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BETA
van Maris, Antonius J. A., ProfessorORCID iD iconorcid.org/0000-0001-5319-7511
Publications (10 of 14) Show all publications
Perez-Zabaleta, M., Guevara-Martínez, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation. Applied Microbiology and Biotechnology, 103(14), 5627-5636
Open this publication in new window or tab >>Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
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2019 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, no 14, p. 5627-5636Article in journal (Refereed) Accepted
Abstract [en]

Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

Place, publisher, year, edition, pages
Springer, 2019
Keywords
Escherichia coli, (R)-3-hydroxybutyrate, acetate, nitrogen limitation, fed batch, BL21.
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251046 (URN)10.1007/s00253-019-09876-y (DOI)000473129900012 ()2-s2.0-85066078742 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency, 70828
Note

QC 20190508

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-08-15Bibliographically approved
Lindroos, M., Hörnström, D., Larsson, G., Gustavsson, M. & van Maris, A. J. A. (2019). Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor. Journal of Hazardous Materials, 365, 74-80
Open this publication in new window or tab >>Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
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2019 (English)In: Journal of Hazardous Materials, ISSN 0304-3894, E-ISSN 1873-3336, Vol. 365, p. 74-80Article in journal (Refereed) Published
Abstract [en]

Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26 +/- 2 treated reactor volumes (39 +/- 3 L). The average effluent concentration during the first 20 h was 0.0018 mM, corresponding to 98.2% removal. Up to 140 +/- 6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low pH regeneration released all chloroquine without apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or manufacturing sites.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Wastewater treatment, Pharmaceuticals, Membrane bioreactor, Adsorption, Surface expression
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-244080 (URN)10.1016/j.jhazmat.2018.10.081 (DOI)000456761000009 ()30412809 (PubMedID)2-s2.0-85055974597 (Scopus ID)
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-10-28Bibliographically approved
Sjöberg, G., Guevara-Martínez, M., van Maris, A. J. A. & Gustavsson, M. (2019). Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome. Journal of Biotechnology, 305, 43-50
Open this publication in new window or tab >>Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
2019 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 305, p. 43-50Article in journal (Refereed) Published
Abstract [en]

In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.

Place, publisher, year, edition, pages
ELSEVIER, 2019
Keywords
3-Hydroxybutyrate, Chromosomal expression, Pathway optimization, F plasmid, Bacterial artificial chromosome, Metabolic engineering
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-261932 (URN)10.1016/j.jbiotec.2019.09.002 (DOI)000487564300007 ()31505217 (PubMedID)2-s2.0-85072060887 (Scopus ID)
Note

QC 20191015

Available from: 2019-10-15 Created: 2019-10-15 Last updated: 2019-12-02Bibliographically approved
Hörnström, D., Larsson, G., van Maris, A. J. A. & Gustavsson, M. (2019). Molecular optimization of autotransporter-based tyrosinase surface display. Biochimica et Biophysica Acta - Biomembranes, 1862(2), 486-494
Open this publication in new window or tab >>Molecular optimization of autotransporter-based tyrosinase surface display
2019 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, no 2, p. 486-494Article in journal (Refereed) Published
Abstract [en]

Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Autotransport, Whole-cell biocatalysis, Tyrosinase, Protein engineering, Escherichia coli
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-244107 (URN)10.1016/j.bbamem.2018.11.012 (DOI)000456764100014 ()30521785 (PubMedID)2-s2.0-85058059281 (Scopus ID)
Funder
Swedish Research Council, VR-621-2014-5293
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-10-28Bibliographically approved
Guevara-Martínez, M., Perez-Zabaleta, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli. Applied Microbiology and Biotechnology, 1-12
Open this publication in new window or tab >>The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
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2019 (English)In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed) Published
Abstract [en]

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Place, publisher, year, edition, pages
Springer, 2019
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-249360 (URN)10.1007/s00253-019-09707-0 (DOI)000464737100008 ()2-s2.0-85062726311 (Scopus ID)
Note

QC 20190509

Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-10-16Bibliographically approved
Marques, W. L., Mans, R., Henderson, R. K., Marella, E. R., Horst, J. T., Hulster, E. D., . . . van Maris, A. J. A. (2018). Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae. Metabolic engineering, 45, 121-133
Open this publication in new window or tab >>Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae
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2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 121-133Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Academic Press Inc., 2018
Keywords
ATP, Chemostat, Facilitated diffusion, Free-energy conservation, Phosphoglucomutase, Yeast physiology, Adenosinetriphosphate, Biomass, Chemostats, Ecology, Energy conservation, Enzyme activity, Fermentation, Free energy, Industrial chemicals, Phosphorylation, Yeast, Facilitated diffusions, Industrial fermentation, Leuconostoc mesenteroides, Phaseolus vulgaris, Specific growth rate, Sucrose phosphorylase, Yeast physiologies, Sugar (sucrose), adenosine triphosphate, disaccharide, glucose 6 phosphate, hydrolase, phosphorylase, sucrose, alkalinization, anaerobic growth, Arabidopsis thaliana, Article, carbon source, cell suspension, cellular distribution, chemical reaction, controlled study, fungus growth, genetic background, metabolic engineering, microscopy, molecular biology, nonhuman, pea, priority journal, protein content, proton motive force, rice, Saccharomyces cerevisiae, sucrose metabolism
National Category
Bioenergy
Identifiers
urn:nbn:se:kth:diva-227102 (URN)10.1016/j.ymben.2017.11.012 (DOI)000424292100013 ()2-s2.0-85038095617 (Scopus ID)
Note

QC 20180515

Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2019-10-28Bibliographically approved
Juergens, H., Niemeijer, M., Jennings-Antipov, L. D., Mans, R., Morel, J., van Maris, A. J. A., . . . Gardner, T. S. (2018). Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics. Scientific Data, 5, Article ID 180195.
Open this publication in new window or tab >>Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics
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2018 (English)In: Scientific Data, E-ISSN 2052-4463, Vol. 5, article id 180195Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Groups, 2018
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-246550 (URN)10.1038/sdata.2018.195 (DOI)000446328900001 ()30280721 (PubMedID)2-s2.0-85054395353 (Scopus ID)
Note

QC 20190319

Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-03-19Bibliographically approved
Valk, L. C., Frank, J., de la Torre-Cortes, P., van 't Hof, M., van Maris, A. J. A., Pronk, J. T. & van Loosdrecht, M. C. M. (2018). Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii". Applied and Environmental Microbiology, 84(18), Article ID UNSP e01370-18.
Open this publication in new window or tab >>Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii"
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2018 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 18, article id UNSP e01370-18Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Society for Microbiology, 2018
Keywords
acetogenesis, enrichment culture, fermentation, galacturonate, metagenomics
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-235115 (URN)10.1128/AEM.01370-18 (DOI)000443291000033 ()29959255 (PubMedID)
Note

QC 20180919

Available from: 2018-09-19 Created: 2018-09-19 Last updated: 2018-09-19Bibliographically approved
Verhoeven, M. D., Bracher, J. M., Nijland, J. G., Bouwknegt, J., Daran, J.-M. G., Driessen, A. J. M., . . . Pronk, J. T. (2018). Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake. FEMS yeast research (Print), 18(6), Article ID foy062.
Open this publication in new window or tab >>Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake
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2018 (English)In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 6, article id foy062Article in journal (Refereed) Published
Abstract [en]

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantarum L-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h(-1) on L-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on L-arabinose in the presence of D-glucose and D-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2(N376T) or Gal(2N376I) substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2(T89I) substitution, which was subsequently also detected in the independently evolved strain IMS0010. In C-14-sugar-transport assays, Gal2(N376S), Gal2(N376T) and Gal(2N376I) substitutions showed a much lower glucose sensitivity of L-arabinose transport and a much higher Km for D-glucose transport than wild-type Gal2. Introduction of the Gal2(N376I) substitution in a non-evolved strain enabled growth on L-arabinose in the presence of D-glucose. Gal2(N376T), T89I and Gal2(T89I) variants showed a lower K-m for L-arabinose and a higher K-m for D-glucose than wild-type Gal2, while reverting Gal2(N376T), T89I to Gal2(N376) in an evolved strain negatively affected anaerobic growth on L-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.

Place, publisher, year, edition, pages
Oxford University Press, 2018
Keywords
yeast, pentose fermentation, L-arabinose, transporter engineering, laboratory evolution, bioethanol, gene duplication
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-239501 (URN)10.1093/femsyr/foy062 (DOI)000449353000011 ()29860442 (PubMedID)
Note

QC 20181128

Available from: 2018-11-28 Created: 2018-11-28 Last updated: 2018-11-28Bibliographically approved
Marques, W. L., Mans, R., Marella, E. R., Cordeiro, R. L., van den Broek, M., Daran, J.-M. G., . . . van Maris, A. J. A. (2017). Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism. FEMS yeast research (Print), 17(1), Article ID fox006.
Open this publication in new window or tab >>Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism
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2017 (English)In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 17, no 1, article id fox006Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Oxford University Press, 2017
Keywords
disaccharide, isomaltase, laboratory evolution, reverse engineering, multiple gene deletion, real-time PCR
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-211631 (URN)10.1093/femsyr/fox006 (DOI)000405634600015 ()
Note

QC 20170809

Available from: 2017-08-09 Created: 2017-08-09 Last updated: 2017-12-07Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-5319-7511

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