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- Kildegaard, K. R., et al.
(författare)
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Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway
- 2016
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the future, oil-and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Results: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 +/- 0.4 g L-1 3HP with a yield of 13 % C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by C-13 metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. Conclusions: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.
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| 4. |
- Rodriguez, A., et al.
(författare)
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Comparison of the metabolic response to over-production of p-coumaric acid in two yeast strains
- 2017
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 44, s. 265-272
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Tidskriftsartikel (refereegranskat)abstract
- The development of robust and efficient cell factories requires understanding of the metabolic changes triggered by the production of the targeted compound. Here we aimed to study how production of p-coumaric acid, a precursor of multiple secondary aromatic metabolites, influences the cellular metabolism of Saccharomyces cerevisiae. We evaluated the growth and p-coumaric acid production in batch and chemostat cultivations and analyzed the transcriptome and intracellular metabolome during steady state in low-and high-producers of p-coumaric acid in two strain backgrounds, S288c or CEN.PK. We found that the same genetic modifications resulted in higher production of p-coumaric acid in the CEN.PK background than in the S288c background. Moreover, the CEN.PK strain was less affected by the genetic engineering as was evident from fewer changes in the transcription profile and intracellular metabolites concentrations. Surprisingly, for both strains we found the largest transcriptional changes in genes involved in transport of amino acids and sugars, which were downregulated. Additionally, in S288c amino acid and protein biosynthesis processes were also affected. We systematically overexpressed or deleted genes with significant transcriptional changes in CEN.PK low and high-producing strains. The knockout of some of the downregulated transporters triggered a 20-50% improvement in the synthesis of p-CA in the CEN.PK high-producing strain. This study demonstrates the importance of transporters in the engineering of cell factories for production of small molecules.
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- Schuette, Moritz, et al.
(författare)
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Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling 44,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.
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| 6. |
- Tiukova, Ievgeniia, 1987, et al.
(författare)
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Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis
- 2019
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Ingår i: FEMS microbiology letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 366:17
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.
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