Glycolytic functions are conserved in the genome of the wine yeast Hanseniaspora uvarum and pyruvate kinase limits its capacity for alcoholic fermentation.

AEM Accepted Manuscript Posted Online 8 September 2017 Appl. Environ. Microbiol. doi:10.1128/AEM.01580-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved.

Glycolytic functions are conserved in the genome of the wine yeast

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Hanseniaspora uvarum and pyruvate kinase limits its capacity for

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alcoholic fermentation Anne-Kathrin Langenberg 1, Frauke J. Bink 1, Lena Wolff 1, Stefan Walter 2, Christian

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von Wallbrunn 3, Manfred Grossmann 3, Jürgen J. Heinisch 1,#, Hans-Peter Schmitz 1,#

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1

Universität Osnabrück, Fachbereich Biologie/Chemie, AG Genetik, Barbarastr. 11, D-49076 Osnabrück, Germany 2

Universität Osnabrück, Fachbereich Biologie/Chemie, Mikroorganismen, Barbarastr. 13, D-49076 Osnabrück, Germany

Genetik

der

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Hochschule Geisenheim, Institut für Mikrobiologie und Biochemie, Von-Lade-Str. 1, D-65366, Germany A.-K.L. and F.J.B. were funded by grants from „Forschungsring des Deutschen Weinbaus“ (FDW).

Running title: Hanseniaspora uvarum alcoholic fermentation and genome

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Keywords:

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# Corresponding authors: Hans-Peter Schmitz and Jürgen J. Heinisch Universität Osnabrück Fachbereich Biologie/Chemie AG Genetik Barbarastr. 11 D-49069 Osnabrück Germany Tel.: +49 (0) 541-969-2290 Fax: +49 (0) 541-969-2349

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Angewandte

E-mails:

enology, genetic markers, chromosomes, ploidy, Apiculatus yeast

[email protected] [email protected]

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Downloaded from http://aem.asm.org/ on September 10, 2017 by FUDAN UNIVERSITY

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ABSTRACT

38

Hanseniaspora uvarum (anamorph Kloeckera apiculata) is a predominant yeast on wine grapes

39

and other fruits and has a strong influence on wine quality, even when Saccharomyces cerevisiae

40

starter cultures are employed. In this work we sequenced and annotated approximately 93% of

41

the H. uvarum genome. Southern and synteny analyses were employed to construct a map of the

42

seven chromosomes present in a type strain. Comparative determinations of specific enzyme

43

activities within the fermentative pathway in H. uvarum and S. cerevisiae indicated that the

44

reduced capacity for ethanol production of the former yeast is primarily caused by an

45

approximately ten-fold lower activity of the key glycolytic enzyme pyruvate kinase.

46

Heterologous expression of the encoding gene HuPYK1 and the two genes encoding the

47

phosphofructokinase subunits, HuPFK1 and HuPFK2, in the respective deletion mutants of S.

48

cerevisiae confirmed their functional homology.

49 50

IMPORTANCE

51

Hanseniaspora uvarum is a predominant yeast species on grapes and other fruits. It contributes

52

significantly to the production of desired as well as unfavorable aroma compounds and thus

53

determines the final product quality, especially in wine. Despite this obvious importance,

54

knowledge on its genetics is scarce. As a basis for targeted metabolic modifications, we here

55

provide the results of a genomic sequencing approach, including the annotation of 3010 protein

56

coding genes, e.g. encoding the entire sugar fermentation pathway, key components of stress-

57

response signaling pathways, and enzymes catalyzing the production of aroma compounds.

58

Comparative analyses suggest that the low fermentative capacity of H. uvarum as compared to

59

Saccharomyces cerevisiae can be attributed to a low pyruvate kinase activity. The data reported

2


37

60

here are expected to aid in establishing H. uvarum as a non-Saccharomyces yeast in starter

61

cultures for wine and cider fermentations.

62


3

INTRODUCTION

64

Alcoholic fermentation in wine industry is generally attributed to the activity of the wine, beer

65

and baker’s yeast Saccharomyces cerevisiae, which in large-scale fermentations is routinely

66

added as starter cultures with a cell density of approximately 106 cells/ml (1). Even without the

67

addition of such starter cultures, i.e. in spontaneous fermentations, S. cerevisiae usually

68

dominates the fungal population after the first few days of vinification, explaining its importance

69

for mankind over thousands of years (2, 3). Besides ethanol S. cerevisiae produces several other

70

compounds which determine the final wine quality, including glycerol, different esters, and fusel

71

alcohols (4). Such compounds may be either desired or deleterious, frequently depending on their

72

concentration. Acetate produced as a by-product of fermentation is generally regarded as

73

unfavourable (5).

74

In contrast to its prevalence in the later stages of fermentation, S. cerevisiae displays a low

75

abundance on the skin of grapes or in freshly prepared musts, with as few as one cell found on

76

1000 grapes (6). Instead, Hanseniaspora uvarum is the predominant yeast species and frequently

77

constitutes more than 80% of the yeast population in the must during early stages of fermentation

78

(7, 8). H. uvarum is also still widely known for its imperfect form, Kloeckera apiculata, which

79

coined the name “apiculate yeasts" due to its lemon-shaped cell morphology. It prevails within

80

the first 48 h of fermentation, after which S. cerevisiae usually takes over and persists until the

81

end (9, 10). Initially, the decline of the H. uvarum population was attributed to its higher

82

sensitivity towards increasing ethanol concentrations in the course of fermentation. However,

83

recent studies indicate that antimicrobial peptides secreted by S. cerevisiae are more important to

84

reduce the population of non-Saccharomyces yeasts (11, 12). Nevertheless, H. uvarum forms a

85

major part of the fungal microbiome in wine fermentations worldwide, including Austria (13),

86

Brazil (14), China (15), France (16), Germany (17), Italy (18, 19), Portugal (20), Slovakia (21) 4


63

and Spain (22). It should be noted that H. uvarum not only inhabits and ferments grapes, but also

88

resides on other fruits like plums (23, 24) and apples, being a major factor in cider production

89

(25, 26). The yeast is also found on more exotic substrates, such as african coffee (27) and in

90

chocolate production (28, 29). H. uvarum has further been suggested to be a useful agent in the

91

biocontrol of molds like Botrytis cinerea, one of the major plant pathogens (30). On the other

92

hand, it is considered a spoilage yeast in orange juice, together with S. cerevisiae (31), and

93

produces a killer toxin active against S. cerevisiae and Candida albicans (32, 33). These

94

examples demonstrate the widespread nature, economical importance and future potential of H.

95

uvarum in food biotechnology.

96

Regarding vinification, different natural isolates of H. uvarum are known for their high capacities

97

to form fruity esters, but are also infamous for producing high levels of acetate and ethyl acetate

98

(28, 34). Since during the first days of wine fermentation H. uvarum can be present at cell

99

densities similar to those of S. cerevisiae, even if the latter is added as starter cultures, its

100

metabolism is expected to contribute significantly to the aroma profile and the final wine quality

101

(5). In fact, after initial classifications as a spoilage yeast, different isolates have been tested for

102

their performance in wine fermentations in mixed cultures with S. cerevisiae, with promising

103

perspectives (35). In addition, a type strain of H. uvarum has been reported to be fructophilic,

104

which could be of special importance regarding stuck fermentations attributed to an imbalance

105

between glucose and fructose (19, 36).

106

Despite this obvious importance of H. uvarum in all kinds of fruit fermentations, data on its

107

genetic make-up remained scarce. Thus, karyotyping approaches suggested the presence of 7-9

108

chromosomes, with high variability between different isolates (37, 38). As expected, H. uvarum

109

also appears in deep-sequencing approaches addressing the microbiome in wine fermentations,

110

since it dominates the yeast population and is readily identified by its rDNA sequences (39, 40). 5


87

However, only a few genes have been isolated and sequenced, such as those encoding pyruvate

112

decarboxylase or actin (40, 41). The mitochondrial genome of H. uvarum has an exceptional

113

structure among fungi, as it is represented by a short, linear DNA molecule (42). Recently, two

114

brief reports on whole genome approaches have appeared for H. uvarum isolates, underlining the

115

growing interest in this yeast, but with limited information regarding the genome annotations

116

((43) and Genbank accession number JPPO00000000).

117

Clearly, determination of its physiological capacities, with the future possibility to employ

118

metabolic design to eliminate undesirable traits, would profit from more detailed genetic studies.

119

We therefore initiated a genome sequencing project with a readily available H. uvarum type

120

strain. Applying next generation sequencing approaches, an estimated 93% of the total genome

121

sequence was deciphered, with >80% assigned to contigs larger than 50 kb. From the annotation

122

of these data and additional manual sequencing of specific PCR products we identified more than

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4000 predicted protein coding genes, 3043 of which were assigned putative functions by

124

comparison with characterized yeast genomes. This included the structure of the highly repetitive

125

rDNA repeats. We applied this information to identify genes coding for hexose transporters, the

126

entire glycolytic pathway, and the fermentative pyruvate decarboxylase and alcohol

127

dehydrogenases. Specific enzyme activities relevant for alcoholic fermentation were determined

128

in crude extracts from H. uvarum and S. cerevisiae. The functionality of genes encoding key

129

enzymes of this pathway was confirmed by complementation studies in the respective S.

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cerevisiae deletion mutants.

131

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MATERIALS AND METHODS

133

Strains, plasmids, media and culture conditions. In this study, the type strain DSM2768 from

134

H. uvarum was obtained from the German collection of microorganisms. It is equivalent to

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ATCC9774 (American Type Culture Collection). Presumably aneuploid derivatives with

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mutations in the HuURA3 gene were obtained by treatment with nocodazole prior to selection on

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medium containing 5-fluoro-orotic acid (5-FOA). Haploid and diploid reference strains from

138

Kluyveromyces lactis employed in FACS analyses were the type strain CBS2359 and KHO70,

139

respectively (44). For preparation of S. cerevisiae chromosomes as a size standard, strain

140

BY4743 (EUROSCARF, Frankfurt, Germany) was used. For comparisons of fermentative

141

enzyme activities we used the diploid S. cerevisiae strain HHD1 (MATa/MATalpha ura3-

142

52/URA3 LEU2/leu2-3,112) as a reference, a derivative of the CEN.PK series previously applied

143

in fermentations for spirit production (45). HD114-8D (MATalpha ura3-52 leu2-3,112 his3-11,15

144

pfk1::HIS3 pfk2::HIS3; (46)) served as a recipient strain for PFK gene expression and VWH3B

145

(MATalpha ura3-52 leu2-3,112 his3Δ1 trp1-289 pyk1::HIS3), also a derivative of the CEN.PK

146

series, was used for expression of genes encoding pyruvate kinase.

147

Standard procedures in the handling of yeast, E. coli strain DH5α and DNA were followed as

148

described previously (47). Genes from H. uvarum were amplified by PCR using the High Fidelity

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Taq polymerase kit from Roche (Mannheim/Germany) and cloned by restriction/ligation into

150

yeast/E. coli shuttle vectors, by either using natural restriction sites or adding the recognition

151

motifs to the 5'-ends of the primer sequences, if required. Plasmids employed were based on the

152

CEN/ARS vector YCplac33 (48) and the 2 µm vector YEp352 (49). Genes were amplified with

153

their flanking regions from genomic DNA preparations by PCR with appropriate primer pairs and

154

cloned by conventional restriction/ligation. Sequences of oligonucleotides and the resulting

155

plasmids are available upon request. Specifically, the PYK1 gene from S. cerevisiae was cloned 7


132

as a BamHI/HindIII fragment into YCplac33 to yield pJJH2137. The gene from H. uvarum

157

(HuPYK1) was also cloned as a BamHI/HindIII fragment into the same vector yielding

158

pJJH2144. To place the open reading frame of HuPYK1 between the promoter and terminator

159

regions of ScPYK1, pJJH2145 was obtained by in vivo recombination using pJJH2137 as a

160

recipient plasmid and a PCR fragment with HuPYK1 and the appropriate flanking regions for

161

recombination. For phosphofructokinase clones, ScPFK1 was cloned as a SphI fragment into

162

YCplac33 to yield YCp111u and ScPFK2 as a SacI/SalI fragment to yield pJJH2203. The latter

163

fragment was also inserted into YCp111u to give pJJH2200, which carries both ScPFK genes on

164

the CEN/ARS vector. PFK genes from H. uvarum were obtained as follows: HuPFK1 was

165

obtained as a SacI/HindIII fragment and cloned into YCplac33 (pJJH2185) and YEp352

166

(pJJH2208). HuPFK2 was obtained as a SacI/PstI fragment and cloned into YCplac33

167

(pJJH1868) and YEp352 (pJJH2209). Plasmids containing both HuPFK genes were obtained by

168

cloning HuPFK2 as a SalI/SphI fragment into pJJH2208 to yield the multicopy plasmid

169

pJJH2211, and by simultaneously cloning HuPFK1 as a BamHI/SacI fragment and HuPFK2 as a

170

SacI/PstI fragment into YCplac33 linearized with BamHI/PstI to yield the CEN/ARS plasmid

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pJJH1856new.

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Rich media were based on 1% yeast extract and 2% bacto peptone (Difco), with 2% glucose

173

(w/v) as a carbon source (YEPD), if not stated otherwise. Synthetic media were based on yeast

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nitrogen base with ammonium sulfate, supplemented with 2% glucose and a mixture of amino

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acids and bases as described (50), omitting compounds for plasmid selection as required.

176

Mixtures of 10% glucose (w/v) plus 10% fructose (w/v) were used to mimick the initial stages of

177

grape must fermentation, and 1% (w/v) of each sugar for later stages.

178 179 8


156

Preparation of genomic DNA and sequencing.

181

The preparation of larger amounts of genomic DNA for sequencing of the H. uvarum genome

182

was carried out with the “DNeasy® Blood and Tissue Kit” (Qiagen, Hilden - Germany)

183

following manufacturer´s instructions. The resulting DNA was eluted in sterile, distilled water

184

and showed absorbance ratios at 260/280 nm ≥ 1.8 and at 260/230 nm ≥ 1.9.

185

LS454 sequencing using GS FLX Titanium Technology (Roche, Basel Switzerland) was done by

186

Microsynth AG (Balgach, Switzerland) and an additional PacBio RS sequencing was performed

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by GATC Biotech (Konstanz, Germany) and data were combined to generate the final assembly.

188

The assembly was performed by GATC Biotech using a hybrid approach for error correction of

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PacBio reads (51) with a final assembly performed by the Celera Assembler (version May 2013).

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Custom Sanger sequencing was performed by GATC Biotech (Cologne/Germany) with

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oligonucleotide primers obtained from Metabion (Munich/Germany).

192 193

Genome annotation and synteny analysis.

194

The main objective of the genome annotation was to accomplish a high data reliability. In a first

195

step

196

http://wolfe.ucd.ie/annotation/). This pipeline employs pre-existing annotations of related species

197

and also includes synteny information. YGAP resulted in a compilation of putative open reading

198

frames (ORFs) and a list of tRNA genes. The YGAP list contained 4300 entries. We decided to

199

further scrutinize this list, since it also contained incomplete ORFs (e.g. those with missing Start-

200

or Stop-codons). In addition, we found that automated annotation of intron-containing ORFs was

201

not reliable and thus excluded all entries of ORFs not being continous between a Start- and a

202

Stop-codon. To this end, a list using GetORF (part of the EMBOSS package; (53)) was generated

203

from the assembly and only entries present in the YGAP and the GetORF list were incorporated

we

therefore

used

the

yeast

genome

9

annotation

pipeline

(YGAP;

(52);


180

in further annotations. This resulted in the annotation of 3010 protein coding sequences. Deduced

205

tRNA genes were then added to the resulting list. Annotation of the ribosomal DNA was

206

performed by a BLAST search (54) using a sequence of H. uvarum rDNA as a query, which was

207

obtained by cloning of PCR amplified DNA fragments and conventional Sanger sequencing.

208

Sequence assembly and annotation were formatted according to the Genbank guidelines. Further

209

analysis of the data were performed using software tools from the EMBOSS package and the R

210

statistical language v 3.3.2 (55). Synteny analysis was performed using MUMmer version 3 (56).

211

The SAMtools (57) package was used for variant analysis.

212 213

Field inversion gel electrophoresis (FIGE) and Southern analysis.

214

Chromosome sizes from FIGE gels were determined using LabImage 1D (Kapelan Bio-Imaging

215

Solutions, Leipzig/Germany). A total of 25 gels obtained from varying electrophoresis conditions

216

were used to determine mean chromosome sizes. For Southern analyses, three different target

217

sequences were chosen from each contig for PCR amplification with the oligonucleotides listed

218

in Table 1. Probes for detection of a number of S. cerevisiae chromosomes were also prepared by

219

PCR with the oligonucleotides listed in Table 1B. Strain BY4743, a diploid derivative of the

220

S288C strain employed for the genome sequencing project, was used as a source for the S.

221

cerevisiae size standard (EUROSCARF collection, Frankfurt, Germany). Probes were prepared

222

with the Hexanucleotide DIG DNA Labeling kit from Roche (Mannheim/Germany).

223 224

Flow cytometry measurements of DNA content

225

Fixation and staining of cells was performed according to (58). Samples were measured on a BD

226

FACSaria II flow cytometer (Beckton Dickinson, Heidelberg, Germany). The R statistical

10


204

227

language v 3.3.2 (55) and flow cytometry packages from Bioconductor (59) were used for data

228

analysis and graphics generation.

229 Determination of specific enzyme activities. To obtain crude extracts for the determination of

231

specific enzyme activities, yeast cells were washed, broken with glass beads and the supernatant

232

obtained after 10 min centrifugation in microfuge at 13.000 × g and 4°C as described previously

233

(60). Specific activities were obtained from coupling the enzyme reactions in question to either

234

NAD(P)H oxidation or reduction with ancillary enzymes as described in (61). Tests were

235

performed in a Beckmann DU800 photometer recording kinetics at 30°C and determining the

236

slope after constant reaction rates were reached. As controls, kinetics were recorded without the

237

presence of substrates prior to starting the reactions. Buffers, concentrations of reagents and

238

ancillary enzymes employed are listed in Table 2. Protein concentrations in crude extracts were

239

determined by the Micro-Biuret method using bovine serum albumin as a standard (62).

240 241

Immunological detection of phosphofructokinase subunits. Crude extracts prepared with glass

242

beads as described above were obtained from cells grown in YEPD, boiled with loading buffer

243

and separated by SDS polyacrylamide gel electrophoresis (PAGE) on gels with 7.5% acrylamide.

244

A polyclonal antiserum raised against PFK from S. cerevisiae was employed for immunological

245

detection of H. uvarum PFK subunits, applied in a dilution of 1:10.000 (63). Since reactivity

246

against the heterologous enzyme is much weaker than that against S. cerevisiae PFK,

247

approximately 250 µg of protein from crude extracts prepared from H. uvarum cells or S.

248

cerevisiae pfk1 pfk2 double deletion mutants transformed with either HuPFK1 or HuPFK2

249

carried on multicopy plasmids were loaded in each lane of the SDS-PAGE. For transformants

250

carrying a multicopy plasmid with both heterologous PFK genes, only approximately 50 µg of 11


230

protein were loaded. This concentration was also used for strains carrying endogenous ScPFK

252

genes. Secondary anti-rabbit antibodies coupled to an infrared dye with fluorescence at 700 nm

253

were then employed for detection of PFK signals using the Odyssey imaging device as described

254

in (64). Contrast, brightness and frames of the images were adjusted using Corel PhotoPaint,

255

which was applied only to entire images shown in the figures, not to single bands.

256 257

Accession numbers. The annotated genome sequence was submitted to Genbank

258

(https://www.ncbi.nlm.nih.gov/genbank/) under the accession number APLS01000000. The two

259

HuPFK sequences were obtained again independently and submitted under the accession

260

numbers MF509744 (HuPFK1) and MF509745 (HuPFK2).

261 262 263

RESULTS AND DISCUSSION

264

Selection of non-flocculating H. uvarum cells from the type strain DSM2768.

265

Basic research on other yeast species, especially on the model yeasts S. cerevisiae and

266

Kluyveromyces lactis, has profited from the use of small sets of laboratory strains after their

267

original isolation from natural sources (44, 65). Therefore, we decided to start the genetic work

268

on H. uvarum using a readily available type strain (DSM2768; DMSZ - German Collection of

269

Microorganisms and Cell Cultures, Braunschweig, Germany). As shown in Fig. 1A (left frame),

270

cells of this strain tended to cluster in liquid cultures, which impedes the use of common

271

microbiological and biochemical techniques, such as the deduction of cell numbers from OD600

272

measurements. In order to avoid this problem, a selection regime was applied, which involved

273

inoculation in liquid YEPD, shaking over night at 30°C, sedimentation without agitation for 1 h

274

at room temperature and re-inoculation from the upper medium phase. This was repeated for 12


251

more than 60 times over a period of several weeks, resulting in a strain producing exclusively

276

non-flocculating single budding cells in liquid cultures (Fig. 1A, right frame), which retained the

277

original cell size and shape and was used for all further investigations. Cells measured

278

approximately 2 x 4 µm and are thus considerably smaller than those of S. cerevisiae, which are

279

in the range of 5 x 7 µm. It should be noted that the smaller cell size of H. uvarum is also

280

reflected in the relation of optical density of the cultures to life-cell platings. Thus, we found that

281

an OD600 of 1 equals approximately 108 colony forming units (cfu) in H. uvarum as opposed to

282

107 cfu for S. cerevisiae. This should be taken into account when using H. uvarum for

283

inoculations in co-cultures with S. cerevisiae for experimental wine fermentations.

284 285

Examination of the genome composition. In order to assess the ploidy of the H. uvarum type

286

strain employed herein, we determined the DNA content per cell using Sybr green stained G0

287

cells with flow cytometry. According to a recent study this method provides the best alternative

288

for ploidy estimation of yeast cells (58). As controls, haploid and diploid strains of the milk yeast

289

Kluyveromyces lactis were employed, which should have a haploid genome size of 10.7 Mbp, i.e.

290

slightly larger than that of H. uvarum with approximately 9 Mbp. As evident from Fig. 1B, the

291

DNA content per cell in the type strain of H. uvarum more closely resembled that of the diploid

292

control cultures, than that of the haploids. Moreover, we were unable to select colonies resistant

293

to 5-FOA, i.e. a mutation in the Huura3 gene, after EMS mutagenesis despite of several attempts.

294

Only after prior induction of chromosome loss by nocodazole treatment, a method inspired by a

295

work on the diploid wine yeast Zygosaccharomyces bailii (66), two Huura3 mutants were

296

obtained in presumed aneuploid derivatives (Fig. 1B). Taken together, these findings suggested

297

that the genome of the H. uvarum type strain employed herein is probably diploid. This

298

assumption is further supported by the genome sequencing data, where variants in 50 +/- 5% of 13


275

the reads were found in 10 % of the annotated open reading frames of sequences with at least 20x

300

read coverage. In addition, Fig. 2 shows that the mean of the fitted normal distribution of the

301

percentage of all variant reads in open reading frames (n= 1717) is very close to 50 %. The fact

302

that these mismatches are distributed over all large contigs assigned to the different chromosomes

303

argues in favor of the yeast being entirely diploid, rather than aneuploid for only some

304

chromosomes.

305

Different natural isolates of H. uvarum have been previously investigated for their chromosomal

306

constitution by pulse-field gel electrophoresis (PFGE), and yielded different chromosome

307

numbers and genome sizes (37). In order to check the adapted type strain described above for

308

these parameters, we used FIGE separations (field inversion gel electrophoresis). As exemplified

309

in Fig. 3A, this revealed that the type strain used herein most likely contains 7 chromosomes,

310

with numbers VI and VII being similar in size, so that they could not be separated (genome

311

analysis data presented below strongly indicate that two different chromosomes form this signal).

312

Electrophoretic mobility in FIGE analyses is inversely proportional to chromosome size, i.e. the

313

larger chromosomes migrated faster. Based on this, all chromosomes detected add up to an

314

approximate genome size of 9 Mbp (Table 3). This correlates well with the 9.6 Mbp genome size

315

calculated for another H. uvarum type strain (37). We further noticed a high degree of variation

316

observed in karyotyping different natural isolates of H. uvarum, with regard to the number of

317

chromosomal bands and the total genome sizes, which we also found in our investigations of

318

isolates from grapes from different locations in Germany and Spain (data not shown).

319

Gels used in karyotyping above were further employed for Southern blot analyses to assign the

320

larger contigs obtained in the genome sequencing described below to specific chromosomal

321

bands. Mixtures of three PCR-generated probes from each contig were used for this purpose

322

(Fig3B). All Southern data obtained are presented in Fig. S1 in supplementary materials. These 14


299

results combined with synteny analyses of the genome sequence (see below) using the YGOB

324

server ((67), ygob.ucd.ie) allowed us to create an approximate chromosome map of H. uvarum

325

(Fig. 4; for detailed descriptions of these analyses see the PhD thesis of A.K.L. (68)). An

326

indication of the validity of these analyses was provided by the finding that outward primers

327

designed for contigs 13 and 16 employed in PCR on genomic DNA of H. uvarum revealed that

328

they were indeed located next to each other and separated only by a small gap of approximately

329

900 bp (Fig. S2).

330 331

Sequence and annotation of the H. uvarum genome. In order to facilitate future genetic

332

manipulations and gain more insight into the phylogenetic position of H. uvarum, we decided to

333

take advantage of the ease of modern sequencing techniques. Since longer reads significantly

334

simplify assembly of de novo sequenced genomes, we utilized a combination of long PacBio RS

335

reads with reads from a GS FLX run used for error correction, as described in materials and

336

methods. As judged from the calculations on chromosome separations reported above, an

337

estimated 93% of the total genome sequence could thus be obtained. In summary, 360 contigs

338

with a total size larger than 9.7 Mb now represent the annotated genome.

339

Regarding its phylogenetic relationships, the genome sequence and synteny analyses indicated

340

that H. uvarum belongs to the group of yeasts not having undergone a whole genome duplication

341

and is most closely related to Kluyveromyces lactis, more distantly to Ashbya gossypii

342

(Eremothecium gossypii) and clearly distinct from the Saccharomyces sensu strictu group of

343

yeasts (Fig. 5). Interestingly, this positions H. uvarum quite close to the whole genome

344

duplication event in phylogenetic trees. It should be noted that our genome sequence available at

345

Genbank (accession number APLS01000000) has recently been included in a highly detailed

346

phylogenetic tree of yeast evolution, which supports these conclusions (69). 15


323

With respect to genome composition, relating the size of chromosome VII with the assigned

348

contigs and the size of a single rDNA repeat determined by our separate analyses of cloned PCR

349

products (Fig. S3A), we calculated a repeat number of approximately 40 rDNA units to be

350

present in the H. uvarum genome, which are located between contigs 1 and 25, presenting

351

overlapping terminal sequences (compare Fig. 4). This is about 30% the number of rDNA units

352

reported for S. cerevisiae and may be attributed to the smaller cell size of H. uvarum (Fig. 1),

353

which could explain the smaller number of ribosomes per cell.

354

Telomeres are also chromosomal structures difficult to assign by genome sequencing approaches

355

due to the large number of short nucleotide repeats. We found 28 small contigs with putative

356

telomeric sequences. Their general composition is depicted in Fig. S3B. This number well

357

exceeds that of 14 telomeres expected from the presence of seven chromosomes and may be

358

explained by the small sizes of the contigs these sequences are located on, owing to their high

359

degree of redundancy, i.e. several of these contigs may in fact constitute parts of the same

360

chromosome ends.

361

As mentioned above, H. uvarum is the sexual form of the anamorphic K. apiculata and should

362

therefore undergo meiosis and produce spores (70). Despite several attempts with the H. uvarum

363

type strain used herein, we were unable to detect any hint of sporulation or sexual reproduction.

364

In fact, the genome analyses did not reveal the presence of a dimorphic mating type locus or of

365

further silenced mating gene copies. Although this lack could be assigned to the 7% of the

366

genome not yet sequenced, alternative explanations are that sexual reproduction has been lost at

367

least in this type strain or that the mating loci escaped detection by homology and synteny

368

alignments due to a high sequence divergence. In this context it should be noted that a high

369

degree of genetic variability was observed in numerous natural isolates of H. uvarum from

16


347

wineries (71). Moreover, loss of sexual reproduction may not be uncommon, as it is frequently

371

found even in strains of S. cerevisiae used by fermentation industries (2).

372

The annotation of 3010 protein coding genes also indicated a number of physiological capacities

373

with implications to wine making, which are compiled in supplementary materials (Tables S1 to

374

S3). Thus, homologs of all genes encoding enzymes necessary for glucose fermentation,

375

including putative hexose transporters, were found. However, as expected from a non-duplicated

376

genome, less redundancy was observed, with a maximum of eight putative hexose transporter

377

homologs as compared to 20 in the S. cerevisiae genome ((72); Table S1). Likewise, only one

378

homolog each of the two hexokinase and two enolase genes present in S. cerevisiae could be

379

identified in the H. uvarum genome. Regarding possible effects on wine quality, we were able to

380

identify 28 genes in H. uvarum with putative functions in production of aroma compounds, which

381

include esterases and glycosidases, but also overlap with the subset involved in central

382

carbohydrate metabolism in the production of glycerol and aldehydes (Table S2). Interestingly, at

383

least six putative genes for xylose degrading activities could be identified, which are only

384

represented by one homolog in either S. cerevisiae or Aspergillus nidulans, supporting the notion

385

that H. uvarum contributes significantly to the production of aroma compounds in the process of

386

vinification. Finally, survival in the vineyard and in wine fermentations requires the presence of

387

various stress response pathways in the yeast cells (73), which we also detected in H. uvarum

388

(Table S3). This includes homologs of most components of the yeast cell wall integrity pathway

389

(CWI), again with less redundancy of components of the downstream mitogen activated protein

390

kinase (MAPK) pathway (74). Interestingly, H. uvarum seems to have a single isoform of protein

391

kinase C (Pkc), which like in S. cerevisiae displays a prototypic structure of Pkc isoforms found

392

in higher eukaryotes, including the regulatory homology domains mediating interaction with a

393

small GTPase located near the N-terminal end (75, 76). In addition, homologs encoding proteins 17


370

of the high osmolarity glycerol (HOG) pathway (77) were identified. Although some

395

intermediary components have not been annotated, the homologs detected indicate that the

396

pathway, like its counterpart in S. cerevisiae is bifurcated in the upstream signaling components.

397

We also found several genes with putative functions in the oxidative stress response (78) (Table

398

S3). Although transcription factors like Yap1 and Skn7 did not appear in this search, the presence

399

of detoxifying enzymes such as Sod1 and Gsh1/Gsh2 indicate that H. uvarum encounters and is

400

well equipped to cope with oxidative stress. Together, these findings suggest that H. uvarum

401

disposes of the necessary mechanisms to respond properly to stresses encountered in the

402

vinification process.

403 404

Comparative investigation of fermentative capacities of H. uvarum and S. cerevisiae.

405

H. uvarum and other non-Saccharomyces yeasts which dominate the first stage of spontaneous

406

wine fermentations have a limited capacity for alcohol production (79). We decided to address

407

this question in more detail by determining the specific activities of all 12 enzymes involved in

408

alcoholic fermentation in H. uvarum under different growth conditions and compare them to

409

those of a diploid laboratory strain of S. cerevisiae (HHD1). The latter is derived from the

410

CEN.PK series (80), has a fermentation capacity and aroma profile similar to commercial yeast

411

strains and is thus also suitable for spirit production (45).

412

As summarized in Table 4, most of the specific enzyme activities measured in extracts from cells

413

grown in the presence of 1% glucose + 1% fructose are in the same range for H. uvarum and S.

414

cerevisiae, i.e. differences are less than two-fold. This indicates a general conservation and a

415

similar capacity of the glycolytic pathways and the subsequent reactions leading to ethanol

416

production in the two species. However, pyruvate kinase shows a striking difference, with H.

417

uvarum having a more than 15-fold lower specific activity than S. cerevisiae. This is especially 18


394

interesting, since pyruvate kinase catalyzes the second irreversible step specific for glycolysis

419

after phosphofructokinase (PFK), thus being an ideal target to control metabolic flux. In fact,

420

pyruvate kinase has been suggested to be a major determinant at the branching point of

421

respiratory and fermentative sugar degradation (81). Of note, fructose-1,6-bisphosphate, the

422

product of the PFK reaction, is a potent allosteric activator of pyruvate kinase in S. cerevisiae,

423

thus connecting the two controlling steps of sugar degradation (82). We conclude that the reduced

424

activity of pyruvate kinase in H. uvarum may be a major factor explaining the lower fermentative

425

capacity of this yeast as compared to S. cerevisiae and also its classification as Crabtree-negative

426

yeast (83). It should be noted that specific pyruvate kinase activities increase both in S. cerevisiae

427

(approximately 2-fold) and in H. uvarum (approximately 10-fold) when cells are grown in the

428

presence of 20% as opposed to 2% sugar, i.e. under conditions similar to must fermentations (Fig.

429

S4). Nevertheless, activities are still three-fold lower in H. uvarum than in S. cerevisiae, and are

430

expected to decrease in the course of wine fermentations, as sugars are degraded. Although the

431

specific activities of several other enzymes tested after growth under high sugar concentrations

432

were also higher, the increase was similar in H. uvarum and in S. cerevisiae, not affecting the

433

ratio of less than two-fold differences between the two species. The same is true for the generally

434

lower specific activities measured after growth on 2% ethanol as a non-fermentable carbon

435

source (Fig. S4).

436

From data obtained for a different H. uvarum isolate grown in chemostat cultures at different

437

dilution rates, it was previously concluded that glucose degradation is limited by a step "before

438

pyruvate formation", which is in good agreement with our findings (84). The authors determined

439

the specific activities of a number of fermentative enzymes and those of pyruvate dehydrogenase

440

and acetyl-CoA-synthase, concluding that the low activity of the latter is a major cause for

441

acetate production. It should be noted that the specific activities of the fermentative enzymes 19


418

reported herein differ considerably from those determined in the work cited, which may be

443

attributed to different assay conditions. Besides the use of batch cultures herein, fructose-2,6-

444

bisphosphate and fructose-1,6-bisphosphate, potent allosteric activators of phosphofructokinase

445

and pyruvate kinase, respectively, were for example apparently not added in the enzymatic assays

446

of (84). Nevertheless, the aim of the present study was the comparison of specific enzyme

447

activities between the H. uvarum type strain and S. cerevisiae. Since the same assay mixtures

448

were used to assess activities in crude extracts from both species, it allowed us to assess the

449

relative contribution of each step to alcoholic fermentation.

450 451

Functional analyses of genes encoding phosphofructokinase and pyruvate kinase.

452

The data obtained above suggested crucial differences in the activity of pyruvate kinase in H.

453

uvarum as opposed to S. cerevisiae. These differences could either be attributed to different

454

regulatory circuits governing PYK1 gene expression in the two yeast species, or inherent

455

properties of the enzyme itself. In order to distinguish between these possibilities, we decided to

456

express the HuPYK1 gene in a pyk1 deletion mutant of S. cerevisiae. Indeed, HuPYK1 introduced

457

on a CEN/ARS vector rescued the glucose-negative growth phenotype of the deletion strain

458

VWH3B (pyk1::HIS3), indicating functional complementation. This was confirmed by enzyme

459

assays of cultures grown in rich medium with 2% glucose, yielding a specific pyruvate kinase

460

activity of 465 ± 100 mU/mg protein, as determined from two independent transformants

461

carrying plasmid pJJH2144 and three technical replicates. This is higher than the specific activity

462

determined for a wild-type H. uvarum strain above under similar growth conditions (193 ± 39

463

mU/mg protein, Table 4), but only approximately 15% of that observed for transformants with a

464

plasmid carrying the ScPYK1 gene (3179 ± 270 mU/mg protein) or the diploid wild-type strain

465

HHD1 (3087 ± 163 mU/mg protein). However, placing the HuPYK1 open reading frame under 20


442

the control of the ScPYK1 promoter yielded 1982 ± 82 mU/mg protein when introduced into

467

VWH3B (pyk1::HIS3), demonstrating that the low specific activity is probably due to a low level

468

of gene expression, rather than being determined by the structure of the heterologously produced

469

enzyme.

470

Finally, the key glycolytic enzyme phosphofructokinase (PFK) of S. cerevisiae is a

471

heterooctamer, composed of four α- and four ß-subunits, which are encoded by the genes PFK1

472

and PFK2, respectively (85). While mutations in any one of the two genes do not prohibit growth

473

on glucose, pfk1 pfk2 double mutants are glucose-negative (63, 86). This has been attributed to a

474

conservation of all catalytic and allosteric domains in each of the encoded subunits, retaining

475

activity of homooligomeric forms in vivo, but not in vitro (87). For H. uvarum, we could also

476

identify two homologous genes in the annotated sequence and designated them HuPFK1 and

477

HuPFK2 based on sequence alignments and synteny analysis. They encode proteins with deduced

478

molecular weights of 103428 Da and 85351 Da, respectively. It should be noted that the

479

annotated genome sequence of HuPFK1 is lacking an adenine nucleotide at position +442

480

relative to the correct ATG translation start codon, as confirmed by cloning and Sanger

481

sequencing (GGAAGCCACCAAAAAGAAAAAAATTGCTG). Thus, the sequence underlined

482

in fact contains five, rather than four nucleotides, which leads to a considerable extension of the

483

5'-end of the predicted HuPFK1 open reading frame. For use in further studies, we therefore

484

obtained the entire coding sequences again by Sanger sequencing of cloned PCR products and

485

submitted the genes under the accession numbers given in materials and methods.

486

When individually introduced on CEN/ARS vectors into a S. cerevisiae pfk1 pfk2 double deletion

487

mutant (HD114-8D, pfk1::HIS3 pfk2::HIS3), either gene from H. uvarum complemented the

488

glucose-negative phenotype, indicating that the subunits are functionally conserved and that they

489

also exert catalysis in vivo. However, like in S. cerevisiae single deletion mutants, PFK activity 21


466

was below detectable levels in enzyme assays of crude extracts from strains carrying either one of

491

the HuPFK genes, even when introduced on multicopy vectors (Table 5). Yet, introducing both

492

heterologous genes together on a CEN/ARS vector restored in vitro PFK activity in the double

493

deletion mutant, which amounted to approximately one-third of the specific activity determined

494

in either H. uvarum or S. cerevisiae wild-type strains. As expected, overproduction by expressing

495

the two genes from a multicopy vector yielded much higher activities. This indicates that PFK of

496

H. uvarum is also an oligomeric enzyme, and may form a heterooctamer like its counterparts in S.

497

cerevisiae and K. lactis (88). The overall domain structure of the subunits, which retain both the

498

catalytic and allosteric substrate binding sites, is consistent with the observation that sufficient

499

glucose can be metabolized in vivo by either HuPfk1 or HuPfk2. Yet, like in S. cerevisiae,

500

heterooligomerization seems to stabilize the enzyme structure, so that in vitro activity can only be

501

observed if both subunits are produced in one cell (87). The lower specific activity measured in

502

the transformants with the two HuPFK genes carried on a CEN/ARS vector as compared to the

503

wild-type strains of both species could be due to a lower expression level caused by the native

504

HuPFK gene promoters in S. cerevisiae, analogous to the data presented above for HuPYK1. It

505

should also be noted that heterooligomeric complexes between subunits of the two species either

506

do not form or are not stable, since transformants of single Scpfk1 or Scpfk2 deletion mutants

507

with the respective HuPFK gene did not restore measurable enzyme activity in crude extracts

508

(data not shown).

509

The enzymatic determinations and sequence analyses were further substantiated by performing

510

Western blot analyses using an antiserum raised against ScPFK, which also detects the subunits

511

of the K. lactis PFK (89). As shown in Fig. 6, the HuPFK subunits migrate according to their

512

deduced molecular weight, with the ß-subunit encoded by HuPFK2 migrating much faster than

513

its counterpart from S. cerevisiae. While PFK subunits from different yeasts and fungi analyzed 22


490

so far all carry an N-terminal elongation of approximately 200 amino acids as compared to their

515

bacterial or mammalian homologs, this extension is missing in the ß-subunit of H. uvarum.

516

Determinations of PFK activities after controlled proteolysis, which cleaved off the N-terminal

517

parts of the S. cerevisiae enzyme, demonstrated that these extensions are actually not required for

518

catalysis (90). It is thus rather surprising, that sequences encoding the N-termini of PFK-subunits

519

should have been eliminated only during evolution of the HuPFK2 gene and not have occurred in

520

other yeasts. With the growing number of genome sequences published on other non-

521

Saccharomyces wine related yeasts, it will be interesting to see if this is indeed a single

522

"evolutionary accident" confined to H. uvarum. If so, this would indicate a selective advantage of

523

larger subunits whose biological significance remains to be elucidated.

524 525 526

CONCLUSIONS

527

We here presented a comprehensive analysis of the genome of a H. uvarum type strain, including

528

an extensive annotation. This and other non-Saccharomyces yeasts are of growing interest for the

529

production of fermented beverages, especially wine, due to their important contribution of

530

desired aroma compounds (5). The genome sequence presented here provides the basis for future

531

manipulations of the underlying pathways, e.g. for increasing the production of desired and

532

reducing that of undesired metabolites. It also allows the assessment of physiological capacities.

533

Thus, our studies indicate that in H. uvarum pyruvate kinase activity could be a limiting step in

534

alcoholic fermentation. This has also implications with regard to climate change and the

535

corresponding increase in grape sugar content. Thus, co-fermentations with non-Saccharomyces

536

yeasts have been suggested as a measure to control increasing ethanol concentrations in wine

537

(91). Although our functional studies of key glycolytic enzymes demonstrate the usefulness of 23


514

the annotated genome sequence, they can be considered just a proof-of-principle. Further

539

functional studies on enzymes involved in the production of aroma compounds, such as esterases

540

or glycosylases will follow. Moreover, the genome sequence provides the basis to study wine-

541

related phenotypes, such as the capacity to flocculate and the signaling pathways involved in

542

coping with environmental stress conditions during vinification (73). All these studies would

543

greatly profit from the development of a system for targeted genetic manipulation of H. uvarum,

544

which is the most urgent subject to be addressed in future research on this yeast.

545 546 547

Acknowledgements:

548

This work was funded by two consecutive grants from the “Forschungsring des Deutschen

549

Weinbaus” (FDW) to J.J.H. We thank Josef Hermann (Veitshöchheim, Germany) for providing a

550

natural isolate of H. uvarum and financial support for a second round of sequencing. We also

551

thank Andrea Murra for excellent technical assistance.

552

24


538

553

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808

37

809

Table 1: Oligonucleotides used to generate contig-specific probes and specific probes for S.

810

cerevisae chromosomes used in Southern analyses

811

A) Probes for H. uvarum DNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25

Forward primer

Reverse primer

TTCAATTCCGCAAGTGATGG CCATACATTTGCCAAGGTTC CGCAAGCCAATGTACCAAAG AACGCTAACAATTCTACAAG ATATTGAAGTACCCGCAATG TCACCAACCACATCCAGTAG ATTCTCCACTTAGGACTTAC GCATCAACTCGTCAAGATAG GTTCCTGTTCTGGTGAAATC TGTTCTCTGTTGGAGGATTC CAACAACCAACTGGTTTCTC TGCCGTTGATAGATATTCTG TTTCTACTTCGCTTAATTGC TGAACATGACCAGGAATTAG AATTCGCTGTGGCAAATTGG TTGCATTAAACAGCGCAAAG TAAGACAGAGTCGTCTAGAG TGGGTGCAAGAAATAGATGG AATCTGCTTCAGGCTGATAC TCTTATCAGCCACATGTCAC CTTGCCTGGGTTGTTGATAG TCTACCATAGCCAATCAAAG GCTTACAGCACCTAAAGATG TTGACATGTTCGAGGTGTTC AACTAAGGCAGTATCCAAAG TGCCTACCTTAAATTCTCAC TGGAATAACGGAGTGAATAC AAATGGCCGAAACCATTGGG ATCATCGCCGTTATTAACAC CCTGCATTGTGAATTTCTTG CGGCAATTTCGGAGTATTTC CATTGCTGCTGGTACTTATG TTAGAGAACCCGCTGTTATC GTCTGAAATATGTGCACTTG CAGAACTGATGCCTTTAATG AGAATCTGCTTCTGCTTCTG AATCTGTTGCCTTGTGTTTG CAGCAACTTGCTCGTTATAC AGGTTGACCTTGAGTGAATG TAGAGTTACCACTGCTGTTC GCCAACGTAGCACTTTCTTC TCAGATGGACCATTCCTTAC CTCTTTCACAAGCTTCTAAC AAAGTTTGCGGATGCTTATG CTACCCAGAAGAATTCAAAG GTTAGAGACGCTTTAAACAG CTGACTTCCAGAAAGTATTG ACCCATGCTGTATCCCATTC TTTGGAATGGACCCATATTG TTGGTTTGAAGACACAAATG TCTATTGGTGGAGCTGAATC GTATTGACCAGGAACAATAC ATATCAAGCCGTTAGTAAGC AAGGTTACTGGAGCTTTGTG ATATACCTGTCCATCCAAAG TGCAGCAGATAACTCAGAAC CCAATGTAAGCACTGTATTC

TATAGAGGCAGGTAACTGAG CTTACCACCAAAGATCATTC CACCGTTACACAACTACC TTAGGCCCTATAGTCAAATC TCTTGGTAACCATGACTAAC AAGCATCTCTTCAACTGTAG ATTCATCACCACCAGTATTG GTAGACAGTGAGGTTGAAAG CTAGGCTCAGTCTCTACTTC GAGTTGCAAGATGCTGTTTC AGCAGCTATGACTGCTAGAG ATGTAGATGCAGAATCTACC TCGGTTACTTCAAGAAATGG TCCAGTATGAGGACATTTC GTCAATCACCTTTGTTATCG ACCAAATCCAACGACCAATG AATTGCGGCTGATGAGTAAG AACGTCTCCACTGCTCTATC ATACCACTTCTTCTGGAATG ATGGACTATTAGGCGATAAC TCCTTCCCTTAGAGAAGTTG CCAGACTTAGAGCTGATGAC GGCTCAAATTTAGCACCATC GATCCCTGCTTTACTTGAC GGTTTAGCAGAATCTTTAGC GCAGCTGATCTATCACTTTG GTCATGTTCTTCACCATACC CTTGTGTTGTTGCTGAATAC GGAAATTAATCCCTGACTTG GCTATATCGCTTGAAGAAGG TTGGTTCCCACAGACCAAAG GCAATTCAGGTGCTCTATAC TACCGTTGTTTGTAACAAAG CATGAACCAACTTGGAATAG GGTATTTAGGCCACAGTATC CTTGGCCCATAAGTAGATTG GTATACCTGTGGAGATATGG ACTGATCCGTACATCGAAAG ATCTGCTTTAGCCAACAAGG CTGGTATTGCCGCCAGTAAC AAAGACCGTTTAATCAGATG ACTCATCATGACCATCTTC TGACTTGTTTGGCTTCATTC AACTTCATCAACAGGCTTAC CCAAAGAACTTGTGGTAGTC AAACGGTTCTAACACCCTTG TCTGCCTTTATATCCATTGC GTACAGTTGTACGAGCATTC ATTGCTGAGTACCAATTGAC GTTCCAAACAAGGCTCATAC CTTGATAGCCTTGTGAATAG ATGAGCATCAAATCCTCATC AAAGTACCAGTGACTCTATC TGTAGATGATGCTGGTATTC AAGATCCTGGCGAATATGTC AACATTGCTTACAGTGTTAG TGCTTCTCTAGCCGCTTCTG

38


Contig number

812

B) Probes for S. cerevisiae DNA Gene

SLA1 TRP1 GEA2 PFK1 GEA1 HXT1 GPM1 MID2 rDNA PFK2 WSC2 non-coding sequence GAL4

gcgtcctgcagCTATTAAGAGCTGATGGAATCATCTTTCGAA CGTGGATAGCGCCGATAAGG GAACCAGTTCACTGGTGGAG CTTAGGGTCGACTCCACCATTTC GCGGCTTGCAGAGCACAGAGG GTCTCCACACCTCCGC gcgtacccgggAAACCACAGCCACATTAAC GGTGCCGTTGGAAATCACTG ggatctcgagTCAATCTCAAGATTCATGCTACGG gagcgctcgagTATTCAGTACCTGGAACG gcgtcgcatgcGGTATAAATGCCACCGTCG CAGCCAAAGACCGCCAATTTGC GGTACCATTGTTTTCCAGGCTGTCGG GCCGGTGAAGGTCAAGAACTAG GAAGCCGCTAGAGCCGGTG GGCAACAGCAGCGGCACCAGC GTGGAACGTTAAAGCACTCG CCTCAAGTGCTGACTCATCTTCCC gcgcgaattcGCTAGTACCGATTGAATGGCTTAG gcgcggatccGATGCGAGAACCAAGAGATCCG ggatctcgagTACTGTTACTACTCCTTTTGTG cagcgctcgagTATTCAGTACCTGGAAC GTGGAATATGCACCTAGATCTC CCACAAACCACACCTACTAC GCGTTTATTGTATCCCTTGAC GGTAGATAGCTTGAGGCAC GGTCTCCGCTGACTAGGGCAC CCCCCTCTATACACCAGGCTCC

Chromosome/ Size ScChr I 0,2 Mb ScChr II 0,81 Mb ScChr IV 1,61 Mb ScChr V 0,61 Mb ScChr VII 1,12 Mb ScChr X 0,745 Mb ScChrVIII 0,56 Mb ScChr XI 0,68 Mb ScChr XII 1,9 Mb ScChr XII 1,9 Mb ScChr XIII 0,915 Mb ScChr XIV 0,785 Mb ScChr XV 1,09 Mb ScChr XVI 0,945 Mb

813 814

Mixtures of three probes for each contig generated by the oligonucleotide pairs indicated were

815

used for Southern analyses on blots from FIGE separated genomic DNA to assign contigs to

816

chromosomes. For S. cerevisiae chromosomes, probes corresponding to the genes and indicated

817

were also obtained by PCR with the oligonucleotide pairs given. Small letters correspond to

818

sequences added to create restriction sites for cloning, which do not hybridize to the target DNA.

819

Sequences are all given in the 5'→ 3' direction.

820

39


BUD14

Forward and reverse primers

821

Table 2: Composition of mixtures for determination of enzyme activities Enzyme Hexokinase

Triosephosphate dehydrogenase

PB2, 0.2 mM NADH, 1 mM ATP, 10 mM MgCl2, 5 mM cysteine, 1U/ml 3-phosphoglycerate kinase

Phosphoglycerate kinase

PB2, 0.2 mM NADH, 10 mM ATP, 10 mM MgCl2, 100 mM KCl, 5 mM cysteine, 1 U/ml glyceraldehyde 3phosphate dehydrogenase IB1, 0.2 mM NADH, 1 mM ADP, 10 mM MgCl2, 0.5 U/ml enolase, 1 U/ml pyruvate kinase, 1 U/ml lactate dehydrogenase PB2, 0.2 mM NADH, 1 mM ADP, 10 mM MgCl2, 100 mM KCl, 1 U/ml pyruvate kinase, 1 U/ml lactate dehydrogenase PB2, 0.2 mM NADH, 0.6 mM ADP, 10 mM MgCl2, 100 mM KCl, 1 mM fructose 1,6-bisphosphate, 1 U/ml lactate dehydrogenase CB3, 0.2 mM NADH, 10 mM MgCl2, 100 mM KCl, 1.7 mM cysteine, 1.6 mM thiamine pyrophosphate, 1 U/ml alcohol dehydrogenase PPB4, 2 mM NAD, 1 mM glutathione ethanol (0.6 mM)

Phosphoglucose isomerase Phosphofructo kinase

Aldolase

Phosphoglycerate mutase Enolase

Pyruvatkinase

Pyruvate dehydrogenase Alcohol dehydrogenase

822 823

Buffers used were:

824

1

IB, 50 mM imidazole buffer, pH 7.0

825

2

PB, 50 mM sodium phosphate buffer, pH 7.0

826

3

CB, 50 mM sodium citrate buffer, pH 7.0

827

4

PPB, 85.5 mM sodium pyrophosphate buffer, pH 7.0 40

Substrate fructose (5 mM) fructose 6phosphate (5 mM) fructose 6phosphate (2.5 mM) fructose 1,6bisphosphate (2 mM) glyceraldehyde 3-phosphate (5 mM) 3-phospho glycerate (4 mM) 3-phospho glycerate (10 mM) 3-phospho glycerate (10 mM) 2-phospho glycerate (10 mM) phosphoenol pyruvate (10 mM) pyruvate (5 mM)


Triosephosphate isomerase

Composition of assay mixture IB1, 0.4 mM NADP, 1 mM ATP, 10 mM MgCl2, 0.2 mM EDTA, 0.3 U/ml glucose 6-phosphate dehydrogenase IB1, 0.4 mM NADP, 1 mM ATP, 10 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 0.3 U/ml glucose 6-phosphate dehydrogenase PB2, 0.2 mM NADH, 1 mM ATP, 1 mM AMP, 10 mM MgCl2, 5 µM fructose 2,6-bisphosphate, 1 U/ml fructose 1,6-bisphosphate aldolase, 0.5 U/ml each of glycerol 3phosphate dehydrogenase and triosephosphate isomerase PB2, 0.2 mM NADH, 5 mM MgCl2, 30 mM KCl, 10 mM NH4Cl, 0.3 mM MnCl2, 0.2 mM Zn-acetate, 0.5 U/ml each of glycerol 3-phosphate dehydrogenase and triosephosphate isomerase IB1, 0.2 mM NADH, 10 mM MgCl2, 0.1 mM EDTA, 0.5 U/ml glycerol 3-phosphate dehydrogenase

828 Assay conditions were modified from the following sources:

830

Hexokinase, glucose isomerase, triosephosphate isomerase, phosphoglycerate kinase and enolase from

831

(92). Phosphofructokinase from (93), aldolase from (94), triosephosphate dehydrogenase from (95),

832

phosphoglycerate mutase from (96), pyruvate kinase from (81), pyruvate dehydrogenase from (97), and

833

alcohol dehydrogenase from (98).

834

Ancillary enzymes and reagents were obtained from Roche/Mannheim, as far as available in

835

ammonium sulfate suspensions and used far beyond the indicated expiration date, partially due to the

836

lack of current comercial resources. Alternatively, enzymes and substrates were obtained from

837

Sigma/Aldrich.

838

41


829

839

Table 3: Chromosome numbers and size as deduced from FIGE-Gels and sequence analysis chromosome

I

II

III

IV

V

VI and VII

[Mbp]

0.67

0.97

1.02

1.36

1.52

1.77

12

0.227

14

0.215

contig size

3

0.564

contig size

contig size

contig size

contig size

2

0.572

7*

0.345

6

0.355

5

0.364

4

0.412

8

0.323

11

0.251

9

0.309

10

0.263

15

0.197

13

0.226

18

0.162

17

0.186

16 *

0.193

contig size

1

0.998

rDNA 0.298

25

0.110

sum

0.44

0.56

0.98

1.09

0.99

1.09

1.41

% coverage

66%

58%

97%

81%

65%

62%

80%

840 841

* Contigs 7 and 16 give an additional signal on chromosome I.

842

% coverage gives the estimated percentage of sequence obtained for each chromosome.

843 844 845

42


contig size

846

Table 4: Comparative analysis of specific enzyme activities relevant for alcoholic fermentation

847

between H. uvarum and S. cerevisiae Abbrev.

Hanseniaspora uvarum

Saccharomyces cerevisiae

Ratio (Sc/Hu)

Hexokinase

Hxk

525 ±24

594 ±38

1.1

Phosphoglucose isomerase

Pgi

1192 ±23

1416 ±118

1.2

Phosphofructokinase

PFK

361 ±22

556 ±84

1.5

Aldolase

Fba

420 ±128

459 ±87

1.1

Triosephosphate isomerase

Tpi

22581 ±2847

26804 ±2329

1.2

Triosephosphate dehydrogenase

Tdh

718 ±305

937 ±297

1.3

Phosphoglycerate kinase

Pgk

753 ±157

1033 ±267

1.4

Phosphoglycerate mutase

Gpm

1379 ±243

1987 ±366

1.4

Enolase

Eno

1251 ±255

2015 ±326

1.6

Pyruvate kinase

Pyk

193 ±61

3263 ±362

16.9

Pyruvate decarboxylase

Pdc

494 ±169

707 ±160

1.4

Alcohol dehydrogenase

Adh

1727 ±201

3802 ±699

2.2

848 849

Specific enzyme activities are given in mU/mg protein at 30°C. The mean of at least three

850

biological and three technical replicates for each enzyme and yeast species was determined, and

851

standard deviations (±) were calculated. Cells were grown in rich medium with 1% glucose and

852

1% fructose (YEPDF) prior to preparation of crude extracts. For a comparison of specific

853

activities after growth on higher sugar concentrations and on ethanol as a carbon source see Fig.

854

S4 in supplementary material.

855

43


Enzyme

856

Table 5: Specific activities of phosphofructokinase Strain (relevant genotype)

PFK gene introduced

Specific PFK activity [mU/mg protein]

H. uvarum (HuPFK1 HuPFK2)

none

none -

413 ±25

HHD1 (PFK1 PFK2)

none

none

326 ±33

HOD114-8D

none

none

Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.

Genome Sequence of the Nonconventional Wine Yeast Hanseniaspora guilliermondii UTAD222.

The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production.

19AF.

The Effect of Proanthocyanidins on Growth and Alcoholic Fermentation of Wine Yeast under Copper Stress.

Yeast Interactions in Inoculated Wine Fermentation.

Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

A Gondwanan imprint on global diversity and domestication of wine and cider yeast Saccharomyces uvarum.

A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling.

Genomic expression program of Saccharomyces cerevisiae along a mixed-culture wine fermentation with Hanseniaspora guilliermondii.

Diversity of yeast strains of the genus Hanseniaspora in the winery environment: What is their involvement in grape must fermentation?

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast.

Yeast glycolytic mRNAs are differentially regulated.

Why, when, and how did yeast evolve alcoholic fermentation?

The proton transfer reactions catalyzed by yeast pyruvate kinase.

Pyruvate kinase from yeast (Saccharomyces cerevisiae).

Hanseniaspora uvarum from Winemaking Environments Show Spatial and Temporal Genetic Clustering.

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

Evolution of phenolic compounds and metal content of wine during alcoholic fermentation and storage.

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy.

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation.

High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

Insights into the bacterial community and its temporal succession during the fermentation of wine grapes.

Phosphorylation of pyruvate kinase and glycolytic metabolism in three human glioma cell lines.