1. |
- Bergenholm, David, 1987, et al.
(author)
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Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids
- 2018
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942
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Journal article (peer-reviewed)abstract
- Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food dditives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized. We engineered Saccharomyces cerevisiae yeast strains toward a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type.
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2. |
- Froslev Nielsen, Jens Christian, 1987, et al.
(author)
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Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species
- 2017
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In: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:6
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Journal article (peer-reviewed)abstract
- Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However, less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were identified, and polyketide synthase and non-ribosomal peptide synthetase based BGCs were grouped into gene cluster families and mapped to known pathways. The grouping of BGCs allowed us to study the evolutionary trajectory of pathways based on 6-methylsalicylic acid (6-MSA) synthases. Finally, we cross-referenced the predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic diversity of Penicillia and highlights the potential of these species as a source of new antibiotics and other pharmaceuticals.
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3. |
- Grijseels, S., et al.
(author)
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Identification of the decumbenone biosynthetic gene cluster in penicillium decumbens and the importance for production of calbistrin
- 2018
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In: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 5:1, s. 1-17
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Journal article (peer-reviewed)abstract
- Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicy-clic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/ Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC ( TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.
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4. |
- Grijseels, S., et al.
(author)
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Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites
- 2016
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6
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Journal article (peer-reviewed)abstract
- A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311(T) = IBT 12289(T)). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species.
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5. |
- Grijseels, S., et al.
(author)
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Physiological characterization of secondary metabolite producing Penicillium cell factories
- 2017
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In: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 4, s. 8-
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Journal article (peer-reviewed)abstract
- Background: Penicillium species are important producers of bioactive secondary metabolites. However, the immense diversity of the fungal kingdom is only scarcely represented in industrial bioprocesses and the upscaling of compound production remains a costly and labor intensive challenge. In order to facilitate the development of novel secondary metabolite producing processes, two routes are typically explored: optimization of the native producer or transferring the enzymatic pathway into a heterologous host. Recent genome sequencing of ten Penicillium species showed the vast amount of secondary metabolite gene clusters present in their genomes, and makes them accessible for rational strain improvement. In this study, we aimed to characterize the potential of these ten Penicillium species as native producing cell factories by testing their growth performance and secondary metabolite production in submerged cultivations.Results: Cultivation of the fungal species in controlled submerged bioreactors showed that the ten wild type Penicillium species had promising, highly reproducible growth characteristics in two different media. Analysis of the secondary metabolite production using liquid chromatography coupled with high resolution mass spectrometry proved that the species produced a broad range of secondary metabolites, at different stages of the fermentations. Metabolite profiling for identification of the known compounds resulted in identification of 34 metabolites; which included several with bioactive properties such as antibacterial, antifungal and anticancer activities. Additionally, several novel species metabolite relationships were found.Conclusions: This study demonstrates that the fermentation characteristics and the highly reproducible performance in bioreactors of ten recently genome sequenced Penicillium species should be considered as very encouraging for the application of native hosts for production via submerged fermentation. The results are particularly promising for the potential development of the ten analysed Penicillium species for production of novel bioactive compounds via submerged fermentations
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6. |
- Froslev Nielsen, Jens Christian, 1987, et al.
(author)
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Comparative Transcriptome Analysis Shows Conserved Metabolic Regulation during Production of Secondary Metabolites in Filamentous Fungi
- 2019
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In: mSystems. - 2379-5077. ; 4:2
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Journal article (peer-reviewed)abstract
- Filamentous 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 the Penicillium genus 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. IMPORTANCE Secondary 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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7. |
- Froslev Nielsen, Jens Christian, 1987, et al.
(author)
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Development of fungal cell factories for the production of secondary metabolites: Linking genomics and metabolism
- 2017
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In: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 2:1, s. 5-12
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Research review (peer-reviewed)abstract
- The genomic era has revolutionized research on secondary metabolites and bioinformatics methods have in recent years revived the antibiotic discovery process after decades with only few new active molecules being identified. New computational tools are driven by genomics and metabolomics analysis, and enables rapid identification of novel secondary metabolites. To translate this increased discovery rate into industrial exploitation, it is necessary to integrate secondary metabolite pathways in the metabolic engineering process. In this review, we will describe the novel advances in discovery of secondary metabolites produced by filamentous fungi, highlight the utilization of genome-scale metabolic models (GEMs) in the design of fungal cell factories for the production of secondary metabolites and review strategies for optimizing secondary metabolite production through the construction of high yielding platform cell factories.
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8. |
- Prigent, Sylvain, 1984, et al.
(author)
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Reconstruction of 24 Penicillium genome-scale metabolic models shows diversity based on their secondary metabolism
- 2018
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:10, s. 2604-2612
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Journal article (peer-reviewed)abstract
- Modeling of metabolism at the genome-scale has proved to be an efficient method for explaining the phenotypic traits observed in living organisms. Further, it can be used as a means of predicting the effect of genetic modifications for example, development of microbial cell factories. With the increasing amount of genome sequencing data available, there exists a need to accurately and efficiently generate such genome-scale metabolic models (GEMs) of nonmodel organisms, for which data is sparse. In this study, we present an automatic reconstruction approach applied to 24 Penicillium species, which have potential for production of pharmaceutical secondary metabolites or use in the manufacturing of food products, such as cheeses. The models were based on the MetaCyc database and a previously published Penicillium GEM and gave rise to comprehensive genome-scale metabolic descriptions. The models proved that while central carbon metabolism is highly conserved, secondary metabolic pathways represent the main diversity among species. The automatic reconstruction approach presented in this study can be applied to generate GEMs of other understudied organisms, and the developed GEMs are a useful resource for the study of Penicillium metabolism, for example, for the scope of developing novel cell factories.
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9. |
- Zhao, Meng, et al.
(author)
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Pathway engineering in yeast for synthesizing the complex polyketide bikaverin
- 2020
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 11:1
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Journal article (peer-reviewed)abstract
- Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.
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10. |
- Bergenholm, David, 1987, et al.
(author)
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Construction of mini-chemostats for high-throughput strain characterization
- 2019
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 116:5, s. 1029-1038
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Journal article (peer-reviewed)abstract
- To achieve large-scale, high-throughput experiments for systems biology research of microorganisms, reliable data from robust cultivation systems are needed. Chemostats are such systems, ensuring reproducibility and quality by providing a stable, well-controlled environment for the cells. However, many of the available chemostat systems require large amounts of media and are complex to set up and expensive to purchase and maintain. To address these concerns, we developed a mini-chemostat (MC) system with 16 reactors, each at a working volume of 40 ml. Sensors measure dissolved oxygen in the reactor, while OD600 is measured in the outflow. We further developed a CO 2 and pH sensor array that can be plugged into the outflow of the reactors. The system was used to characterize yeast physiology at four metabolically different conditions: limitations of glucose, both aerobic and anaerobic, nitrogen, and ethanol. The physiology of yeast cells grown at the four different conditions in the MC system was compared with the yeast cells grown in a DASGIP 1 L system using RNAseq analysis. The results show that the MC system provides the same environmental conditions as the DASGIP system and that the MC system is reproducible between different runs. The system is built to be easily scalable with more reactors and to include more sensors, if available. Our study shows that a robust, reproducible chemostat system for high-throughput and large-scale experiments can be built at low costs.
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