| 1. |
- Bracher, J. M., et al.
(författare)
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The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae
- 2018
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
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| 2. |
- Bracher, J. M., et al.
(författare)
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Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations
- 2017
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 83:16
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Tidskriftsartikel (refereegranskat)abstract
- Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ= 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.
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| 3. |
- Gabba, M., et al.
(författare)
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Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane
- 2020
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 118:2, s. 422-434
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Tidskriftsartikel (refereegranskat)abstract
- We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.
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| 4. |
- Marques, W. L., et al.
(författare)
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Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae
- 2018
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Ingår i: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 45, s. 121-133
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Tidskriftsartikel (refereegranskat)abstract
- Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.
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| 5. |
- Juergens, H., et al.
(författare)
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Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics
- 2018
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Ingår i: Scientific Data. - : Nature Publishing Groups. - 2052-4463. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous ‘Materials and Methods’ sections in scientific journals, thus promoting reproducibility and reusability of published studies.
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| 6. |
- Valk, Laura C., et al.
(författare)
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Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures : Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii"
- 2018
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 84:18
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Tidskriftsartikel (refereegranskat)abstract
- Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of D-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with D-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H-2 as coproducts. The observed molar ratio of acetate and the combined amounts of H-2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during D-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for D-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under D-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual D-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source. IMPORTANCE This study on D-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, D-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of D-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of D-galacturonate under different cultivation regimes.
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| 7. |
- Cueto-Rojas, Hugo F., et al.
(författare)
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Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae
- 2017
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Ingår i: BMC Systems Biology. - : BIOMED CENTRAL LTD. - 1752-0509. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Microbial production of nitrogen containing compounds requires a high uptake flux and assimilation of the N-source (commonly ammonium), which is generally coupled with ATP consumption and negatively influences the product yield. In the industrial workhorse Saccharomyces cerevisiae, ammonium (NH4+) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH4+ ion, resulting in ATP expenditure to maintain the intracellular charge balance and pH by proton export using the plasma membrane-bound H+ -ATPase. Results: To decrease the ATP costs for nitrogen assimilation, the Mep genes were removed, resulting in a strain unable to uptake the NH4+ ion. Subsequent analysis revealed that growth of this Delta mep strain was dependent on the extracellular NH3 concentrations. Metabolomic analysis revealed a significantly higher intracellular NHX concentration (3.3-fold) in the Delta mep strain than in the reference strain. Further proteomic analysis revealed significant up-regulation of vacuolar proteases and genes involved in various stress responses. Conclusions: Our results suggest that the uncharged species, NH3, is able to diffuse into the cell. The measured intracellular/extracellular NHX ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NHx compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the Delta mep strain than in the reference strain, which suggests that the lower biomass yield of the Delta mep strain was related to higher turnover rates of biomass components.
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| 8. |
- Marques, Wesley Leoricy, et al.
(författare)
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Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae : a platform strain for engineering sucrose metabolism
- 2017
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h(-1) after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.
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| 9. |
- Papapetridis, Ioannis, et al.
(författare)
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Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae
- 2018
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 18:6
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Tidskriftsartikel (refereegranskat)abstract
- Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose-xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83(G673T) allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L-1 glucose and 10 g L-1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.
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| 10. |
- Papapetridis, Ioannis, et al.
(författare)
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Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae
- 2017
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD(+)-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP(+)-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1 Delta gpd2 Delta strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP(+)-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.
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