Publications by authors named "Antonius J A van Maris"

88 Publications

Laboratory Evolution and Reverse Engineering of for Growth on Glucose and Fructose.

Appl Environ Microbiol 2021 04 13;87(9). Epub 2021 Apr 13.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden

The native ability of to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of , as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 g g, respectively, compared to 0.15 ± 0.01 g g for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 ± 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 ± 0.01 h and resulted in immediate growth on glucose at 0.24 ± 0.01 h Additional introduction of a G-to-V mutation at position 148 in resulted in immediate growth on fructose at 0.32 ± 0.03 h These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems , (iii) allow experiments at >120 g liter soluble substrate concentration, or (iv) reduce costs for labeling studies.
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http://dx.doi.org/10.1128/AEM.03017-20DOI Listing
April 2021

Characterization of volatile fatty-acid utilization in Escherichia coli aiming for robust valorisation of food residues.

AMB Express 2020 Oct 17;10(1):184. Epub 2020 Oct 17.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.

Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well-studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight-chain VFAs (C-C) in the mixture were readily consumed simultaneously. At a dilution rate of 0.1 h, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon from the supplied VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
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http://dx.doi.org/10.1186/s13568-020-01121-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568742PMC
October 2020

A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks.

ACS Synth Biol 2020 05 2;9(5):1117-1128. Epub 2020 Apr 2.

Fischell Department of Bioengineering, University of Maryland, 3102 A. James Clark Hall, 8278 Paint Branch Drive, College Park, Maryland 20742, United States.

There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by , -(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.
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http://dx.doi.org/10.1021/acssynbio.9b00469DOI Listing
May 2020

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane.

Biophys J 2020 01 27;118(2):422-434. Epub 2019 Nov 27.

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands. Electronic address:

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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http://dx.doi.org/10.1016/j.bpj.2019.11.3384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976801PMC
January 2020

Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome.

J Biotechnol 2019 Nov 7;305:43-50. Epub 2019 Sep 7.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbiotec.2019.09.002DOI Listing
November 2019

Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation.

Appl Microbiol Biotechnol 2019 Jul 18;103(14):5627-5639. Epub 2019 May 18.

School of Engineering Sciences in Chemistry, Biotechnology, and Health (CBH), Department of Industrial Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE-10691, Stockholm, Sweden.

Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia 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 five times less acetic acid per mole 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 h) and the highest 3HB concentration (16.3 g L) 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.
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http://dx.doi.org/10.1007/s00253-019-09876-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597613PMC
July 2019

The role of the acyl-CoA thioesterase "YciA" in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli.

Appl Microbiol Biotechnol 2019 May 5;103(9):3693-3704. Epub 2019 Mar 5.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, SE 10691, Stockholm, Sweden.

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 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.
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http://dx.doi.org/10.1007/s00253-019-09707-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469607PMC
May 2019

Molecular optimization of autotransporter-based tyrosinase surface display.

Biochim Biophys Acta Biomembr 2019 02 4;1861(2):486-494. Epub 2018 Dec 4.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, SE 10691 Stockholm, Sweden. Electronic address:

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 coli 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.
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http://dx.doi.org/10.1016/j.bbamem.2018.11.012DOI Listing
February 2019

Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor.

J Hazard Mater 2019 03 29;365:74-80. Epub 2018 Oct 29.

KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Industrial Biotechnology, AlbaNova University Center, 114 21, Stockholm, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jhazmat.2018.10.081DOI Listing
March 2019

Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics.

Sci Data 2018 10 3;5:180195. Epub 2018 Oct 3.

Riffyn, Inc., 360 17th Street, Suite 100, Oakland, CA 94612, USA.

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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http://dx.doi.org/10.1038/sdata.2018.195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169258PMC
October 2018

Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains.

FEMS Yeast Res 2019 01;19(1)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.
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http://dx.doi.org/10.1093/femsyr/foy104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240133PMC
January 2019

Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter.

Yeast 2018 12 15;35(12):639-652. Epub 2018 Oct 15.

School of Food Engineering, University of Campinas, Campinas, Brazil.

Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (μ = 0.19 h and μ = 0.08 h , respectively). All five mutants displayed sucrose-induced proton uptake (13-50 μmol H (g biomass)  min ). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1 ) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h ) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.
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http://dx.doi.org/10.1002/yea.3357DOI Listing
December 2018

Identification of novel genes involved in acetic acid tolerance of Saccharomyces cerevisiae using pooled-segregant RNA sequencing.

FEMS Yeast Res 2018 12;18(8)

Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.

Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN.PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.
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http://dx.doi.org/10.1093/femsyr/foy100DOI Listing
December 2018

Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains.

FEMS Yeast Res 2018 12;18(8)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

d-Glucose, d-xylose and l-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three 'specialist' Saccharomyces cerevisiae strains. A d-glucose- and l-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented d-xylose in the presence of 20 g L-1 of d-glucose and l-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose non-fermenting laboratory strain, rapidly and simultaneously converted d-glucose and l-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved 'generalist' strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1d-glucose, 10 g L-1d-xylose and 5 g L-1l-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.
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http://dx.doi.org/10.1093/femsyr/foy075DOI Listing
December 2018

Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp. nov. "Candidatus Galacturonibacter soehngenii".

Appl Environ Microbiol 2018 09 31;84(18). Epub 2018 Aug 31.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

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 species, for which the name " Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H as coproducts. The observed molar ratio of acetate and the combined amounts of H and formate deviated significantly from 1, which suggested that some of the hydrogen and CO 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 " 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. 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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http://dx.doi.org/10.1128/AEM.01370-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121984PMC
September 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 Res 2018 09;18(6)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-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 Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I 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 Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 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.
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http://dx.doi.org/10.1093/femsyr/foy062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044391PMC
September 2018

A Simulator-Assisted Workshop for Teaching Chemostat Cultivation in Academic Classes on Microbial Physiology.

J Microbiol Biol Educ 2017 4;18(3). Epub 2017 Oct 4.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands.

Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state. In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/or chemostat cultivation.
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http://dx.doi.org/10.1128/jmbe.v18i3.1292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976038PMC
October 2017

Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae.

FEMS Yeast Res 2018 09;18(6)

Delft University of Technology, Department of Biotechnology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

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 GAL83G673T 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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http://dx.doi.org/10.1093/femsyr/foy056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6001886PMC
September 2018

The transporter AraT enables high-affinity, glucose-insensitive l-arabinose transport in .

Biotechnol Biofuels 2018 13;11:63. Epub 2018 Mar 13.

1Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the 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 strains.

Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus 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 strain in which the endogenous l-arabinose transporter, , had been deleted. Sugar transport assays indicated that this fungal transporter, designated as AraT, is a high-affinity ( = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing strain in which was replaced by 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 and 1.8 g L, 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 AraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing strain enabled growth in media supplemented with both 20 g L l-arabinose and 20 g L 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 AraT a valuable transporter for application in metabolic engineering strategies aimed at engineering strains for efficient conversion of lignocellulosic hydrolysates.
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http://dx.doi.org/10.1186/s13068-018-1047-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848512PMC
March 2018

Optimizing anaerobic growth rate and fermentation kinetics in strains expressing Calvin-cycle enzymes for improved ethanol yield.

Biotechnol Biofuels 2018 25;11:17. Epub 2018 Jan 25.

1Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Background: Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast . Anaerobic cultures of wild-type require formation of glycerol to maintain the intracellular NADH/NAD balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in was shown to enable reoxidation of NADH with CO as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.

Results: Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the GroEL/GroES chaperones and expression of PRK from the anaerobically inducible promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of , which encodes an isoenzyme of NAD-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.

Conclusions: The metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of , with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.
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http://dx.doi.org/10.1186/s13068-017-1001-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784725PMC
January 2018

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae.

Metab Eng 2018 01 2;45:121-133. Epub 2017 Dec 2.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

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 ± 0.02h (LmSPase) and 0.06 ± 0.01h (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01h and 0.08 ± 0.00h, 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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http://dx.doi.org/10.1016/j.ymben.2017.11.012DOI Listing
January 2018

A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.

FEMS Yeast Res 2017 12;17(8)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.
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http://dx.doi.org/10.1093/femsyr/fox085DOI Listing
December 2017

Saccharomyces cerevisiae strains for second-generation ethanol production: from academic exploration to industrial implementation.

FEMS Yeast Res 2017 08;17(5)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

The recent start-up of several full-scale 'second generation' ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.
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http://dx.doi.org/10.1093/femsyr/fox044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812533PMC
August 2017

Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations.

Appl Environ Microbiol 2017 08 1;83(16). Epub 2017 Aug 1.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of 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). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of , which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of 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 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h). Mutations in the membrane transporter genes and/or were found in several of the evolved strains. Deletion of and in a -overexpressing strain increased its specific growth rate to 0.25 h The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that 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. Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.
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http://dx.doi.org/10.1128/AEM.00892-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541218PMC
August 2017

Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in S.

Biotechnol Biofuels 2017 26;10:107. Epub 2017 Apr 26.

Industrial Microbiology Section, Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in , 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: encodes one of two 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 , yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L 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 provides an attractive strategy for improving product yields of acetate-reducing 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 , 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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http://dx.doi.org/10.1186/s13068-017-0791-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406903PMC
April 2017

Membrane potential independent transport of NH in the absence of ammonium permeases in Saccharomyces cerevisiae.

BMC Syst Biol 2017 04 17;11(1):49. Epub 2017 Apr 17.

Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands.

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 (NH) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH 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 NH ion. Subsequent analysis revealed that growth of this ∆mep strain was dependent on the extracellular NH concentrations. Metabolomic analysis revealed a significantly higher intracellular NH concentration (3.3-fold) in the ∆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, NH, is able to diffuse into the cell. The measured intracellular/extracellular NH ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NH compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the ∆mep strain than in the reference strain, which suggests that the lower biomass yield of the ∆mep strain was related to higher turnover rates of biomass components.
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http://dx.doi.org/10.1186/s12918-016-0381-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392931PMC
April 2017

Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis.

Sci Rep 2017 04 12;7:46155. Epub 2017 Apr 12.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca/Mn ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.
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http://dx.doi.org/10.1038/srep46155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388867PMC
April 2017

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism.

FEMS Yeast Res 2017 01;17(1)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.

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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http://dx.doi.org/10.1093/femsyr/fox006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424818PMC
January 2017

Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae.

FEBS J 2017 02 1;284(4):654-665. Epub 2017 Feb 1.

Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.

NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO , and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc ) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc S. cerevisiae on glucose, and this capacity was dependent on the availability of CO . On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc strains from C auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C compounds and redox shuttling in plant cells.
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http://dx.doi.org/10.1111/febs.14013DOI Listing
February 2017

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations.

Biotechnol Biofuels 2016 12;9:173. Epub 2016 Aug 12.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Background: Acetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor.

Results: Preadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50-55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive.

Conclusions: A novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.
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http://dx.doi.org/10.1186/s13068-016-0583-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983051PMC
August 2016