EC Accepted Manuscript Posted Online 12 June 2015 Eukaryotic Cell doi:10.1128/EC.00064-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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A minimal set of glycolytic genes reveals strong

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redundancies in Saccharomyces cerevisiae central

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metabolism.

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Daniel Solis-Escalantea, Niels G. A. Kuijpersa, Nuria Barrajon-Simancas, Marcel van den Broek,

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Jack T. Pronk, Jean-Marc Daran and Pascale Daran-Lapujade #.

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

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Running title: A yeast minimal glycolysis.

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#

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E-mail: [email protected].

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Corresponding author: Dr. Pascale Daran-Lapujade, Tel.: +31 15 2789965, fax: +31 15 2782355,

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These authors contributed equally to this work.

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Abstract

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As a result of ancestral whole genome and small-scale duplication events, the genome of

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Saccharomyces cerevisiae’s, and of many eukaryotes, still contains a substantial fraction of

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duplicated genes. In all investigated organisms, metabolic pathways, and more particularly

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glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the

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Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an

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important role leading to S. cerevisiae current lifestyle favoring fermentative metabolism even

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in the presence of oxygen and characterized by a high glycolytic capacity. In modern S.

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cerevisiae, the 12 glycolytic reactions leading to the biochemical conversion from glucose to

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ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of

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this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (MG

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strain). Remarkably, a combination of quantitative, systems approach and of semi-quantitative

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analysis in a wide array of growth environments revealed the absence of phenotypic response

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to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication

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of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative

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capacities that are characteristic for S. cerevisiae and essential for many of its industrial

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applications, and argues against gene dosage effects as a means for fixing minor glycolytic

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paralogs in the yeast genome. MG was carefully designed and constructed to provide a robust,

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prototrophic platform for quantitative studies, and is made available to the scientific

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community.

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Introduction

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Gene duplication plays a key role in evolution by providing DNA templates for evolutionary

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innovation, while preventing interference with the cellular function of the original genes (1-3).

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Immediately after gene duplication, the resulting paralog pairs are usually identical and,

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therefore, functionally redundant. Unless duplication confers a selective advantage, either via

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gene-dosage effects or via mutational acquisition of modified or new functions, duplicated

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genes will eventually be pseudogenized and/or lost from the genome (2; 3).

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Gene duplication played a key role in the evolutionary history of the yeast

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Saccharomyces cerevisiae, an intensively investigated model in eukaryotic evolutionary biology.

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A whole-genome duplication event (WGD) in an ancestor of S. cerevisiae, ca. 100 My ago, was

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followed by loss of ca. 90% of the resulting gene duplications (4; 5). Despite the long time

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interval that separates current S. cerevisiae strains from the WGD, many surviving paralog pairs

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still exhibit a substantial degree of functional redundancy, as indicated by neutral or weak

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phenotypes of single-paralog deletion mutants (6-9). However, the selective pressures that

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caused the long-term retention of these functionally redundant paralogs remain elusive (1; 10).

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The Embden-Meyerhof-Parnas (EMP) pathway, the main route for oxidation of glucose

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to pyruvate in all eukaryotes and in many other organisms, is among the most slowly evolving

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metabolic pathways (11; 12). In all taxa, paralog families are found for the structural genes that

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encode the EMP enzymes (12-14). Under conditions of oxygen limitation and/or sugar excess, S.

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cerevisiae couples the EMP pathway to the fermentative production of ethanol via pyruvate

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decarboxylase and NAD+-dependent alcohol dehydrogenase (15; 16). In this publication, we use

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the term ‘glycolysis’ to indicate the set of twelve enzyme-catalyzed reactions in yeast that

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convert intracellular glucose to ethanol.

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In S. cerevisiae, no fewer than eight of the twelve enzyme reactions in glycolysis are

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represented by multiple paralogous genes (Fig. 1). This incidence represents a significant

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overrepresentation (p = 1.9 x 10-10 , based on hypergeometric distribution analysis) relative to

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the ca. 26% of the yeast genome that consists of parologous combinations (17). The WGD event

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and resulting duplication of genes involved in central metabolism have been implicated in the

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appearance of several key physiological characteristics of S. cerevisiae. In particular, the

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duplication of glycolytic genes has been proposed to have contributed to the strong tendency of

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S. cerevisiae to produce ethanol under aerobic conditions (Crabtree effect) and its high

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glycolytic capacity (18-20). However, the impact of reducing the number of glycolytic paralogs

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on these and other physiological characteristics of S. cerevisiae has not been systematically

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explored. In all paralogous gene sets in yeast glycolysis, with the notable exception of

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phosphofructokinase, gene expression and gene deletion studies support the definition of a

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single, major paralog and one to four minor paralogs (Fig. 1). Except for the pseudogenes GPM2

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and GPM3, all paralogs have retained their original catalytic function, although their context-

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dependent expression profiles differ (Fig. 1). Deletion of minor paralogs for individual glycolytic

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enzymes has minor effects on enzyme activities in cell extracts and on specific growth rate

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under standard laboratory conditions (usually shake-flask cultivation on yeast extract, peptone

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and glucose, Fig. 1 and (21-25)). Several hypotheses have been forwarded to explain the neutral

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effect of paralog deletion (6; 26-31). These include experimental limitations such as the poor

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sensitivity of fitness screens (32) and the narrow range of cultivation condition tested (generally

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complex medium) (33). Additionally, analysis of deletion mutants in which paralogs that encode

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a single glycolytic enzyme are inactivated, cannot reveal synergistic effects of the minor

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paralogs of different glycolytic enzymes. Such synergistic effects might, for example, arise from

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the well documented phenomenon of distribution of metabolic control over multiple enzymes

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in metabolic pathways (34; 35) or from other regulatory or catalytic interactions.

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Insight into the importance, under laboratory conditions, of the minor glycolytic paralogs

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is essential for understanding an apparent genetic redundancy in a key, ubiquitous metabolic

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pathway in an important model organism and industrial platform. In addition, analyzing the

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extent of genetic redundancy in central metabolism is highly relevant for the complete redesign

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and construction of entirely synthetic yeast genomes, as pursued in the Synthetic Yeast 2.0

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initiative (36). Moreover, if complexity in yeast glycolysis can be significantly reduced by

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eliminating ‘redundant’ isoenzymes, this could eliminate uncertainties and, thereby, facilitate

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the formulation and validation of mathematical models that describe the kinetics of this key

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metabolic pathway (37; 38).

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The goals of the present study are to experimentally explore genetic redundancy in yeast

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glycolysis by cumulative deletion of minor paralogs and to provide a new experimental platform

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for fundamental yeast research by constructing a yeast strain with a functional ‘minimal

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glycolysis’. To this end, we deleted 13 minor paralogs, leaving only the 14 major paralogs for the

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S. cerevisiae glycolytic pathway. The cumulative impact of deleting all minor paralogs was

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investigated by two complementary approaches. A first, quantitative analysis focused on the

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impact on glycolytic flux under a number of controlled cultivation conditions that, in wild-type

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strains, result in different glycolytic fluxes. These quantitative growth studies were combined

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with transcriptome, enzyme-activity and intracellular metabolite assays to capture potential

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small phenotypic effects. A second, semi-quantitative characterization explored the phenotype

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of the ‘minimal glycolysis’ strain under a wide array of experimental conditions to identify

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potential context-dependent phenotypes.

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Materials and Methods

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Strains and strain construction.

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Plasmid propagation and isolation were performed with chemically competent Escherichia coli

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DH5α (Z-competentTM transformation kit, Zymo research, Orange, CA) cultivated in Lysogeny

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broth (LB) media (39; 40), supplemented with 100 mg L-1 ampicillin (LBAmp) when required. All

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yeast strains are derived from the CEN.PK family (41-43) and are listed in SI, Table S3. All strains

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were stored at -80° C in 1 mL aliquots of 30% glycerol and the appropriate medium. CEN.PK102-

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12A was selected as parental strain for the minimal glycolysis strain (MG). The order of gene

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deletions that led to the final minimal glycolysis strain IMX372 was GLK1, HXK1, TDH1, TDH2,

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GPM2, GPM3, ENO1, PYK2, PDC5, PDC6, ADH2, ADH5 and ADH4. The genes GLK1, HXK1, TDH1

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and TDH2 were deleted using the auxotrophic and dominant markers Sphis5 (44; 45), KlLEU2

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(45; 46), KanMX (47; 48) and hphNT1 (49; 50); respectively. These markers remained in the

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genome during all the strain construction process. GPM2 to ADH4 deletions were performed

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using a strategy of selection and counter-selection with the KlURA3 / 5-FOA system (51) for the

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recovery of the marker module. KlURA3 was removed seamlessly as previously described (52;

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53). The dominant marker modules KanMX and nphNT1 were removed using deletion cassettes

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containing KlURA3 and amdSYM, respectively. These markers were sequentially removed as

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reported previously (54). To obtain a prototrophic strain, a cassette containing the marker

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module KlURA3 was integrated in the TDH1 locus, this generated the MG strain. All genetic

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modifications were confirmed by PCR and later by whole-genome sequencing.

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Molecular biology techniques.

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All integrative cassettes were constructed using Phusion Hot Start II High Fidelity Polymerase

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(Thermo Scientific, Landsmeer, The Netherlands) following manufacturer’s recommendations

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and the primer pairs listed in SI, Table S4 with the plasmids in SI, Table S5 as templates. Correct

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integrations, deletions and marker excision were confirmed by PCR using Dreamtaq polymerase

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(Thermo Scientific) and following manufacturer’s recommendations. Primers used for

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confirmation are listed in SI, Table S4. Genomic DNA that served as template for PCR was

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obtained by extraction with 0.05N NaOH directly from single colonies or by purification using

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YeaStar Genomic DNA kit (Zymo research, Orange, CA) following manufacturer’s

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recommendations. All PCR products were loaded on gels containing 1% (w/v) agarose (Thermo

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Scientific) and 1xTAE buffer (Thermo Scientific). Integrative cassettes were gel-purified using

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Zymoclean™ Gel DNA Recovery Kit (Zymo research). Yeast strain transformations were

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performed with the lithium acetate protocol as previously described (55). Plasmids were

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isolated from E. coli with GenElute Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO).

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Media

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Complex and non-selective media for grow rate determination and propagation contained 10 g

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L-1 yeast extract, 20 g L-1 peptone and 20 g L-1 glucose (YPD). When selection was required, YPD

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was supplemented with 200 mg L-1 G418 (YPD+G418) or 200 mg L-1 hygromycin (YPD+Hyg).

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Synthetic media (SM) containing 6.6 g L-1 K2SO4, 3 g L-1 KH2PO4, 0.5 g L-1 MgSO4.7H2O, 5 g L-1

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(NH4)2SO4, 1 mL L-1 of a trace element solution and 1 mL L-1 of a vitamin solution as previously

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described (56). SM was supplemented with 20 g L-1 glucose (SMG) for propagation, growth rate

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determination and batch cultivations. When auxotrophic strains were cultivated, SMG was 8

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supplemented with 150 mg L-1 uracil, 125 mg L-1 histidine and/or 500 mg L-1 leucine, according

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to strain auxotrophy (57). Selection of strains lacking the KlURA3 marker was done in SMG

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supplemented with 150 mg L-1 uracil and 1 g L-1 5-fluoroorotic acid (SMG-5FOA). Solid media

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were obtained by addition of 20 g L-1 agar or agarose. Unless otherwise stated, liquid cultures

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were performed in 500 mL little shake flasks with a working volume of 100 mL and incubated at

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30° C and 250 rpm, the targeted starting OD being 0.5. unless otherwise stated cultures on solid

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media were incubated for 3 days at 30° C.

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Semi-quantitative phenotypic characterization on plates

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Growth on MG and of the prototrophic control strain CEN.PK113-7D was performed on agar

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plates containing SMG (2% glucose), SMGal (2% galactose), SMMal (2% maltose), SMSuc (2%

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sucrose), YPD, SMEtOH (3%(v/v) ethanol), SMGlyc (6% glycerol), SMHG (5% glucose), SMLG

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(0.5% glucose), SMG-Lit (2.5, 5 or 10 mM LiCl), SMGSa (200 or 500 mM NaCl), SMGCad (50, 100

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or 200 µM CdCl2), SMGSor (1, 1.5 or 2 M Sorbitol), SMGpH (4, 6 or 7.5 pH) or SMGOx (1mM

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H2O2). Spot plates were inoculated with 101 to 105 cells obtained by serial dilutions of liquid

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cultures grown to exponential phase on SMG. All plates were incubated at 30°C for 3 days,

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except for plates containing SMGlyc which were incubated for 6 days.

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Quantitative characterization of MG’s physiology in shake flask.

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Growth rate determination was performed by OD660 measurement of cultures grown in 500 mL

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shake flasks containing 100 mL of YPD, SMG or SMEtOH. Cultures for growth rate measurements

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were inoculated with pre-cultures grown until late exponential phase in identical conditions (i.e.

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same medium and temperature). 9

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Glucose/Ethanol/Glucose switches were started by inoculation of SMG shake-flasks with a pre-

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culture grown overnight on the same medium. At mid-exponential phase (OD of ca. 4) samples

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were taken, washed twice with sterile demineralized water and used to inoculate shake flasks

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containing 100 mL of SMEtOH. When reaching an OD of 3, a sample from the culture was

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washed twice with sterile demineralized water and used to inoculate a 500 mL shake flask

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containing 100 mL SMG. Growth was then followed until late exponential phase.

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To evaluate tolerance of MG to oxidative stress, cells grown to exponential phase in shake flasks

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containing SMG were transferred to a shake flask containing SMG supplemented with 4.5 mM

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H2O2. Samples were then taken every 20 min for 1 h, diluted and plated on SMG plates to reach

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approximately 100 cells per plate. Colony forming units (CFU) were used to determine the

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percentage of survival cells.

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Quantitative characterization of MG’s physiology in bioreactor.

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Aerobic and anaerobic batch cultivations were performed in 2 L laboratory fermenters

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(Applikon, Schiedam, The Netherlands) with 1 L working volume. SM medium was used and set

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at pH 5 before autoclaving at 121° C. Antifoam Emulsion C (Sigma) was added to a concentration

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of 0.2 g L-1 from a 20% (v/v) solution autoclaved separately (121° C). Prior to inoculation, glucose

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was added to a final concentration of 20 g L-1 from a sterile (110° C) 50% glucose solution and 1

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mL of a vitamin solution (56). Anaerobic cultivations were supplemented with 0.01 g L-1

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ergosterol and 0.42 g L-1 Tween 80 dissolved in ethanol as previously described (56). The pH was

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maintained at 5 by the automatic addition of 2 M KOH. Compressed air or gaseous nitrogen

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(quality 5.0, >99.999% vol N2,

A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism.

As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a ...
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