A squalene–hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene–tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353–358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene–hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

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A squalene–hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol–independent anaerobic growth

10.1101/2021.03.17.435848 ◽

2021 ◽

Author(s):

Jonna Bouwknegt ◽

Sanne J. Wiersma ◽

Ra&uacutel A. Ortiz-Merino ◽

Eline S.R. Doornenbal ◽

Petrik Buitenhuis ◽

...

Keyword(s):

Unsaturated Fatty Acids ◽

Yeast Species ◽

Early Report ◽

Anaerobic Growth ◽

Fungal Cell ◽

Putative Gene ◽

Yeast Cultures ◽

Cell Factories ◽

Lipid Fractions ◽

Synthetic Media

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep–branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol–independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen–independent cyclization of squalene by a squalene–tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report (Bulder (1971), Antonie Van Leeuwenhoek, 37, 353–358) that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop–22(29)–ene, hop–17(21)–ene, hop–21(22)–ene and hopan–22–ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene–hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and supported ergosterol–independent anaerobic growth, thus confirming that one or more of the hopanoids produced by SjShc1 can act as ergosterol surrogate in anaerobic yeast cultures. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast sterol–independent anaerobic growth of Sch. japonicus is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

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Enzymatic Hydrolysate of Cinnamon Waste Material as Feedstock for the Microbial Production of Carotenoids

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18031146 ◽

2021 ◽

Vol 18 (3) ◽

pp. 1146

Author(s):

Stefano Bertacchi ◽

Stefania Pagliari ◽

Chiara Cantù ◽

Ilaria Bruni ◽

Massimo Labra ◽

...

Keyword(s):

Industrial Sector ◽

Waste Material ◽

Fungal Cell ◽

Enzymatic Hydrolysate ◽

Carbon And Nitrogen ◽

Microbial Cell Factories ◽

Cell Factories ◽

Separate Hydrolysis And Fermentation ◽

Additional Processing ◽

Microbial Cultivation

In the context of the global need to move towards circular economies, microbial cell factories can be employed thanks to their ability to use side-stream biomasses from the agro-industrial sector to obtain additional products. The valorization of residues allows for better and complete use of natural resources and, at the same time, for the avoidance of waste management to address our needs. In this work, we focused our attention on the microbial valorization of cinnamon waste material after polyphenol extraction (C-PEW) (Cinnamomum verum J.Presl), generally discarded without any additional processing. The sugars embedded in C-PEW were released by enzymatic hydrolysis, more compatible than acid hydrolysis with the subsequent microbial cultivation. We demonstrated that the yeast Rhodosporidium toruloides was able to grow and produce up to 2.00 (±0.23) mg/L of carotenoids in the resulting hydrolysate as a sole carbon and nitrogen source despite the presence of antimicrobial compounds typical of cinnamon. To further extend the potential of our finding, we tested other fungal cell factories for growth on the same media. Overall, these results are opening the possibility to develop separate hydrolysis and fermentation (SHF) bioprocesses based on C-PEW and microbial biotransformation to obtain high-value molecules.

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Influence of the method of disintegration of biomass of microalga Chlorella sorokiniana on the production of lipid fraction

Butlerov Communications ◽

10.37952/roi-jbc-01/19-59-7-85 ◽

2019 ◽

Vol 59 (7) ◽

pp. 85-91

Author(s):

Yulia A. Smyatskaya ◽

◽

Natalia A. Politaeva ◽

Amira Toumi ◽

◽

...

Keyword(s):

Fatty Acids ◽

Cell Wall ◽

Microwave Radiation ◽

High Speed ◽

Unsaturated Fatty Acids ◽

Lipid Fraction ◽

Chlorella Sorokiniana ◽

Lipid Fractions ◽

Significant Difference ◽

Selection Of

This article discusses the effect of the disintegration of the cell wall of the microalgae Chlorella sorokiniana on the output of the lipid fraction. The biomass of the microalgae Chlorella sorokiniana was grown under laboratory conditions in special photobioreactors at a temperature of 25 °C, with a constant aeration of a mixture of carbon dioxide and air at a rate of 1.5 liters/min, illumination 2200-2800 Lx. Nutrient medium for cultivation contained macro – and micronutrients for high-speed growth of microalgae. Selection of optimal cultivation parameters allows obtaining biomass with desired properties. Disintegration was carried out with the homogenization of biomass and under the influence of microwave radiation. Extraction of lipids was carried out on a semi-automatic extractor according to the Randall method, using organic solvents. The output of the lipid fraction without treatment was 10.18% after the destruction of the cell wall 14.45% with the homogenization of biomass and 13.85% under the influence of microwave radiation. A qualitative analysis of the lipid fraction, carried out under gas chromatography, obtained under various conditions showed that there was no significant difference in composition from the disintegration method. Lipid fractions (more than 50%) in both cases consist mainly of unsaturated fatty acids, of which irreplaceable unsaturated fatty acids constitute more than 18% for both samples. The residual biomass formed after the extraction of the lipid fraction can be used as fertilizer in the plant, for the manufacture of sorption materials for the purification of industrial water and as a biofuel. The purpose of this study was to study the effect of cell wall disintegration on the output of the lipid fraction and qualitative composition.

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12. Fungal cell factories and their applications

Microbial Applications ◽

10.1515/9783110412789-014 ◽

2016 ◽

Author(s):

Shafiquzzaman Siddiquee

Keyword(s):

Fungal Cell ◽

Cell Factories

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Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02284-19 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 5

Author(s):

Kenya E. Fernandes ◽

Kerry Weeks ◽

Dee A. Carter

Keyword(s):

Amphotericin B ◽

Galleria Mellonella ◽

Comprehensive Evaluation ◽

Yeast Species ◽

Phenotypic Diversity ◽

Antifungal Drugs ◽

Fungal Cell ◽

Content Type ◽

Iron Saturation ◽

Capsule Size

ABSTRACT Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 μg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus. Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

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Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production

Biotechnology Advances ◽

10.1016/j.biotechadv.2019.107449 ◽

2019 ◽

Vol 37 (8) ◽

pp. 107449 ◽

Cited By ~ 2

Author(s):

Antoine Vassaux ◽

Loïc Meunier ◽

Micheline Vandenbol ◽

Denis Baurain ◽

Patrick Fickers ◽

...

Keyword(s):

Genome Mining ◽

Nonribosomal Peptides ◽

Heterologous Production ◽

Fungal Cell ◽

Cell Factories

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Comparative Transcriptome Analysis Shows Conserved Metabolic Regulation during Production of Secondary Metabolites in Filamentous Fungi

mSystems ◽

10.1128/msystems.00012-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 3

Author(s):

Jens Christian Nielsen ◽

Sylvain Prigent ◽

Sietske Grijseels ◽

Mhairi Workman ◽

Boyang Ji ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Secondary Metabolite ◽

Filamentous Fungi ◽

Metabolic Regulation ◽

Fungal Species ◽

Efficient Production ◽

Fungal Cell ◽

Fungal Metabolism ◽

Cell Factories

ABSTRACTFilamentous fungi possess great potential as sources of medicinal bioactive compounds, such as antibiotics, but efficient production is hampered by a limited understanding of how their metabolism is regulated. We investigated the metabolism of six secondary metabolite-producing fungi of thePenicilliumgenus during nutrient depletion in the stationary phase of batch fermentations and assessed conserved metabolic responses across species using genome-wide transcriptional profiling. A coexpression analysis revealed that expression of biosynthetic genes correlates with expression of genes associated with pathways responsible for the generation of precursor metabolites for secondary metabolism. Our results highlight the main metabolic routes for the supply of precursors for secondary metabolism and suggest that the regulation of fungal metabolism is tailored to meet the demands for secondary metabolite production. These findings can aid in identifying fungal species that are optimized for the production of specific secondary metabolites and in designing metabolic engineering strategies to develop high-yielding fungal cell factories for production of secondary metabolites.IMPORTANCESecondary metabolites are a major source of pharmaceuticals, especially antibiotics. However, the development of efficient processes of production of secondary metabolites has proved troublesome due to a limited understanding of the metabolic regulations governing secondary metabolism. By analyzing the conservation in gene expression across secondary metabolite-producing fungal species, we identified a metabolic signature that links primary and secondary metabolism and that demonstrates that fungal metabolism is tailored for the efficient production of secondary metabolites. The insight that we provide can be used to develop high-yielding fungal cell factories that are optimized for the production of specific secondary metabolites of pharmaceutical interest.

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Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics

Applied and Environmental Microbiology ◽

10.1128/aem.69.1.113-121.2003 ◽

2003 ◽

Vol 69 (1) ◽

pp. 113-121 ◽

Cited By ~ 71

Author(s):

Eric Rosenfeld ◽

Bertrand Beauvoit ◽

Bruno Blondin ◽

Jean-Michel Salmon

Keyword(s):

Saccharomyces Cerevisiae ◽

Oxygen Consumption ◽

Stationary Phase ◽

Respiratory Chain ◽

Unsaturated Fatty Acids ◽

De Novo ◽

Anaerobic Growth ◽

Fermentation Rate ◽

Fermentation Kinetics ◽

Oxygen Pulse

ABSTRACT The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.

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