cofermentation of glucose, xylose, and cellobiose by the...

Cofermentation of glucose, xylose, and 1

cellobiose by the beetle-associated yeast, 2

Spathaspora passalidarum 3

Running title: Cofermentation of glucose, xylose, and cellobiose 4

Tanya M. Long‡1, Yi-Kai Su‡1,2, Jennifer Headman1,3†, Alan Higbee1††, Laura B. Willis1, 3, 4, and 5

*Thomas W. Jeffries1, 3, 4 6

7

1DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI 53706 ; 8

2Department of Biological Systems Engineering, University of Wisconsin, Madison, WI 53706; 9

3Department of Bacteriology, University of Wisconsin, Madison, WI 53706; 4U.S. Department 10

of Agriculture, Forest Products Laboratory, Madison, WI 53590 11

‡These authors contributed equally to the research reported herein. 12

†Current address: Verdezyne, 2715 Loker Avenue West, Carlsbad CA 92010 13

††Current address: University of Wisconsin, Department of Chemistry, 1101 University Avenue, 14

Madison WI 53706-1322 15

*Correspondence should be addressed to: Thomas W. Jeffries ([email protected]) at USDA, 16

Forest Products Laboratory, 1 Gifford Pinchot Drive, Madison, WI 53726; Tel: 608-231-9453 17

Prepared for submission to Applied and Environmental Microbiology 18

Version of Friday, May 18, 2012 19

Classifications: Microbial physiology and Biotechnology 20

21

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00374-12 AEM Accepts, published online ahead of print on 25 May 2012

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Cofermentation of glucose, xylose, and cellobiose

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Abstract 22

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food vs. fuel 23

conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and 24

xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous 25

cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because 26

glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-27

associated yeast, Spathaspora passalidarum, which ferments xylose and cellobiose natively, can 28

also coferment these two sugars in the presence of 30 g/l glucose. S. passalidarum will 29

simultaneously assimilate glucose and xylose aerobically; it will simultaneously coferment 30

glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol 31

production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, 32

an adapted strain of S. passalidarum produced 39 g/l ethanol with a yield of 0.37 g/g sugars from 33

a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the 34

fermentations of glucose and xylose showed that the flux of glycolytic intermediates is 35

significantly higher on xylose than on glucose. High affinity of its xylose reductase activities for 36

NADH and xylose combined with allosteric activation of glycolysis probably account in part for 37

its unusual capacities. These features make S. passalidarum very attractive for studying 38

regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts. 39

Keywords: Metabolic regulation; SSF; simultaneous saccharification and fermentation, passalid 40


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Introduction 41

Cofermentation of glucose and xylose is critical for bioconversion because they are present in 42

virtually all enzymatic hydrolysates of pretreated lignocellulose (4, 47). Their sequential 43

utilization extends fermentation times and can result in incomplete substrate consumption if 44

products reach inhibitory levels before slowly utilized sugars are consumed (14). Moreover 45

cofermentation of xylose with cellobiose could reduce enzyme costs and facilitate simultaneous 46

saccharification and fermentation. Glucose represses utilization of xylose, cellobiose, or other 47

carbon sources in most microbes (11, 12). This is a problem that can be addressed through 48

metabolic engineering or strain selection. 49

Placing genes for xylose or cellobiose assimilation under the regulation of constitutive 50

promoters, or altering transcriptional activators (42), can relieve glucose repression (21). For 51

example, co-utilization of glucose and xylose (10, 25), and of cellobiose and xylose (35) by 52

engineered Saccharomyces cerevisiae can produce 60 g/l ethanol in 72 h from pure sugars (13). 53

However, even when heterologous genes for assimilation are expressed under constitutive 54

promoters, co-utilization with glucose is problematic because the affinities of yeast 55

monosaccharide transporters are 8 to 100 times higher for glucose than for xylose (36, 37). This 56

problem can be addressed by introducing heterologous transporters with higher affinity for 57

xylose. For example, S. cerevisiae TMB34006, engineered with the glucose/xylose facilitator 58

Gxf1 from Candida intermedia (34), shows 1.2- to 2-fold higher rates of xylose uptake when 59

initial glucose concentration is less than 5 g/l (10). To accommodate higher rates of xylose 60

transport, downstream reactions in the xylose metabolic pathway must be overexpressed, but 61

cofactor imbalances limit xylose assimilation in the presence of glucose (25) and reduce its 62

contribution to metabolic flux (10). Cofermentation of xylose and cellobiose is less difficult 63


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because cellobiose, a disaccharide, does not compete with xylose transport (13). 64

Systematic modeling and metabolic engineering can modify S. cerevisiae to address these 65

problems, but this yeast does not provide a useful model for how overall metabolic flux is 66

natively regulated during cultivation on xylose. In fact, aside from a few studies of genes and 67

enzymes induced during the assimilation of xylose and the induction of pyruvate decarboxylase 68

under oxygen limitation (31), very little is known about how native xylose-fermenting yeasts 69

mediate the simultaneous metabolic demands for cell growth and product formation during 70

ethanol production on xylose. Most native xylose-fermenting yeasts such as Scheffersomyces 71

(Pichia) stipitis (26) do not co-utilize glucose and xylose, largely because glucose represses the 72

genes for xylose assimilation (16). However, this does not appear to be the case for all yeasts. 73

In the present work we report that an unusual native xylose-fermenting yeast, Spathaspora 74

passalidarum NN245 (15, 30, 46), will coassimilate xylose and glucose aerobically, use xylose 75

faster than glucose when the sugars are presented individually, and coferment glucose, xylose, 76

and cellobiose from mixtures of pure sugars or hydrolysates under oxygen-limiting conditions. 77

These features make S. passalidarum potentially useful for simultaneous saccharification and 78

fermentation (SSF) and for studying enzymatic and regulatory mechanisms enabling 79

simultaneous utilization of cellulosic and hemicellulosic sugars. 80

Materials and Methods 81

Organism, medium, and culture conditions 82

Spathaspora passalidarum NN245 (=NRRL Y-27907, CBS 10155, MYA-4345) (30) was used 83

for all experiments and adaptations reported herein. Cultures were maintained on YPX agar 84

plates containing 10 g/L yeast extract, 10g/L peptone, 20 g/L xylose, and 20g/L agar. Inocula 85


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were cultivated overnight in 50 ml of YP broth containing 20 g/L yeast extract, 10 g/L peptone, 86

plus 20 to 60 g/l glucose (G), xylose (X), or cellobiose (Cb). The cultivation was then transferred 87

to defined minimal medium and grown to an appropriate cell density. All flask cultivations were 88

carried out at 30 °C in 125-ml polycarbonate flasks (GeneMate) fitted with membrane filters for 89

aeration. Aerobic and oxygen-limited inoculum preparation conditions were 200 rpm, ≤0.2 g/l 90

cell dry weight (cdw), and 100 rpm, ≥1 g/l cdw, respectively. Defined minimal medium (CBS) 91

containing nitrogen, trace metal elements, and vitamins was used in all bioreactor cultivations. It 92

contained 2.4 g/L urea, 3 g/L KH2PO4, 0.5 g/L MgSO4 7•H2O, 1 ml L–1 trace element solution, 93

(ml L–1) vitamin solution, and 0.05 mL/L antifoam 289 (Sigma A-8436) (modified from (44)). 94

Carbon sources consisted of D-glucose, D-xylose, or a mixture of xylose with glucose and/or 95

cellobiose. Maple hemicellulose hydrolysate (MHH) medium consisted of 9:1 (vol:vol) mixture 96

of MHH (41) and 10-fold concentrated CBS basal defined minimal medium. Supplemental 97

glucose was added to the hydrolysate to attain 65 g/l xylose and 35 g/l glucose. Control medium 98

contained the same sugars in CBS defined minimal medium. The starting inoculum was 0.8 mg 99

cdw/ml. AFEX hydrolysate medium (27, 47) supplemented AFEX hydrolysate with 2.4 g/L urea, 100

1 g/L KH2PO4, 1 g/L K2HPO4,50 mg/L FeSO4•7H2O, 5.5 mg/L ZnSO4• 7H2O, and 8 mg/L 101

CuSO4 •5H2O. Modified vitamin solution (40) deleted riboflavin, folic, and thiotic acids. 102

Bioreactor fermentations were performed in duplicate New Brunswick Scientific Bio Flo 110 3-l 103

reactors with working volumes of 2 l. Bioreactor temperature was maintained at 25 °C (39, 40), 104

and pH was kept constant at 5.0 ± 0.1 by automatic addition of 5 N KOH. All fermentation 105

kinetics and metabolite levels are reported as averages of biological duplicates. The oxygen 106

transfer rate (OTR) was calculated using the gassing out method based on kinetic oxygen 107

measurements (38). The dissolved oxygen level (dO2) was monitored using an InPro 6800 108


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oxygen sensor. Oxygen transfer rates were measured independently in four bioreactors at two 109

agitation rates using air and a 90:10 mixture of N2 and air (Table S1). The bioreactor was 110

sparged with air at 0.5 l/min (0.25 vvm, 500 rpm). Full air saturation (100% of scale) was set 111

after reaching equilibrium. Under these conditions the oxygen transfer rate (OTR) was estimated 112

as 13.56 ± 2.33 mmol O2/l•h. The zero level was set following equilibrium when sparging with 113

nitrogen. Fully aerobic cultures (Figure 1) were sparged with 1 volume/volume minute (vvm) air 114

and agitated at 700 rpm. For oxygen-limited fermentation of glucose or xylose, the air flow was 115

set to 0.25 vvm while a dissolved oxygen (dO2) controller (New Brunswick) maintained agitation 116

in the range between 150 to 300 rpm to attain 10% of saturation (~2.1% dO2). Once the growth 117

phase was complete (A), air was replaced with a mixture of 90% N2 and 10% air to attain a mole 118

fraction of O2 in the gas phase. The DO controller was set to a range of 300 to 500 rpm, and 119

ethanol production ensued (Figure 2). In the glucose, xylose, and cellobiose cofermentation 120

(Figure 4) and in the MHH fermentation (Figure 5), the bioreactors were equilibrated with 90% 121

N2 and 10% air (2.1% dO2) from the start, and the dO2 controller was set to a range of 400 to 500 122

rpm. 123

Adaptation and fermentation 124

For cofermentation S. passalidarum was propagated first in four 50-ml aliquots of YPX/Cb 125

medium (45 g/l X, 25 g/l Cb) in 125-ml Erlenmeyer flasks shaken at 200 rpm at 30 °C. After 24 126

h, these cultures were used to inoculate four 200 ml volumes of CBS X/Cb medium in 2-l 127

Erlenmeyer flasks, which were incubated at 150 rpm, 30 °C for 24 h. Each bioreactor was 128

inoculated with 200 ml of S. passalidarum broth into a final volume of 2.0 l of CBS X/Cb or 129

CBS G/X/Cb medium. S. passalidarum strain AF2 was adapted by two passages (total of ~11 130

doublings) of NRRL Y-27907 in 50 ml wood hydrolysate medium under oxygen-limiting 131


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conditions (20). It was then adapted to grow in AFEX corn stover hydrolysate under oxygen-132

limiting conditions for ~5 doublings. S. passalidarum E7 was derived from NRRL Y-27907 by 133

cultivation in wood hydrolysate corn stover hydrolysates for 2 months (54 doublings) under 134

oxygen-limiting conditions. Spent broth plus cells of strain E7 was directly transferred from an 135

adaptation flask containing stover hydrolysate into 500-ml Erlenmeyer flasks containing 120 ml 136

MHH plus CBS medium to create inocula for 2-l bioreactors. Inocula were cultured at 150 rpm 137

and 30 °C, then cells plus whole broth were added to Bio Flo 110 3-l bioreactors for a total 138

working volume of 1.2 l. The inoculum grown in MHH was transferred into bioreactors 139

containing CBS medium plus MHH, and the inoculum grown with synthetic medium was 140

transferred into bioreactors containing CBS synthetic medium plus sugars. The bioreactors were 141

sparged at 0.3 l/min (0.25 vvm) with a mixture of nitrogen and air with 9:1 ratio to attain ~2.1% 142

O2 saturation. Agitation was controlled between 400~500 rpm to maintain saturation. After 12 h, 143

500 rpm agitation could not meet the oxygen demand of the growing culture, and measurable O2 144

levels declined to near zero. 145

Sampling, extraction and metabolite analysis 146

Each 5 ml culture sample was rapidly withdrawn and injected by syringe into 20 ml of 60:40 147

methanol:H2O in a 50-ml conical centrifuge tube that had been equilibrated at –80 °C. The 148

cell/medium/methanol mixture was immediately vortexed thoroughly, held briefly (<4 min) in an 149

acetone dry ice bath, then centrifuged for 12 min at –9 °C. The methanol water supernatant 150

solution was decanted, and the frozen cell pellet was stored at –80 °C until extracted. Metabolite 151

analysis followed the method of Rabinowitz and Kimball (33). Nucleotide phosphates, organic 152

acids, sugar phosphates, and phosphorylated intermediates of glycolysis were analyzed by ion 153

chromatography coupled to electrospray ionization–tandem mass spectrometry (ESI–MS/MS) 154


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using minor modifications to a previously described method (43). The column (IonPac AS-155

11HC, Dionex, Inc.) was equilibrated with 0.5 mM NaOH, and metabolites were eluted with 100 156

mM NaOH. An Agilent 1200 HPLC system with gradient pump, micro vacuum degasser, and 157

chilled autosampler (4 °C) was employed. The same equipment was used to analyze the extract 158

in positive ionization mode to quantify 21 amino acids by the method of Bajad et al. (1). 159

Instrumental precision was estimated by comparing five replicate injections of a mixture of 160

twelve of the reported compounds (pyruvate, malate, succinate, glucose-6-phosphate, 2-161

ketoglutarate, fumarate, citrate, isocitrate, fructose-1,6-diphosphate, AMP, ADP, and ATP). 162

Relative standard deviations ranged from 22.0% to 2.6% with an average of 8.9% for all twelve 163

compounds. 164

For routine sugar consumption and end product analysis 1.0 ml of sample from each bioreactor 165

was used for sugars, xylitol, glycerol and ethanol determinations by high performance liquid 166

chromatography using a refractive index detector and Bio Rad Aminex HPX-87 Lead-column 167

(300 × 7.8 mm) at 85°C. The mobile phase was distilled deionized H2O at flow rate of 0.6 168

ml/min. 169

Results 170

Aerobic co-utilization of glucose and xylose. The capacity of S. passalidarum for xylose 171

fermentation compared favorably with that of Scheffersomyces stipitis in shake flask studies (9), 172

so we decided to characterize it further. Fully aerobic cultivation in defined minimal medium 173

with a very low initial cell density (0.014 mg cdw/ml) resulted in little growth for the first 20 to 174

36 h. Subsequently, however, S. passalidarum used glucose or xylose at essentially the same 175

rates when present individually or in a 50:50 mixture (Figure 1). No ethanol was formed under 176


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aerobic conditions. Cell yield was ~20% higher on glucose (0.67 g cdw/g) than on xylose (0.56 g 177

cdw/g). 178

Preferential fermentation of xylose. Prior studies with S. stipitis have shown that native xylose-179

fermenting yeasts induce ethanol production under oxygen limitation (23). The very long lag 180

time observed with fully aerobic conditions led us to use higher initial cell densities and lower 181

initial aeration rates. We strove to achieve the same initial oxygen-limited fermentative 182

conditions in the bioreactor as were present in the shake flask used for inoculum propagation. 183

We compared sugar consumption and ethanol production rates by S. passalidarum on CBS 184

medium in duplicate bioreactors with either glucose or xylose as carbon sources (Figure 2AB). 185

Immediately following inoculation, cell mass was 1.23 mg/ml, and with air sparging, 150 rpm 186

was sufficient to maintain dO2 at the set point of 2.1% O2 saturation. Initial growth rates during 187

the air-sparging phase were 0.19 and 0.14 h–1 for glucose and xylose, respectively. Specific 188

ethanol production rates were very low (glucose) or undetectable (xylose). After 7 h, cell 189

densities reached 4.45 and 3.15 g/l for glucose and xylose, respectively. At that point, the 190

sparging gas was switched to 90% N2, 10% air. With agitation capped at 500 rpm and inlet 191

oxygen at 2.1%, dO2 decreased to low or undetectable levels as cell densities increased. 192

Following the switch to oxygen limitation, specific growth rates fell by 2.5- to 5-fold, while 193

specific ethanol production rates increased dramatically (Table 1). During the oxygen-limited 194

phase, cell yield on xylose was only about half of that observed with glucose, but specific 195

ethanol formation rate was three times higher. This resulted in significantly higher ethanol 196

production with xylose even though cell yield was notably lower (c.f. Figure 2A, 2B). 197

Cofermentation of glucose, xylose, and cellobiose. During enzymatic saccharification and 198

fermentation of pretreated lignocellulosics, the major sugars present are xylose and glucose, 199


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xylan oligosaccharides (from hydrolysis of hemicellulose), and cellobiose (from enzymatic 200

saccharification of pretreated cellulosic solids). We therefore examined the cofermentation of 201

cellobiose and xylose. In duplicate shake flasks, the xylose consumption rate was much greater 202

than that for cellobiose, but after cells attained a density of ≤5 g/l, oxygen-limitation induced 203

ethanol production. Cellobiose and xylose were co-utilized at 0.49 and 0.94 g/l•h, respectively, 204

and volumetric ethanol production rate was 0.56 g/l•h (Figure 3). In bioreactors, cellobiose and 205

xylose were cofermented from the outset at very similar rates in the presence (Figure 4A) or 206

absence (Figure 4B) of 20 g/l glucose. Prior to inoculation, dO2 was 2.1%, but this declined to 207

near zero shortly after inoculation. During the fermentation phase, dO2 occasionally rose slowly 208

to ~0.2–0.5% without apparent effect on ethanol production or growth. In the absence of glucose, 209

xylose and cellobiose were cometabolized at essentially similar rates until xylose was depleted at 210

48 h (Figure 4A). The rate of ethanol production declined significantly thereafter, but cell growth 211

continued. In the presence of glucose, co-utilization of cellobiose and xylose was delayed 212

slightly in the first 15 h, but all three sugars were co-utilized at very similar rates from 15 to 68 213

h, at which point all sugars were consumed and ethanol production ceased (Figure 4B). 214

Maximum ethanol production rate from a mixture of xylose and cellobiose was 1.07 g/l•h, and 215

from all three sugars it was 0.73 g/l•h. Ethanol yields during the phase of maximum production 216

rate were 0.43 g/g and 0.42 g/g for xylose/cellobiose and glucose/xylose cellobiose mixtures, 217

respectively. Overall volumetric productivities and rates (0-72 hr) were 0.53 g/L-h for the 218

glucose, xylose and cellobiose mixture and 0.58 g/L-h for the xylose and cellobiose mixture. 219

Hydrolysate fermentation. The maple hemicellulose hydrolysate (MHH) contained 1.8 times 220

more xylose than glucose, and the AFEX hydrolysate contained ~2.3 times more glucose than 221

xylose. Unlike the AFEX-treated enzymatic hydrolysate of corn stover, MHH did not contain 222


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significant amounts of acetic acid or cellobiose, and the glucose:xylose ratio was 35:65 at the 223

start of the fermentation. Fermentation of MHH (Figure 5A) was compared to a sugar mixture 224

(SM) designed to mimic the hydrolysate (Figure 5B). Almost from the outset, glucose and xylose 225

were cometabolized in the MHH, but utilization of xylose was delayed by ~12 h in the SM. 226

During the bulk of the fermentation glucose and xylose were co-utilized at similar rates in both 227

the MHH and SM media. Fermentation of the SM proceeded more rapidly to attain a peak of 40 228

g/l ethanol in 38 h, whereas fermentation of the MHH required 59 h to attain 38 g/l ethanol. 229

During the fermentative phase (19.5 to 49 h), cell and ethanol yields were 0.19 and 0.34 g/g 230

sugar consumed from the MHH and 0.25 and 0.37 g/g consumed from the SM respectively. 231

Fermentation time with AFEX was relatively long (Figure 6A). Its initial acetic acid 232

concentration was about 1.4 g/l. Cells grew under these conditions but did not exhibit significant 233

ethanol production until acetic acid was depleted at 3 d (Figure 6B). Xylose utilization was 234

largely delayed while glucose was consumed, but after 6 d, Strains adapted to growth in 235

hydrolysate rapidly converted 84% of the xylose into 23 g/l ethanol with a yield of 0.45 and 16% 236

into xylitol. 237

Metabolome analysis. To better understand the regulatory and metabolic basis for S. 238

passalidarum’s preferential utilization and co-utilization of xylose, we analyzed samples for 239

relative metabolite abundance in cells cultivated on glucose or xylose at the ends of the aerobic 240

growth phase and during the ethanol production phase (arrows 1 and 2, Figure 2). At the end of 241

the initial growth phase (arrow 1), levels of glucose 1-phosphate (G1P), glucose 6-phosphate 242

(G6P), fructose 6-phosphate (F6P), fructose 1,6 bis-phosphate (F16P), 2- and 3-243

phosphoglycerate (2+3PG), phospho-enol-pyruvate (PEP), and pyruvate were all significantly 244

higher when cells had been cultivated on glucose than on xylose (Figure 7). Likewise, 6-245


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phosphogluconate (6PG), which is at the entry to the oxidative pentose phosphate pathway 246

(PPP), and α-ketoglutarate (αKG), which is derived from the tricarboxylic acid cycle (TCA), 247

were higher on glucose at that time. Amino acids derived from these intermediates followed 248

essentially the same patterns as their glycolytic or PPP precursors (Figure S1). 249

In contrast to these metabolites, levels of pentose phosphate (PP), sedoheptulose 7-phosphate 250

(S7P), and particularly erythrose 4-phosphate (E4P) were higher at the end of the growth phase 251

when cells were cultivated on xylose than when they were cultivated on glucose. Interestingly, 252

cellular levels of phenylalanine, tyrosine, and tryptophan, which are formed from E4P and PEP, 253

all followed the profile exhibited by E4P rather than that of PEP (Figure S1). In contrast, 254

concentrations of histidine (which is derived from ribose 5-phosphate) followed the profile of 255

6PG rather than that of PP. Our analytical procedure was not able to distinguish among ribose 5-256

phosphate (Ri5P), ribulose 5-phosphate (Ru5P), and xylulose 5-phosphate (Xu5P), but although 257

Ri5P is derived directly from 6PG, Xu5P is derived from phosphorylation of xylulose, so the 258

pool of Ri5P might not be directly affected by growth on xylose as are the concentrations of S7P 259

ad E4P. Alanine also attained relatively high levels (~50 µmol/g cdw) that were on the same 260

order of magnitude as glutamate, glutamine, and arginine (Figure S2). The greatest observed 261

differences in metabolite levels occurred during the oxygen-limited fermentative phase (arrow 2, 262

Fig. 2). With the exception of G1P, which is tied closely to growth through synthesis of cell wall 263

glucans, levels of virtually all glycolytic, pentose phosphate and TCA intermediates (except 264

pyruvate) and the amino acids derived from them were higher on xylose than on glucose. ATP 265

and ADP levels were higher on glucose than on xylose during the aerobic growth phase, but 266

under oxygen limitation, ATP levels were about three times higher on xylose than on glucose 267

(Figure S3). Ratios of reduced glutathione to oxidized glutathione stayed almost constant for 268


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each of the four conditions, but intracellular concentrations increased about 3.5-fold on xylose 269

relative to glucose during the oxygen-limited fermentative phase. 270

Discussion 271

S. passalidarum was originally isolated from the mid-gut of a passalid beetle that preferentially 272

inhabits white-rotted hardwoods (30). The mid-gut of wood-boring beetles is hypothesized to be 273

oxygen limited, so evolution for oxygen limited growth on mixtures of cellulosic and 274

hemicellulosic sugars might be expected. S. passalidarum NN245 is the sole known 275

teleomorphic isolate within the species, but other species in the Spathaspora genus and related 276

anamorphic species such as Candida jeffriesii have been isolated and characterized. Spathaspora 277

arborariae, which was isolated from rotting wood in the Brazilian Atlantic Rain Forest, is also 278

capable of fermenting xylose (6, 7), but other members of the Spathaspora clade are not (2, 5). 279

The yeast clade to which Spathaspora belongs is related to but distinct from that of 280

Scheffersomyces (26), which also includes a number of xylose-fermenting yeasts, including S. 281

stipitis (16). The capacity for producing ethanol from cellobiose is infrequent but occurs over a 282

wide taxonomic range within the yeasts. Even so, the traits for xylose and cellobiose 283

fermentation are variable within genera and even species. Although the capacity to ferment both 284

xylose and cellobiose is rare among yeasts, the abilities to ferment xylose faster than glucose and 285

to simultaneously cometabolize and ferment glucose, xylose, and cellobiose are believed to be 286

unprecedented. Fermentation rates and yields for xylose and cellobiose fermentations by native 287

strains of Spathaspora species exceed those of Saccharomyces cerevisiae strains engineered for 288

these traits (Table S2). 289

Under fully aerobic conditions, S. passalidarum grew poorly at first on glucose or xylose. Once 290

started, however, sugar utilization and cell growth rates were similar and aerobic co-utilization of 291


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glucose and xylose were essentially simultaneous. We inferred that the cells underwent 292

physiological adaptation to achieve full aerobic growth. Under typical low aeration conditions, 293

cells would grow and ferment rapidly, so the long lag under fully aerobic conditions was 294

atypical. Cell yields on xylose, however, were only about 84% of those on glucose under fully 295

aerobic conditions. Higher efficiency of cell growth on glucose was even more apparent under 296

O2 limitation (Figure 2A). Cell yield on xylose was only 55% of that on glucose (Figure 2B); 297

however, ethanol yield was 32% higher on xylose than on glucose, and the specific fermentation 298

rate on xylose was 3.4-fold higher. This was in contrast to the nearly simultaneous co-utilization 299

of both sugars in a mixture under aerobic conditions where no ethanol production was observed. 300

A lower net ATP yield for cells grown on xylose could account for the higher glycolytic flux 301

under O2 limitation. Hou reported a lower cell yield with S. passalidarum growing on xylose 302

than on glucose (15) and suggested that S. passalidarum might use low-affinity, low-capacity 303

facilitated diffusion for growth under O2 limitation and the high-capacity, high-affinity system 304

during aerobic growth (28). Previous studies have shown that Scheffersomyces stipitis induces 305

respirofermentative growth and pyruvate decarboxylase under oxygen-limiting conditions (23, 306

31). It seems likely that similar mechanisms for oxygen sensing could regulate transport as well. 307

We observed maximal volumetric fermentation rates of 1.14 and 0.72 g/l•h for xylose and 308

glucose, and our maximal rates for xylose and cellobiose consumption in the presence of glucose 309

were 0.55 and 0.57 g/l•h, respectively, in minimal medium with urea as the nitrogen source. The 310

specific rate of xylose fermentation was more than 3 times greater than the glucose fermentation 311

rate under conditions that allowed for the induction of transcripts and enzymatic activities 312

important for anaerobic or oxygen-limited metabolism. 313


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Metabolite levels measured during oxygen limitation support the observation that glycolytic flux 314

is greater in cells cultivated on xylose than on glucose. Levels of glucose derived metabolites 315

were higher during the initial aerobic growth phase, but they dropped dramatically as cells 316

shifted to fermentative conditions. By comparison, xylose-derived intermediates were 317

significantly higher on xylose under either aerobic or O2-limited conditions. Cells cultivated 318

under O2-limited conditions showed higher levels of ATP on xylose than on glucose. There are 319

several possible reasons: (1) Glycolytic flux is greater on xylose, (2) ATP demand is lower, (3) 320

Net ATP yield is higher, or (4) Regulation of ATP biosynthesis is different on glucose and 321

xylose. Because cell yield was significantly lower in xylose than on glucose, a decreased ATP 322

demand is probably the dominant effector. 323

The XR of Scheffersomyces stipitis has about 70% as much activity with NADH as it does with 324

NADPH (45). The capacity to recycle NADH to NAD+ under anaerobic or oxygen-limiting 325

conditions can reduce xylitol production and increase the capacity for xylose assimilation (3, 17, 326

19, 22, 24, 29, 32). Recently, Hou (19) reported that crude XR activity of S. passalidarum has a 327

1.8-fold higher affinity for NADH than for NADPH. By comparison, the XR of S. stipitis shows 328

1.6-fold higher affinity for NADPH. Moreover the affinity of the crude XR activity for xylose is 329

3-fold higher with NADH than with NADPH. The xylitol dehydrogenase (XDH) activity of S. 330

passalidarum also has a much higher affinity for xylitol than does the corresponding enzyme 331

from S. stipitis (15). These kinetic characteristics are much more favorable than the best aldose 332

reductase genes previously engineered for high selectivity and activity with NADH (19, 22, 29), 333

and they could account in large part for the high PPP, F6P, and F16P metabolite levels on xylose. 334

The S. passalidarum genome contains two orthologs each for XYL1 and XYL2 (46), so it is not 335


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possible to determine from these data which of these contribute more to the cofactor affinities as 336

measured in crude homogenates. 337

Most glycolytic regulation occurs “metabolically” via allosteric mechanisms to determine the 338

overall metabolic flux, rather than “hierarchically,” through changes in gene expression (8). 339

Thus, relative affinities of enzymes for cofactors and substrates along with enzyme activities at 340

modulated steps determine the overall flow of metabolites. Pyruvate kinase, which catalyzes the 341

final step in glycolysis, is allosterically activated by F16P and is important for controlling levels 342

of ATP, GTP, and glycolytic intermediates (18). The affinities of NADH-coupled XR and 343

NAD+-coupled XDH, would tend to drive both the oxidoreductase pathway for xylose 344

assimilation and the reduction of acetaldehyde to ethanol. The strong xylose assimilation would 345

likewise increase levels of F16P, thereby activating glycolysis via pyruvate kinase. This does not 346

fully explain the higher cell yield and lower glycolytic activity observed with glucose-grown 347

cells, but it does offer a model for further experimentation, and it begins to offer insight to how 348

we might go about engineering rapid co-utilization of these sugars. 349

We describe what we believe to be the first report of an organism capable of cofermentation of 350

xylose and cellobiose in the presence of glucose. This ability makes S. passalidarum a 351

potentially useful organism for SSF and an interesting organism for unraveling the regulatory 352

mechanisms enabling bioconversion of lignocellulosic materials by yeasts. 353

Acknowledgments 354

This work was funded in part by the DOE Great Lakes Bioenergy Research Center (DOE Office 355

of Science BER DE-FC02-07ER64494) and by a grant from the Heart of Wisconsin (Wisconsin 356

Rapids) to the University of Wisconsin, Madison. TWJ is supported by the USDA, Forest 357


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Products Laboratory. The authors thank Haibo Li and Amanda Prichard for assistance in 358

performing fermentations and analyses; Prof. Thomas E. Amidon, State Univ. of New York, 359

Environmental Science and Forestry, Syracuse, for samples of pretreated maple hemicellulose 360

hydrolysate; Wes Marner (GLBRC Madison, WI) for providing AFEX-pretreated, cellulase-361

digested corn stover and Prof. Meredith Blackwell, Louisiana State Univ., for providing original 362

culture of S. passalidarum. 363

Author Contributions 364

TWJ conceived the project, directed the work, and wrote the manuscript. Y-K S, TML, and JH 365

performed the fermentation experiments, contributed to experimental designs, and analyzed 366

results. LBW contributed to conceptualization, experimental designs, performed fermentation 367

experiments, data analysis, and editing. AH developed and conducted analytical techniques. All 368

coauthors contributed to writing the manuscript. 369

370


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United States of America 108:13212-13217. 528

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532

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Tables 534

Yield (g/g sugar) Rate (g/l•h) Qp (g/g cdw•h)a

Cells Ethanol Ethanol Cells Ethanol

Glucose 0.22 0.31 0.72 0.05 0.05

Xylose 0.12 0.41 1.14 0.05 0.17

Table 1. Spathaspora passalidarum product yields and specific formation rates on glucose or 535

xylose during the oxygen-limited phase. 536

aEthanol yields, specific growth rates and ethanol productivities were calculated from t=0 to t=50 537

h for glucose and from t = 0 to t=36 for xylose. 538

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Figures 540

541

Figure 1. Spathaspora passalidarum cultivated under fully aerobic conditions. Cells were 542

cultivated at 30 °C in 2000 ml of defined minimal medium (CBS) with glucose (A), xylose (B), 543

or a 50:50 mixture of the two sugars (C). Cultures were aerated with 1 vvm air and an agitation 544

rate of 700 rpm. Symbols: glucose, open triangles; xylose, solid squares; cell mass, circles; 545

ethanol, diamonds. 546

0

20

40

60

80

100

0 20 40 60 80

Glu

cose

(g/l

)

0

20

40

60

80

100

0 20 40 60 80

Xyl

ose

(g/l

)

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20

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547

548

Figure 2. Spathaspora passalidarum cultivated under oxygen-limited conditions. Cells were 549

cultivated in duplicate 3-l bioreactors at 30 °C in 2000 ml of defined minimal medium (CBS) 550

with either (A) glucose (open symbols) or (B) xylose (closed symbols). Glucose, triangles; 551

xylose, squares, ethanol, diamonds, cell dry weight, circles. First metabolomics sample and shift 552

to 2.1% O2 (arrow 1); Second metabolomics sample (arrow 2) Averages values from duplicate 553

runs are shown. Gray bars depict range of values. 554

0

5

10

15

20

25

30

35

40

45

50

0

10

20

30

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100

0 12 24 36 48

Eth

ano

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Dry

Cel

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(g/L

)

Glu

cose

(g/L

)

Time (h)

12

A

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ose

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Time (h)

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555

Figure 3. Cofermentation of cellobiose and xylose under oxygen limitation in triplicate shake 556

flasks. Symbols: xylose, solid squares; cellobiose, open squares; ethanol, diamonds; cell mass, 557

circles. Minimal defined medium (CBS) was employed. Flasks were incubated at 30 °C and 558

shaken at 200 rpm. 559

560

0

10

20

30

0

5

10

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30

35

40

45

0 12 24 36 48 Et

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/l)

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ose

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cello

bio

se (g

/l)

Time (h)


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561

Figure 4. Co-utilization of glucose, xylose, and cellobiose by Spathaspora passalidarum 562

Symbols: glucose, open triangles; xylose, closed squares, cellobiose, open squares, ethanol, 563

diamonds; cell mass, circles. 564

0

15

30

45

0

10

20

30

40

50

60

0 24 48 72

Cel

ls o

r Et

han

ol (

g/l

)

Glu

cose

, xyl

ose

or

cello

bio

se (g

/l)

Time (h)

0

15

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45

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60

0 24 48 72

Cel

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ose

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Cel

lob

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(g/l

) A

B


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565

Figure 5. Fermentation of a maple hemicellulose hydrolysate (A) and a synthetic sugar mixture 566

(B) by Spathaspora passalidarum E7 in defined minimal medium (CBS). Symbols the same as 567

Figure 4. Averages of two bioreactors is shown with range of values (grey bars). 568

0

6

12

18

24

30

36

42

0

10

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0 12 24 36 48 60 Et

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/l)

Glu

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Xyl

ose

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Time (h)

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42

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ose

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569

570

Figure 6. Fermentation of an AFEX hydrolysate by Spathaspora passalidarum AF2 in defined 571

minimal medium (Slininger). Fermentation was conducted in duplicate shake flasks (50 ml/125 572

shaken at 100 rpm). AF2 was cultured in a 125-ml Erlenmeyer flask containing 50 ml of AFEX 573

hydrolysate medium at 100 rpm and 30 °C for 5 d (OD above 40) and then transferred directly 574

from adapted flask into another flask containing the same AFEX hydrolysate medium. The initial 575

targeted OD600 was 0.5. Duplicate flask fermentations were conducted at 30 °C with rotary 576

shaking at 100 rpm. The starting cell density was 0.1 mg cdw/ml. Symbols: (A) glucose, open 577

triangles; xylose, closed squares, cellobiose, open squares, ethanol, gray diamonds; cell mass, 578

gray circles; (B) acetic acid, open diamonds; xylitol open circles. Averages of two shake flasks 579

are shown. 580

0

5

10

15

20

25

0

10

20

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50

0 1 2 3 4 5 6 7 8 9

Eth

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cell

dry

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, xyl

ose

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bio

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/l)

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0.0

0.5

1.0

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2.5

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Ace

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ol (

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581

Figure 7. Intracelluar levels of glycolytic metabolites and amino acids in Spathaspora 582

passalidarum cultivated on glucose or xylose under aerobic (blue bars) and oxygen-limited (red 583

bars) conditions (cf. Figure 2AB) All units are µmol/g cdw except for 6P-gluconate and 584

sedoheptulose-7P, which are expressed as area units/g cdw. 585

586

Triose-P

1

2

3

4

Glucose Xylose

Glucose-6P

0.2 0.4 0.6 0.8 1.0 1.2

Glucose Xylose

Fructose-6P

0.10

0.20

0.30

0.40 Fructose-1,6 bP

Glucose Xylose

0.05 0.10 0.15 0.20 0.25 0.30

Glucose Xylose

PEP

-

1 2 3 4 5 6

Glucose Xylose

Pyruvate

1

2

3

4

5

Glucose Xylose

2+3 P-Glycerate

100

200

300

400

Glucose Xylose

6P-Gluconate

1,000

2,000

3,000

4,000

Glucose Xylose

Sed-7P

2 4 6 8

10 12

Glucose Xylose

Erythrose 4P

1

2

3

4 Pentose P

Glucose Xylose

Area u

Area u

XyloseGlucose

1

2

3

4

Glucose Xylose

α-Ketoglutarate

1

2

3

4

5

Glucose Xylose

Succinate

0.5 1.0 1.5 2.0 2.5

Glucose Xylose

Fumarate

2 4 6 8

10

Glucose Xylose

Malate

2

4

6

8

Glucose Xylose

Citrate + isocitrate

Acetyl CoA

Ethanol


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0

20

40

60

80

100

0 20 40 60 80

Glu

cose

or

CD

W (

g/l

)

0

20

40

60

80

100

0 20 40 60 80

Xy

lose

or

CD

W (

g/l

)

0

20

40

60

80

100

0 20 40 60 80

Glu

cose

, x

ylo

se o

r C

DW

(g

/l)

Time (h)


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0

5

10

15

20

25

30

35

40

45

50

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48

Etha

nol o

r Dry

Cel

l Wt (

g/L)

Glu

cose

(g/L

)

Time (h)

12

A

0

5

10

15

20

25

30

35

40

45

50

0

10

20

30

40

50

60

70

80

90

100

0 12 24 36 48

Etha

nol o

r Dry

Cel

l Wt (

g/L)

Xylo

se (g

/L)

Time (h)

1

2

B


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0

10

20

30

0

5

10

15

20

25

30

35

40

45

0 12 24 36 48

Eth

an

ol

or

cell

dry

wt

(g/l

)

Xy

lose

or

cell

ob

iose

(g

/l)

Time (h)


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0

15

30

45

0

10

20

30

40

50

60

0 24 48 72

Ce

lls

or

Eth

an

ol

(g/l

)

Glu

cose

, xy

lose

or

cell

ob

iose

(g

/l)

Time (h)

0

15

30

45

0

10

20

30

40

50

60

0 24 48 72

Ce

lls

or

Eth

an

ol

(g/l

)

Glu

cose

, xy

lose

or

Ce

llo

bio

se (

g/l

) A

B on May 23, 2018 by guest

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0

6

12

18

24

30

36

42

0

10

20

30

40

50

60

70

0 12 24 36 48 60

Eth

an

ol

or

Dry

Ce

ll W

t (g

/l)

Glu

cose

or

Xy

lose

(g

/l)

Time (h)

0

6

12

18

24

30

36

42

0

10

20

30

40

50

60

70

0 12 24 36 48 60

Eth

an

ol

or

Dry

Ce

ll W

t (g

/l)

Glu

cose

or

Xy

lose

(g

/l)

Time (h)

A B


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0

5

10

15

20

25

0

10

20

30

40

50

0 1 2 3 4 5 6 7 8 9

Eth

an

ol

or

cell

dry

wt

(g/l

)

Glu

cose

, xy

lose

or

cell

ob

iose

(g

/l)

Time (d)

0.0

0.5

1.0

1.5

2.0

2.5

0 1 2 3 4 5 6 7 8 9

Ace

tic

aci

d o

r x

yli

tol

(g/l

)

Time (d)

A B


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.org/D

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.org/D

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cofermentation of glucose, xylose, and cellobiose by the...

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