| 1. |
- Bergenholm, David, 1987, et al.
(författare)
-
Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids
- 2018
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942
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Tidskriftsartikel (refereegranskat)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.
(författare)
-
Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species
- 2017
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:6
-
Tidskriftsartikel (refereegranskat)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.
(författare)
-
Identification of the decumbenone biosynthetic gene cluster in penicillium decumbens and the importance for production of calbistrin
- 2018
-
Ingår i: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 5:1, s. 1-17
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)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.
(författare)
-
Physiological characterization of secondary metabolite producing Penicillium cell factories
- 2017
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Ingår i: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 4, s. 8-
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Comparative Transcriptome Analysis Shows Conserved Metabolic Regulation during Production of Secondary Metabolites in Filamentous Fungi
- 2019
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Ingår i: mSystems. - 2379-5077. ; 4:2
-
Tidskriftsartikel (refereegranskat)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.
(författare)
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Development of fungal cell factories for the production of secondary metabolites: Linking genomics and metabolism
- 2017
-
Ingår i: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 2:1, s. 5-12
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Forskningsöversikt (refereegranskat)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.
(författare)
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Reconstruction of 24 Penicillium genome-scale metabolic models shows diversity based on their secondary metabolism
- 2018
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:10, s. 2604-2612
-
Tidskriftsartikel (refereegranskat)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.
(författare)
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Pathway engineering in yeast for synthesizing the complex polyketide bikaverin
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Construction of mini-chemostats for high-throughput strain characterization
- 2019
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 116:5, s. 1029-1038
-
Tidskriftsartikel (refereegranskat)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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| 11. |
- Brunk, Elizabeth, et al.
(författare)
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Recon3D enables a three-dimensional view of gene variation in human metabolism
- 2018
-
Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 36:3, s. 272-281
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Tidskriftsartikel (refereegranskat)abstract
- Genome-scale network reconstructions have helped uncover the molecular basis of metabolism. Here we present Recon3D, a computational resource that includes three-dimensional (3D) metabolite and protein structure data and enables integrated analyses of metabolic functions in humans. We use Recon3D to functionally characterize mutations associated with disease, and identify metabolic response signatures that are caused by exposure to certain drugs. Recon3D represents the most comprehensive human metabolic network model to date, accounting for 3,288 open reading frames (representing 17% of functionally annotated human genes), 13,543 metabolic reactions involving 4,140 unique metabolites, and 12,890 protein structures. These data provide a unique resource for investigating molecular mechanisms of human metabolism. Recon3D is available at http://vmh.life.
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| 12. |
- Liu, Quanli, 1988, et al.
(författare)
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Modular Pathway Rewiring of Yeast for Amino Acid Production
- 2018
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Ingår i: Methods in Enzymology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 608, s. 417-439
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Amino acids find various applications in biotechnology in view of their importance in the food, feed, pharmaceutical, and personal care industries as nutrients, additives, and drugs, respectively. For the large-scale production of amino acids, microbial cell factories are widely used and the development of amino acid-producing strains has mainly focused on prokaryotes Corynebacterium glutamicum and Escherichia coli. However, the eukaryote Saccharomyces cerevisiae is becoming an even more appealing microbial host for production of amino acids and derivatives because of its superior molecular and physiological features, such as amenable to genetic engineering and high tolerance to harsh conditions. To transform S. cerevisiae into an industrial amino acid production platform, the highly coordinated and multiple layers regulation in its amino acid metabolism should be relieved and reconstituted to optimize the metabolic flux toward synthesis of target products. This chapter describes principles, strategies, and applications of modular pathway rewiring in yeast using the engineering of L-ornithine metabolism as a paradigm. Additionally, detailed protocols for in vitro module construction and CRISPR/Cas-mediated pathway assembly are provided.
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| 13. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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| 14. |
- Froslev Nielsen, Jens Christian, 1987, et al.
(författare)
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Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:22, s. 8237-8248
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Tidskriftsartikel (refereegranskat)abstract
- The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates and glucose uptake rates, while commercial AFA had no effect in concentrations relevant for defoaming purposes. Industrial AFA were further tested in laboratory scale simulations of the Brazilian ethanol production process and proved to decrease cell viability compared to the control, and the effects were intensified with increasing AFA concentrations and exposure time. Transcriptome analysis showed that AFA treatments induced additional stress responses in yeast cells compared to the control, shown by an up-regulation of stress-specific genes and a down-regulation of lipid biosynthesis, especially ergosterol. By documenting the detrimental effects associated with chemical AFA, we highlight the importance of developing innocuous systems for foam control in industrial fermentation processes.
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| 15. |
- Froslev Nielsen, Jens Christian, 1987
(författare)
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Systems Biology of the Secondary Metabolism in Filamentous Fungi
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Filamentous fungi constitute a rich reservoir of pharmaceutically relevant bioactive small molecules. These compounds, commonly referred to as secondary metabolites, are widely used as antibiotics for the treatment of microbial infections, but also as other pharmaceuticals such as immunosuppressors, cholesterol lowering agents and anticancer drugs. Although fungal derived antibiotics have been known for almost a century, genome sequencing has revealed that the biosynthetic potential of fungi is not fully exhausted.The Penicillium genus consists of around 350 accepted species, and many of these are well-known producers of pharmaceuticals and industrially exploited for this. The genus as a whole, however, is grossly understudied at the genomic level. To assess the potential for secondary metabolite biosynthesis in the Penicillium genus, we sequenced the genomes of ten species that produce diverse arrays of secondary metabolites in culture. One of the sequenced isolates was described as a new species, and we mapped secondary metabolites detected in culture to the corresponding biosynthetic gene clusters. The ten sequenced genomes were analyzed together with published Penicillium genomes, altogether 24, and we developed a pipeline to group biosynthetic gene clusters and map them to known pathways. We found a large untapped potential for biosynthesis of secondary metabolites, encoded in the genomes of these species, that potentially could fill the drug discovery pipeline. Based on our predictions, we experimentally identified a novel compound from the antifungal class of antibiotics called yanuthones.Since heterologous expression of secondary metabolite pathways has proved troublesome, the ten genome-sequenced Penicillium species were evaluated as cell factories in controlled bioreactor fermentations. Compared to an industrially relevant strain, the ten Penicillium species showed growth characteristics that encourage further exploration of their industrial potential. Transcriptome analysis of six of the species enabled the identification of a metabolic network that is responsible for precursor formation of secondary metabolites. This network provides important insight into the further industrial development of Penicillium cell factories, and could be used in designing metabolic engineering strategies for optimization of secondary metabolite production.Altogether this thesis provides novel insights into genetic and metabolic aspects of fungal secondary metabolism. Our findings propose that industrial production of secondary metabolites can be effectively established on the basis of native producers. Penicillium species constitute a rich source of drug leads, and possess promising physiological characteristics to be established as industrial production platforms.
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| 16. |
- Kovatcheva-Datchary, Petia, et al.
(författare)
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Simplified Intestinal Microbiota to Study Microbe-Diet-Host Interactions in a Mouse Model
- 2019
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 26:13
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Tidskriftsartikel (refereegranskat)abstract
- The gut microbiota can modulate human metabolism through interactions with macronutrients. However, microbiota-diet-host interactions are difficult to study because bacteria interact in complex food webs in concert with the host, and many of the bacteria are not yet characterized. To reduce the complexity, we colonize mice with a simplified intestinal microbiota (SIM) composed of ten sequenced strains isolated from the human gut with complementing pathways to metabolize dietary fibers. We feed the SIM mice one of three diets (chow [fiber rich], high-fat/high-sucrose, or zero-fat/high-sucrose diets [both low in fiber]) and investigate (1) how dietary fiber, saturated fat, and sucrose affect the abundance and transcriptome of the SIM community, (2) the effect of microbe-diet interactions on circulating metabolites, and (3) how microbiota-diet interactions affect host metabolism. Our SIM model can be used in future studies to help clarify how microbiota-diet interactions contribute to metabolic diseases.
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| 17. |
- Li, Xiaowei, et al.
(författare)
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Harnessing xylose pathways for biofuels production
- 2019
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Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 57, s. 56-65
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Forskningsöversikt (refereegranskat)abstract
- Energy security, environmental pollution, and economic development drive the development of alternatives to fossil fuels as an urgent global priority. Lignocellulosic biomass has the potential to contribute to meeting the demand for biofuel production via hydrolysis and fermentation of released sugars, such as glucose, xylose, and arabinose. Construction of robust cell factories requires introducing and rewiring of their metabolism to efficiently use all these sugars. Here, we review recent advances in re-constructing pathways for metabolism of pentoses, with special focus on xylose metabolism in the most widely used cell factories Saccharomyces cerevisiae and Escherichia coli. We also highlight engineering advanced biofuels-synthesis pathways and describes progress toward overcoming the challenges facing adoption of large-scale biofuel production.
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| 18. |
- Liu, Zhengtao, et al.
(författare)
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Pyruvate kinase L/R is a regulator of lipid metabolism and mitochondrial function
- 2019
-
Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 52, s. 263-272
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Tidskriftsartikel (refereegranskat)abstract
- The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) has been associated with altered expression of liver-specific genes including pyruvate kinase liver and red blood cell (PKLR), patatin-like phospholipase domain containing 3 (PNPLA3) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Here, we inhibited and overexpressed the expression of these three genes in HepG2 cells, generated RNA-seq data before and after perturbation and revealed the altered global biological functions with the modulation of these genes using integrated network (IN) analysis. We found that modulation of these genes effects the total triglycerides levels within the cells and viability of the cells. Next, we generated IN for HepG2 cells, identified reporter transcription factors based on IN and found that the modulation of these genes affects key metabolic pathways associated with lipid metabolism (steroid biosynthesis, PPAR signalling pathway, fatty acid synthesis and oxidation) and cancer development (DNA replication, cell cycle and p53 signalling) involved in the progression of NAFLD and HCC. Finally, we observed that inhibition of PKLR lead to decreased glucose uptake and decreased mitochondrial activity in HepG2 cells. Hence, our systems level analysis indicated that PKLR can be targeted for development efficient treatment strategy for NAFLD and HCC.
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| 19. |
- Liu, Zihe, 1984, et al.
(författare)
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Synthetic Biology of Yeast
- 2019
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 58:11, s. 1511-1520
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Forskningsöversikt (refereegranskat)abstract
- With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.
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| 20. |
- Ma, Tian, et al.
(författare)
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Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene
- 2019
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 52, s. 134-142
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil–triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2 mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5 mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37 g/L and 73.3 mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.
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| 21. |
- Yu, Tao, 1986, et al.
(författare)
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Strategies and challenges for metabolic rewiring
- 2019
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Ingår i: Current Opinion in Systems Biology. - : Elsevier BV. - 2452-3100. ; 15, s. 30-38
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Forskningsöversikt (refereegranskat)abstract
- Metabolic engineering is often centred on rewiring cellular metabolism to improve the production of chemicals. Turning cells into efficient factories is challenging because of ubiquitous and tightly regulated metabolic interactions. Tools and strategies from different disciplines, including systems biology, synthetic biology and evolutionary engineering, have been integrated into metabolic engineering to overcome challenges with rewiring metabolism for cell factory development. In this review, we summarise the recent development of tools and strategies for dynamic pathway regulation, compartmentalisation, modular and evolutionary engineering. In addition, we describe how systems biology tools benefit metabolic rewiring for advancing the development of cell factories.
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| 22. |
- Zhang, Yueping, et al.
(författare)
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A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.
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