Conversion of pentoses by yeasts

Conversion of Pentoses by Yeasts

CHENG-SHUNG GONG, TANYA A. CLAYPOOL, LINDA D. MCCRACKEN, CHRISTINE M. MAUN,

PEAR P. UENG, and GEORGE T. TSAO, Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907

summary

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: 1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed vanes with the yeast strains used. The most commonly detected product is xylitol. 2) The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. 3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. 4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. 5) Of the four substrates examined, ~-xylulose was the preferred substrate, followed by D - X Y ~ O S ~ , L-arabinose, and xylitol. 6 ) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.

INTRODUCTION

Both D-xylose and L-arabinose are the constituent pentoses of hemicellu- losic materials. Traditionally, these sugars have been considered nonfer- mentable by yeasts. Many bacteria metabolize pentoses to produce a variety of metabolic products including ethanoL2 Some mycelial fungi such as Fu- s a r i ~ r n , ~ Mucor, and Monilia4 also ferment pentoses to produce ethanol; however, the slow pentose conversion rate in mycelial fungi renders the fermentation of pentoses to ethanol by such organisms impractical.

Many yeasts metabolize pentoses aerobically5 to produce corresponding polyols (e.g., xylitol and arabitol) as metabolic by-product^,^,' and in some cases small amounts of ethanol are produced.8

Recently, the use of yeasts and yeast mutants to produce ethanol from D-xylose has been reported. A yeast originally isolated from wood sulfur li- quor, Pachysolen tannophilus, has been reported to produce ethanol from ~ - x y l o s e . ~ ~ ' ~ Candida tropicalis ATCC 1369 has also been reported to produce ethanol from D-xylose, but only when specific fermentation conditions are used." Furthermore, a mutant strain of yeast, Candida sp. XF 217, was

Biotechnology and Bioengineering, Vol. XXV, Pp. 85-102 (1983) 0 1983 John Wiley & Sons, Inc. CCC 0006-8592/83/010085-18$02.80

86 GONG ET AL.

reported to produce ethanol from D-xylose at the expense of xylitol, l2 the nor- mal metabolic product from D-xylose.

There are two possible metabolic routes by which microorganisms asimi- late pentoses. In prokaryotes, the early step of pentose metabolism involves the induction of synthesis of uptake enzymes followed by the isomerization of D-xylose to D-xylulose. Subsequently, D-xylulose is phosphorylated to D - x ~ ~ u - lose-5-phosphate, the key intermediate of D-xylose metabolism. l3 On the other hand, eukaryotic microorganisms such as mycelial fungi and yeasts, metabolize pentoses mainly through reduction of pentoses to pentitols, followed by the oxidation of pentitols to pentuloses. 14-16 Again, D-xylulose-5- phosphate is the key intermediate resulting from the phosphorylation of pentulose to pentulose-5-phosphate.

Yeasts are not able to produce ethanol from pentoses in significant quantities. Many yeasts, however, including those not normally able to metabolize pentoses (e.g., S. cerevisiae), ferment pentulose (e.g., D-xylulose) to ethanol readily.8J7-19 In some cases, the yield of ethanol can reach 90% of the theo- retical yield (two moles of ethanol per mole of sugars Results indicate that yeasts and mycelial molds possess the enzyme systems that are required to produce ethanol from pentoses.

As part of the research projects undertaken in our laboratory while study- ing the conversion of pentoses to liquid fuels, we have conducted experiments to survey the ability of selected yeast strains to metabolize pentoses and pentose metabolites.

MATERIALS AND METHODS

Microorganisms

The majority of yeast cultures were purchased from American Type CA- ture Collection (Rockville, MD). Yeast mutants were obtained by UV muta- genesis. Cultures were maintained on peptone, malt-extract, yeast-extract, and glucose agar (YMA-Difco) slants.

Substrates

D-Xylose, L-arabinose, xylitol, D-arabitol, and L-arabitol were purchased from Sigma Chemical Company (St. Louis, MO). The D-xylulose was prepared from D-xylose through enzymatic isomerization by D-xylose isomerase using the methods described by Gong et aL8 Pure concentrated D-xylulose syrup was obtained by the methods described by Chiang et aL21 The purity of D-xylulose is shown in Figure 1.

Media and Cultures

Yeast extract-malt-peptone (YMP) media was used for growth and fermentation and contained the following composition (in g/L): Bacto-yeast

CONVERSION OF PENTOSES BY YEASTS 87

a)

X

C)

X

d) 1

I

L Fig. 1. Chromatograms of D - X Y I O S ~ and D-XYIUIOS~ at different stages of preparation: (a)

xylose; (b) xylose and xylulose, after enzymatic isomerization; (c) xylulose and xylose, after ethanol precipitation of D-xylose; (d) xylulose, after differential microbial fermentation. For detail see Chiang et al., ref. 21.

extract, 3; Bacto-malt, 3; Bacto-peptone, 5. Concentration of carbon source in growth media was 10 g/L and for fermentation experiments was 50 g/L. Media and substrates were autoclaved separately and mixed after sterilization. Sterilization was accomplished by autoclaving at 121°C for 15 min.

Fermentation

Shake-flask experiments were conducted in 50-mL Erlenmeyer flasks, each containing 10 mL of media with the appropriate substrates. Inocula were prepared by growing the yeasts in flasks in the same media for 20-48 h depending on the time required for maximal growth. Yeast cells were har- vested by centrifugation. After washing the yeast pellets with sterile water, a cell density of (1-3) X los cells/mL was inoculated into fresh YMP media with substrates. Cells were incubated for 3 days at 30°C on an incubator- shaker at 200 rpm (New Brunswick).

Samples were then taken to determine and quantify substrate consumption and product formation. Flask cultures used for aerobic incubation were capped with milk filters and aluminum foil. For oxygen-limited fermentative incubation, Erlenmeyer flasks were capped with rubber stoppers; the carbon dioxide produced was allowed to escape through a hypodermic needle (18 gauge).

88 GONG ET AL.

Sugar Consumption and Product Formation

The sugars consumed and the nonvolatile products formed were analyzed and quantified by low-pressure liquid chromatography as described by La- disch and Tsao.22 The column packing material was Aminex 50W-X4 (Bio- Rad) in the Ca2+ form and the operating conditions were those described earlier. Figure 2 shows the typical liquid chromatography retention times for the compounds of interest. The actual retention times varied slightly from one column to another. The concentration of ethanol produced was mea- sured by gas chromatography. Sugar alcohols produced were identified and verified by paper chromatography. 23

RESULTS

Conversion of D-Xylose

Candida

A total of 20 strains of Candida yeasts belonging to 11 species were examined for D-xylose conversion in flask cultures. The extent of D-xylose utilization and product formation under aerobic and fermentative incubation conditions are shown in Table I. All yeast strains utilized D-xylose readily, especially under aerobic incubation conditions. Candida friedrichii, C. melibiosica, and C. pseudotropicalis were poor D-xylose utilizers under fermentative conditions.

1

6 12 I

Fig. 2. Liquid chromatography retention times for the compounds of interest: (X) xylose; (Xu) xylulose; (G) glycerol; (AO) arabitol; (XO) xylitol.

TAB

LE I

C

onve

rsio

n of

D-X

YIO

S~

by C

andi

da s

pp.

Prod

ucts

(To)

' ln

cuba

tiona

X

ylos

eb

Org

anis

m

ATC

C N

o.

cond

ition

co

nsum

ed (

70

) xy

litol

ar

abito

l et

hano

l

C. b

lank

ii 18

735

C. jr

iedr

ichi

i 22

970

C. I

usiia

niae

26

287

C. m

elib

iosic

a 18

738

C. m

ogii

1836

4

C. p

arap

silo

sis

2a47

4

C. p

arap

silo

sis

3407

8

C. p

seud

otro

pica

lis

4135

C. s

olan

i 13

234

C. s

teat

oiyt

ica

1882

4

C. i

ropi

calis

20

240

C. t

ropi

calis

20

215

C. u

tilis

22

023

aA-a

erob

ic;

N-f

erm

enta

tive.

bI

nitia

l D-X

Y~O

W co

ncen

tratio

n w

as 5

% (w

/v).

'Per

cent

, w

eigh

t by

volu

me.

A N A N A N A N A N A N A N A N A N A N A N A N A N

4.55

2.

78

3.09

0.

9 4.

36

1.7

4.26

0.

85

4.74

1.

41

5.0

4.8

2.9

1.5

4.26

0.

82

5.0

2.4

4.26

2.

17

5.0

2.96

5.

0 1.

64

4.49

1.

82

1.82

0.

92

1.1

0 1.96

0.

61

1.22

0.

08

2.82

1.

04

3.32

2.

96

1.26

1.

08

0.04

0.

28

0.26

1.

44

0.13

0.

84

2.8

1.72

2.

31

0.64

1.

28

1.2

0.09

0.

08

0.37

0.

2 0 0 1.

35

0 0.26

0 0.

42

0.28

0.

09

0.04

0.

04

0 0.13

0 0.

31

0.24

0 0.

8 0 0 0 0

0.51

0.

41

0.27

0 0.

11

0.11

0 0 0 0 0 0.

27

0 0 0.22

0 0.

15

0.27

0 0.

2 0 0.

34

0 0.27

0 0

90 GONG ET AL.

Product analysis shows that, generally, xylitol was the predominant product, followed by D-arabitol and ethanol. The relative amounts of products produced varied depending on the strains employed. Small amounts of glycerol and other unidentified products were formed by some strains. The good xylitol producers were C. parapsilosis (28474) and all five strains of C. tropi- cafis examined. Products formed from D-xylose were utilized as substrates when the supply of D-xylose was depleted. Thus, xylitol is the major product of catabolism of D-xylOSe in yeasts, and yeasts utilize xylitol upon depletion of D-xylose resulting in increased cell density.

Utilization of D-xylose and the products formed from D-xylose under fermentative conditions were similar to those formed under aerobic conditions (see Table I). The slower rates of D-xylose utilization and product formation under fermentative conditions are due to reduced yeast growth rates.

Most of the yeast strains produced less than 1% D-arabitol from D-xylose; however, C. melibiosica produced 1.35% D-arabitol under aerobic conditions. Some strains produced low levels of ethanol under both aerobic and fermentative conditions, but no consistent product pattern was observed. The amounts of ethanol produced could be increased through mutation and selection in these yeast strains. Enhanced ethanol production at the expense of xylitol production was reported in Candida sp. mutant, XF217.I2

Saccharomyces

A total of 21 strains of Saccharomyces yeasts belonging to eight species were examined for D-xylose conversion. The results of D-xylose utilization and conversion by selected strains of yeasts under aerobic and fermentative incubation conditions are shown in Tables I1 and 111. Since all of the yeast strains of the genus Saccharomyces except S. cerevisiae (24553) grow poorly when D-xylose was used as a carbon and energy source; D-glucose was used to grow the yeast cultures for subsequent pentose fermentation analyses.

TABLE I1 Conversion of D-XYIOW by Saccharomyces spp.

Xylose consumed Xylitol produced (%)

Organism ATCC No. A N A N

S. bailii 8766 1.24 0.97 0 0.47 S. diastaticus 28338 0.7 0.47 0.13 0.44 S. rouxii 32901 1 .O 0.9 0.35 0.4 S. saki 2642 1 0.5 0.24 0.31 0.14

S. uvarium 24556 0.74 1.54 0.22 0.09 S. uvarium 26602 4.84 1.03 2.1 0.44

s. sp. 764 0.26 0.36 0.1 0.28

aInitial D-xylose concentration was 5% (w/v): A-aerobic; N-fermentative.


TABLE 111 Conversion of D-Xylose by Saccharomyces cerevisiae

Xylose consumeda Xylitol produced (70 ) ("lo)

Strains (ATCCNo.) A N A N

4126 4132 9763

24553 24857 24859 26497 26603 26785

0.2 0.57 0.2 0.5 0.65 0.5 3.25 1.04 0.68

1.18 0.71 0.84 0.88 1.35 1.32 1.32 0.51 1.28

0.1 0.1 0.1 0.2 0.33 0.33 0.68 0.17 0.3

0.31 0.16 0.2 0.25 0.52 0.84 0.52 0.1 0.58

aInitial D-xylose concentration was 5% (w/v): A-aerobic; N- fermentative.

Xylitol was the metabolic product formed from D - X ~ ~ O S ~ by these Saccharo- myces yeasts, however, a small amount of D-arabitol (< 0.1%) instead of xylitol was'produced by S. bailii (8766) under aerobic conditions. In general, Saccharomyces yeasts utilized xylose poorly with the exception of S. uvarum 26602 (Table 11) and S. cerevisiae 26497 (Table 111).

Schizmaccharomyces pombe

Eight strains of yeasts belonging to the species S. pombe were examined for D-xylose utilization under aerobic and fermentative conditions. Like the Saccharomyces yeasts, the strains of S. pombe grew poorly when D-xylose was the carbon source. As a result, they were grown in media with glucose as the carbon source.

The results of D-xylose consumption and product formation are shown in Table IV. The results suggest that most strains of S. pombe utilize xylose readily, especially under aerobic conditions. The majority of strains tested produced xylitol and ethanol from D-xylose. In addition to xylitol and ethanol, S. pombe 2476 and 16979 produced organic acids under both aerobic and fermentative conditions.

Conversion of D-Xylulose

The yeast cultures for these experiments were grown as previously described in liquid media containing D-xylose or n>-glucose as the carbon source.

Candida

The pattern of D-xylulose utilization and product formation by Caizdida yeasts differed from the pattern observed when D-xylose was used as the sub-

92 GONG ET AL.

TABLE IV Conversion of D-Xylose by Schizosaccharomyces pombe

Products (TO) Xylose consumed

(%)a Xylitol EtOH Strains

(ATCC No.) A N A N A N

2476 2478 16979 20130 2475 1 26189 26192 26760

3.91 3.43 3.48 3.54 3.7 3.29 4.05 3.23

2.88 2.07 2.15 2.18 1.9 2.21 2.04 2.08

0.07 0 0 0 0.12 0.09 0 0.07

0.16 0.14 0 0.14 0.11 0.35 0.21 0.29

0 0.31 0 0.5 0 0.11 0.1 0.5 0 0.43 0 0.14 0 0.39 0 0.17

aInitial D-xylose concentration was 5% (w/v): A-aerobic; N-fermentative.

strate. Most yeasts utilized D-xylulose readily under aerobic and fermentative conditions with the exception of C. blankii which utilized D-xylulose poorly. Table V shows that the products formed from xylulose by Candida yeasts in- clude ethanol, xylitol, and D-arabitol. The amount and type of products formed depends on the strains of yeast. The results summarized in Table V indicate that C. tropicalis 20240 produced predominantly ethanol. Similar results were obtained from C. tropicalis 750, 1369, and 20215. Both C. mogii 18364 and C. pseudotropicalis 4135 were good D-arabitol producers under both aerobic and fermentative conditions, while C. parapsilosis 28474 produced predominantly xylitol under aerobic conditions and D-arabitol under fermentative conditions.

Saccharomyces

Most of the yeast strains belonging to the genus Saccharomyces utilized xylulose readily under both aerobic and fermentative conditions (Table VI). Succharomyces cerevisiae 4126 and S. cerevisiae 24859 utilized D-xylulose poorly and S. saki 26421 did not utilize D-xylulose under either incubation condition. The products formed from D-xylulose by Saccharomyces yeasts are ethanol, xylitol, and D-arabitol. The amount and type of products formed by this group of yeasts varied widely depending on the yeast strain and incubation conditions employed. For example, s. uvarum 24556 produced mostly ethanol under fermentative conditions, but produced mainly arabitol under aerobic conditions; D-arabitol is the major product of S. cerevisiae 26497 under both aerobic and fermentation conditions. S. cerevisiae 4132 produced approximately equal amounts of xylitol and D-arabitol under aerobic conditions, but produced mostly xylitol under fermentative conditions.

TA

BL

E V

C

onve

rsio

n of

D-X

ylul

ose

by C

andi

da s

pp.

0 0

z Pr

oduc

ts (7

’0)

s z 5 C

. bla

nkii

1873

5 A

0.

39

0 0.

1 0

z N

0.

36

0 0.

2 0.

1 C

. mog

ii 18

364

A

3.66

0

0.56

1.

0 z

N

4.49

1.

17

1.01

1.

25

C. s

teat

olyt

ica

1882

2 A

2.

1 0.

7 0.

4 0.

1 4

2.

15

1.12

0

0.1

2 N

C

. par

apsi

losi

s 28

474

A 4.

87

0.29

1.

6 0.

9 (n

z N

3.69

0.

51

0.4

1.25

C

. pse

udot

ropi

calis

41

35

A

5.

0.19

0

0.67

N

5.

1.

54

0.8

1.22

s

C. t

ropi

calis

20

240

A

5.

1.04

0.

2 0.

45

% N

4.67

2.

04

0.2

0.2

2

Xyl

ulos

eb

Org

anism

A

TCC

No.

C

ondi

tions

a co

nsum

ed (7

’0)

EtO

H

Xyl

itol

Ara

bito

l

z m

aA-a

erob

ic;

N-f

erm

enta

tive.

bI

nitia

I D-x

ylul

ose c

once

ntra

tion

was

5%

(w

/v).

TA

BL

E V

I C

onve

rsio

n of

D-X

ylul

ose b

y Sa

ccha

rom

yces

spp

.

Xyl

ulos

eb

Prod

ucts

(%)

cons

umed

O

rgan

ism

ATC

C N

o.

Con

ditio

nsa

("lo

) E

tOH

X

ylito

l A

rabi

tol

S. c

erev

iriae

41

26

A

0.74

0

0.1

0 N

0.

6 0.

34

0 0

4132

A

4.

56

0.39

0.

46

0.48

0

N

4.28

1.

02

1.93

0.

31

2649

7 A

4.

01

0.1

0.99

1.

14

N

4.07

0.

94

0.68

1.

36

3 S.

sak

i 26

421

A

0 0

0 0

* N

0

0 0

0 r

s. sp

. 76

4 A

3.

99

0 0.

1 1.

17

N

4.17

1.

02

0.04

2.

35

S. u

varu

m

2455

6 A

2.75

0

0.61

2.

45

N

4.95

2.

27

0.66

0

S. u

varu

m

2660

2 A

3.

97

0.69

0.

2 0.

23

N

4.78

1.

61

0.26

0.

87

aA-a

erob

ic;

N-fe

rmen

tativ

e.

bIni

tiaI D

-xyl

ulos

e con

cent

ratio

n w

as 5

% (w

/v).


Schizmaccharomyces pombe

All strains of S. pombe are excellent ethanol producers from D-xylulose with more ethanol being produced under fermentative than under aerobic conditions (Table VII). The reduced yield of ethanol under aerobic conditions is due in part to the utilization of ethanol by yeasts. Small amounts of xylitol and D-arabitol are also produced by these yeast strains.

Yeasts of other genera

Table VIII summarizes product formation from D-xylulose for yeasts belonging to other genera. Hansenula anomala 34080 and Kluyveromyces fra- gilis 12424 were good ethanol producers under fermentative conditions, while Wingea robertii 22312 was a good D-arabitol producer under both aerobic and fermentative conditions. Organic acids were produced under aerobic conditions by Brettanomyces claussenii 20323, Dekkera intermedia 24196, and Pachysolen tannophilus 32691.

Conversion of Xylitol

In general, xylitol was not a good substrate when compared to either D-

xylose or D-xylulose. Table IX summarizes the results of xylitol utilization

TABLE VII Conversion of ~-Xylulose by Schizosaccharomyces pombea

Products (70) Strains

(ATCC No.) Conditionsb EtOH Xylitol Arabitol

2476

2478

16979

20130

24751

26189

26192

26760

A N A N A N A N A N A N A N A N

1.27 2.33 1.46 1.85 1.56 2.13 1.17 2.04 1.27 1.94 1.36 1.94 1.36 2.04 1.46 2.23

0.92 0.1 0.92 0.81 0.59 0.86 0.7 0.65 0.76 0.97 0.54 0.7 1.13 0.76 1.03 0.6

0.32 0.27 0.14 0.09 0.18 0.23 0.1 0.14 0.23 0.27 0.14 0.23 0.23 0.27 0.14 0.18

aInitial D-xylulose concentration was 5% (w/v). After incubation, all

bA-aerobic: N-fermentative. of the D-XYIUIOS~ were consumed by yeasts.

TA

BL

E V

III

Con

vers

ion

of D

-Xyl

ulos

e by

Yea

sts

Prod

ucts

(7

0)

Xyl

ulos

e O

rgan

ism

ATC

C N

o.

Con

ditio

ns

cons

umed

(%)

EtO

H

Xyl

itol

Ara

bito

l A

cids

a

Han

senu

la a

nom

ala

3408

0 A

N

Kl

uyve

rom

yces

frag

ilis

1242

4 A N

Toru

lasp

ora

hens

enii

2012

4 A N

W

inge

a ro

bert

ii 22

312

A

N Br

etta

nom

yces

cla

usse

nii

2032

3 A N

Dek

keru

int

erm

edia

24

196

A

N

N Pa

chys

olen

tun

noph

ilus

3269

1 A

3.24

4.

53

5 5 5 5 5 3.76

3 3.

3 1.

56

4.43

5

0.1

2.3

1.23

2.

05

1.23

1.

79

0.3

0.58

0.

39

1.06

0 1.

56

1.26

-

0.6

0.1

0 0.1

0.34

0.

1 1.

3 0.

3 0.

15

0.1

0.1

0 0.58

-

0.28

0.

5 0.

95

0

0.99

8

0.28

m

1.

46

c3 > r

1.04

2.

26

0.46

+

0.71

0

+ 1.

23

0.91

+

-

aAm

ount

s of

acid

pro

duce

d w

ere

not q

uant

ified

.

CONVERSION OF PENTOSES BY YEASTS

TABLE IX Conversion of Xylitol by Yeasts

97

Xylitol consumed EtOH produced (70)" (YO)


S. cerevisiae 26785 0 0 0 0 C. parapsilosis 34078 0.6 0.6 0 0 Hunsenulu anomula 34080 1.84 1.34 0 0 Wingea robertii 22312 4.96 2.71 0 0 C. tropicalis 750 1.97 1.19 0 0.23 Kluyromyces frugilis 12424 0.71 1.16 0 0.4 C. pseudotropicalis 4135 2.95 1.51 0.4 0.5

"Initial xylitol concentration was 5 % (w/v): A-aerobic; N-fermentative.

and product formation by selected yeasts. Small amounts of ethanol were produced by some yeasts such as C. pseudotropicalis 4135.

Conversion of L-Arabinose

Most yeast strains examined utilized L-arabinose under both aerobic and fermentative conditions: L-arabitol is the major metabolic product. Table X summarizes the results of L-arabinose conversion by selected yeasts. In some cases, small amounts of ethanol were produced (e.g., Schizosaccharomyces pombe 26192).

Yeast Mutants With Modified D-Xylose Conversion

Previously, we reported that the mutant strain, XF 217, derived from Can- dida sp., produces ethanol from D-xylose at the expense of xylitol production.12 We have obtained other mutants from the original strain with differ-

TABLE X Conversion of L-Arabinose by Yeasts

Arabinose consumeda Arabitol produced (%) (%)


Hansenula polymorphu 14754 0 0 0 0 Schizosaccharomyces

pombe 26192 1.34 1.5 0.7 0.12 Puchysolen tannophilus 32691 3.04 2.9 1.8 0.59 C. tropicalis 20215 4.2 0.52 2.7 0.4

'Initial L-arabinose concentration was 5% (w/v): A-aerobic; N-fermentative.

98 GONG ET AL.

ences in metabolic product patterns. Figure 3 compares the time course of D-xylose utilization and product formation of the wild-type strain and several mutants. The wild-type strain, Candida sp., produced predominantly xylitol from D-xylose with small amounts of glycerol and ethanol [Fig. 3(A)]. A slight increase in glycerol and ethanol production was observed in a mutant, C210 [Fig. 3(B)]. Increased ethanol production at the expense of xylitol was observed in mutant strain, XF 217 [Fig. 3(C)]. Production of D-arabitol was introduced at the expense of xylitol production in yet another mutant C26 [Fig. 3(D)J. These observations indicate that the activity of enzymes involved in D-xylose metabolic pathways can be modified through mutation and selection to result in the altered product patterns.

Mutants with an enhanced ability to produce ethanol were obtained from yeasts belonging to genera other than Candida. Figure 4 shows the time courses of D-xylose utilization and product formation by the parent strain, Pachysolen tannophilus 32691, and a mutant strain, PT 15. Enhanced etha-

A

mFc 0

C

'0 24 48 ?2

K

D

k 24 48 76

HOURS

Fig. 3. Aerobic conversion of D-xylose [6.5% (w/v)] by Candida sp. and mutants: (A) wild- type; (B) C210; (C) XF217; (D) C26; (X) xylose; (XO) xylitol; (E) ethanol; (G) glycerol; (A) arabitol.


2

I

- -1 \ (3

-1 - P

0 1 HOURS

Fig. 4. Conversion of D-xylose [8% ( w J v ) ] by Pachysolen tannophilus ATCC 32691 and PT 15: (A) P. tannophilus ATCC 32691, aerobic; ( B ) P. tannophilus ATCC 32691, fermentative; (C) PT 15, aerobic: (D) PT 15, fermentative.

no1 production was observed in F T 15 under both aerobic and fermentative incubation conditions [Figs. 4(C) and 4(D)]. Figure 5 shows the time course of ethanol and xylitol production from D-xylose by a mutant yeast derived from a S. cerevkiae strain incapable of producing ethanol from D-xylose.

DISCUSSION

The results of pentose metabolic studies presented in this article demon- strate that xylitol is the most common metabolite produced by yeasts from either D-xylose or D-xylulose. The production of xylitol confirms the commonly accepted idea that oxidoreduction reactions form the basis for the predominant pentose catabolic pathway in yeasts.

In the majority of prokaryotes the isomerization of D-xylose to D-xylulose is the first step in D-xylose metabolism. Although for some yeasts, such as C. ~ t i l k ~ ~ and Rhodotorula gracilis l5 the presence of inducible D-xylose isomerase has been demonstrated. Our attempts to measure the activity of this enzyme in many yeasts have proved unsuccessful.

Some yeasts, such as C. blankii, and C. tropicalis (Table I), produce small

100 GONG ET AL.

0 5t HOUR

Fig. 5. Conversion of D-xylose [ lo% (w/v)] by Saccharomyces cerevisiae SC138: (A) aerobic; (B) fermentative; (X) xylose; (XO) xylitol; (E) ethanol.

quantities of ethanol from D-xylose. This observation, in conjunction with the fact that many yeasts quantitatively metabolize D-xylulose readily to form ethanol, indicates that once D-xylose is converted to D-xylulose, the xylulose is phosphorylyzed to xylulose-5-phosphate and channelled into the pentose phosphate pathway to produce pyruvate as the key intermediate.B In Fusarium oxysporium (resting cells), the metabolism of D-xylose is believed to result in the production of 2-carbon and 3-carbon intermediates26 via a pathway similar to that for lactic acid production in bacteria.2 This type of metabolism is not evident in the majority of yeasts.

The production of arabitol from either D-xylulose or D-xylose is interesting in that ribulose-5-phosphate is the precursor of arabitol, 27 and ribulose-5- phosphate can be derived from xylulose-5-phosphate by phosphoketopentoepimerase (~-xylulose-5-phosphate-3-epimerase). C. melibiosica produces significant quantities of arabitol, an indication that this yeast could be high in phosphoketopentoepimerase, D-nbulose-5-phosphatase and arabitol dehydro- genase.28 Enzymatic studies of this yeast could confirm this assumption.

The presence of oxygen enhances the rate of D-xylose metabolism but has no significant effect on D-xylulose utilization. A similar oxygen-related effect,


the “Kluyer Effect” has been described by Sim and B a n ~ e t t ~ ~ for the utilization of oligosaccharides and D-galactose by yeasts. This oxygen effect could result from either a direct or indirect oxygen requirement for the entry of D-xylose or for the activity of an initial catabolic enzyme.

The ability of C. blankii to utilize D-xylose more effectively than D-xylulose is intriguing since in general D-xylulose is a better substrate for yeasts than D-xylose. One possible explanation is that C. blankii is lacking a D-xylulose uptake carrier. Most yeasts also utilize D-xylulose at a faster rate than D-xylose. This fact and the response of C. blankii to D-xylulose indicate that yeasts possess different carriers for D-xylose and D-xylulose transport.

It is of particular interest to note that many yeast strains belonging to the species S. pombe (Table IV) and the genus Saccharomyces (Table 111) are able to convert D-xylose to products such as xylitol and ethanol even though they cannot utilize it effectively for cell growth. In addition, these yeasts are not able to accumulate D-xylose when pulse-labelled experiments with 1 4 C - ~ - xylose were conducted (unpublished observation). A possible explanation for this behavior is that S. pombe and some strains of Saccharomyces take up D-xylose and rapidly convert it to products, such as ethanol and xylitol, and that these products cannot serve effectively as carbon sources. It deserves mention that at least one additional unknown metabolic product is produced by some strains of S. pombe under both aerated and fermentative conditions. This product may be an intermediate of D-xylose metabolism in S. pombe.

The results of mutant studies indicate that mutations resulting in different metabolic product patterns can be induced in yeasts. These mutants can be employed to study the metabolic regulation of pentose utilization in yeasts. As a final note, yeast could be classified into groups with respect to pentose utilization and types of products formed. However, this information cannot be used as a basis for taxonomical classification of yeasts.

The authors express their appreciation to both Savannah Foods and Industries, Inc. and United States Sugar Corp. for sponsoring this research effort. They also wish to thank Mr. Allen Anderson, Ms. Cecilie Hunter, Ms. Mary Vanderby, and Mr. David Goldhamer for their tech- nical assistance.

References

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Accepted for Publication June 21, 1982

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Conversion of pentoses by yeasts