaction and enzyme databases, new computational methods and software tools for data-driven rational biosynthesis design have emerged in recent years. With the coming of the era of big data, particularly in the bio-medical field, data-driven rational biosynthesis design could potentially be useful to construct target-oriented chassis organisms. Engineering the complicated metabolic systems of chassis organisms to biosynthesize target molecules from inexpensive biomass is the main goal of cell factory design. The process of data-driven cell factory design could be divided into several parts: (1) target molecule selection; (2) metabolic reaction and pathway design; (3) prediction of novel enzymes based on protein domain and structure transformation of biosynthetic reactions; (4) construction of large-scale DNA for metabolic pathways; and (5) DNA assembly methods and visualization tools. The construction of a one-stop cell factory system could achieve automated design from the molecule level to the chassis level. In this article, we outline data-driven rational biosynthesis design steps and provide an overview of related tools in individual steps.

scholarly journals Proteomic analysis of Saccharomyces cerevisiae grown on glucose or glycerol

2021 ◽  
Author(s):  
◽  
Hannah D. Hoang
Keyword(s):  
Mass Spectrometry ◽  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Triosephosphate Isomerase ◽  
Lower Amount ◽  
Protein Abundance ◽  
Two Dimensional ◽  
Yeast Saccharomyces Cerevisiae ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis

<p>The goal of this research was to use two-dimensional electrophoresis to examine changes in abundance of enzymes of the glycolytic pathway in the yeast Saccharomyces cerevisiae grown on carbon sources that support either fermentation to ethanol or oxidative metabolism. Large-scale profiling of protein abundances (expression proteomics) often detects changes in protein abundance between physiological states. Such changes in enzyme abundance are often interpreted as evidence of metabolic change although most textbooks emphasise control of enzyme activities not enzyme amount. Two-dimensional difference gel electrophoresis (2DDIGE) was therefore used to examine differences in protein abundance between S. cerevisiae strain BY4741 grown on either glucose (fermentation) or glycerol. Growth on 2% glucose, but not on glycerol, was accompanied by extensive production of ethanol. Doubling times for growth were 2 h 5 min in glucose and 9 h 41 min in glycerol. Conditions for extraction and two-dimensional electrophoresis of proteins were established. One hundred and seventy nine proteins were identified by MALDI mass spectrometry of tryptic digests of protein spots excised from Coomassie stained gels. All of the enzymes for conversion of glucose to ethanol, except for the second enzyme of glycolysis phosphoglucose isomerase, were identified using twodimensional electrophoresis of 100 μg of protein from cells grown on 2% glucose. Identification of proteins excised from the DIGE gels was more challenging, partly because of the lower amount of protein. Eight of the proteins that showed statistically significant differences in abundance (≥ 2-fold, p ≤ 0.01) between glucose and glycerol were identified by mass spectrometry of proteins excised from the 2DDIGE gels, and a further 18 varying proteins were matched to proteins identified from the Coomassie stained gels. Of these total 26 identified or matched proteins, subunits of five of the enzymes for conversion of glucose to ethanol were more abundant from the fermentative cells grown on glucose. The more abundant glycolytic enzymes were phosphofructokinase 2, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase and enolase, plus pyruvate decarboxylase that was required for conversion of the glycolytic product pyruvate to acetaldehyde. The alcohol dehydrogenases Adh1 and Adh4 that convert acetaldehyde to ethanol were detected but did not vary significantly between growth on glucose or glycerol. The results confirmed that in this case changes in abundance of some enzymes were consistent with the altered metabolic output. Future studies should examine whether changes in the abundance and activity of these enzymes are responsible for the differences in metabolism.</p>

scholarly journals Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics

2021 ◽  
Author(s):  
Balázs Kakuk ◽  
Roland Wirth ◽  
Gergely Maróti ◽  
Szuhaj Márk ◽  
Gábor Rakhely ◽  
...  
Keyword(s):  
Microbial Community ◽  
Large Scale ◽  
Methanogenic Archaea ◽  
Redox Balance ◽  
Early Response ◽  
Limiting Factor ◽  
Cell Factory ◽  
Microbial Composition ◽  
Cell Factories ◽  
Power To Gas

Abstract Background. The detailed molecular machinery of the complex microbiological cell factory of biogas/biomethane production is not fully understood. One of the main puzzling process control elements is the formation, consumption and regulatory role of hydrogen (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly important in the Power-to-Gas renewable energy conversion and storage technologies. Results. The early response of the mixed mesophilic microbial community to H2 gas injection was investigated with the goal of uncovering the first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min H2 injection by excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition and taxonomic abundance of the biogas producing anaerobic community did not change appreciably two hours after the H2 treatment, indicating that this time period was too short to display differences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. The importance of syntrophic cross-kingdom interactions in H2 metabolism and the effects on the related Power-to-Gas process are discussed. Conclusion s. External H2 regulates the functional activity of certain Bacteria and Archaea. Mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-off and turn-on of the Power-to-Gas microbial cell factories.

scholarly journals INHIBITION OF THE GROWTH OF TOLERANT YEAST Saccharomyces cerevisiae STRAIN I136 BY A MIXTURE OF SYNTHETIC INHIBITORS

2017 ◽  
Vol 18 (1) ◽  
pp. 17
Author(s):  
Eny Ida Riyanti ◽  
Edy Listanto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Carbon Sources ◽  
Resistance Mechanism ◽  
Lag Phase ◽  
Cell Factory ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fermentation Products ◽  
Hydroxymethyl Furfural ◽  
Cell Biomass

<p>Biomass from lignocellulosic wastes is a potential source for biobased products.  However, one of the constraints in utilization of biomass hydrolysate is the presence of inhibitors. Therefore, the use of inhibitor-tolerant microorganisms in the fermentation is required. The study aimed to investigate the effect of a mixture of inhibitors on the growth of Saccharomyces cerevisiae strain I136 grown in medium containing synthetic inhibitors (acetic acid, formic acid, furfural, 5-hydroxymethyl furfural/5-HMF, and levulinic acid) in four different concentrations with a mixture of carbon sources, glucose  (50 g.l-1) and xylose (50 g.l-1) at 30oC. The parameters related to growth and fermentation products were observed. Results showed that the strain was able to grow in media containing natural inhibitors (BSL medium) with µmax of 0.020/h. Higher level of synthetic inhibitors prolonged the lag phase, decreased the cell biomass and ethanol production, and specific growth rate. The strain could detoxify furfural and 5-HMF and produced the highest ethanol (Y(p/s) of 0.32 g.g-1) when grown in BSL. Glucose was utilized as its level decreased in a result of increase in cell biomass, in contrast to xylose which was not consumed. The highest cell biomass was produced in YNB with Y (x/s) value of 0.25 g.g-1. The strain produced acetic acid as a dominant side product and could convert furfural into a less toxic compound, hydroxyl furfural. This robust tolerant strain provides basic information on resistance mechanism and would be useful for bio-based cell factory using lignocellulosic materials. </p>

scholarly journals Analysis of lipid composition reveals mechanisms of ethanol tolerance in the model yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
M Lairón-Peris ◽  
S. J. Routledge ◽  
J. A. Linney ◽  
J Alonso-del-Real ◽  
C.M. Spickett ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Responses ◽  
Ethanol Tolerance ◽  
Yeast Species ◽  
Culture Media ◽  
Sugar Content ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Tolerant Strain ◽  
Cell Factories

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyse five representatives with varying ethanol tolerances. The most tolerant strain, AJ4, was dominant in co-culture at 0% and 10% ethanol. Unexpectedly, although it does not have the highest NIC or MIC, MY29 was the dominant strain in co-culture at 6% ethanol, which may be linked to differences in its basal lipidome. Whilst relatively few lipidomic differences were observed between strains, a significantly higher PE concentration was observed in the least tolerant strain, MY26, at 0% and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggests a limited set of membrane compositions that diverse yeast species use to achieve this. Importance Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and non-resistant isolates for future applications.

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