Development on ethanol production from xylose by recombinant Saccharomyces cerevisiae

doi:10.4236/ns.2009.13028

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Vol.1, No.3, 210-215 (2009)

doi:10.4236/ns.2009.13028

SciRes

Natural Science

Development on ethanol production from xylose by

recombinant Saccharomyces cerevisiae

Jin-Ying Yang, Jian-Ren Lu*, Hong-Yue Dang, Yan Li, Bao-Sheng Ge

Center for Bioengineering and Biotechnology of China University of Petroleum, Qingdao, China

Received 26 August 2009; revised 28 September 2009; accepted 30 September 2009.

ABSTRACT

Xylose is the second major fermentable sugar

present in lignocellulosic hydrolysates, so its

fermentation is essential for the economic con-

version of lignocellulose to ethanol. However,

the traditional ethanol production strain Sacch-

aromyces cerevisiae does not naturally use xy-

lose as a substrate. A number of different ap-

proaches have been used to engineer yeasts to

reconstruct the gene background of S. cerevi-

siae in recent years. The recombinant strains

showed better xylose fermentation quality by

comparison with the natural strains. This review

examines the research on S. cerevisiae strains

that have been genetically modified or adapted

to ferment xylose to ethanol from three aspects

including construction of xylose transportation,

xylose-metabolic pathway and inhibitor toler-

ance improvement of S. cerevisiae.

Keywords: Sacchromyces cerevisiae; Xylose;

Ethanol; Metabolic Engineering

1. INTRODUCTION

Rising concerns over the cost of petroleum and the

prospect of global warming are driving the development

of technologies for the production of alternative fuels

such as ethanol [1]. Cellulosic biomass is an attractive

feedstock for fuel ethanol production since it is readily

available, e.g., as a waste from the pulp and paper or

agricultural industries, and also due to the fact that it is

renewable with cycles many orders of magnitude shorter

compared with those of fossil fuels. Hydrolysates of

cellulosic biomass will contain mixtures of sugars, inclu-

ding glucose, galactose, mannose, xylose and arabinose,

and other constituents in variable proportions depending

on the source [2]. Successful industrial production of

ethanol from lignocellulosic hydrolysate depends on the

quantitative conversion of carbon present in the biomass.

It is well known that one of the most effective etha-

nol-producing organisms for hexose sugars is Sac-

charomyces cerevisiae, which shows high ethanol pro-

ductivity, high tolerance to ethanol, and tolerance to in-

hibitory compounds present in the hydrolysate of ligno-

cellulosic biomass [3-5]. However, S. cerevisiae does not

naturally use xylose as a substrate. Only a few yeasts

such as Pichia stipitis [6] and Pachysolen tannophilus [7]

are able to ferment xylose. Genetic engineering can be

used to enable S. cerevisiae to transport and ferment

xylose including modeling, mutation, deletion and so on.

The pentose phosphate pathway (PPP) [8] is a process

that serves to generate NADPH for reductive biosynthe-

sis reactions within cells and the synthesis of pentose

(5-carbon) sugars for the synthesis of the nucleotides and

nucleic acids. There are two distinct phases in the path-

way. The oxidative phase converts the hexose, D-glucose

6P, into the pentose, D-ribulose 5P, plus CO2 and

NADPH. The non-oxidative phase converts D-ribulose

5P into D-ribose 5P, D-xylulose 5P, D- sedoheptulose 7P,

D-erythrose 4P, D-fructose 6P and D- glyceraldehyde 3P.

D-Xylose and L-arabinose enter the PPP through

D-xylulose (Figure 1)

In bacteria, xylose is directly isomerized to xylulose

by xylose isomerase (XI) before entering pentose phos-

phate pathway. In xylose-fermenting yeasts, xylose is

first reduced to xylitol by xylose reductase (XR) and

then oxidized to xylulose by xylitol dehydrogenase

(XDH) [9]. Xylulokinase (XK) phosphorylates xylulose

to xylulose 5-phosphate, which is then metabolized

through the PPP and glycolysis (Figure 2). S. cerevisiae

is not able to metabolize xylose due to the lack of XR

and XDH activity, but it can utilize the isomeric form

xylulose.

2. ENGINEERING YEASTS FOR XYLOSE

METABOLISM

2.1. Xylose Uptake

Xylose is not readily fermentable in wild-type strains of

S. cerevisiae. To circumvent this problem, different

J. Y. Yang et al. / Natural Science 1 (2009) 210-215

211

Engineering pentose metabolism in yeasts. The pentose phosphate pathway (PPP) in yeasts contains the oxidative phase, which

consists of glucose 6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), and the

non-oxidative phase, which is carried out by D-ribulose-5-phosphate 3-epimerase (RPE1), ribose-5-phosphate ketol-isomerase

(RKI1), transketolase (TKL1) and transaldolase (TAL1).

Figure 1. The pentose phosphate pathway.

metabolic engineering strategies have been applied to

enable xylose metabolism, and pentose-fermenting str-

ains of S. cerevisiae have been created principally by

engineering the pathways for converting xylose to xylu-

lose-5-phosphate [10,11]. However, fermentation of xy-

lose still remains significantly less efficient than that of

glucose by these strains. The uptake of xylose into the

cell is one of the reasons.

The S. ceresiave genome contains 20 genes that en-

code for hexose transporters but does not contain genes

for xylose-specific transport system like natural xy-

lose-utilizing yeasts [12]. Uptake of xylose by S. cere-

visiae has been proposed to be mediated more or less

unspecifically by its hexose-transport system. This is

composed of a large family of 18 related transporter

proteins called Hxts and additional sugar transporters

with broader substrate specificity [13,14].

Hamacher et al. [12] found that after deletion of all of

the 18 hexose-transporter genes, the ability of S. cere-

visiae cells to take up and to grow on xylose was lost.

Re-introduction and constitutive expression of individual

HXT genes in strain TMB3201 revealed that at 2% xy-

lose concentrations, high- (Hxt7 and Gal2) and interme-

diate-affinity (Hxt4 and Hxt5) glucose transporters are

required for xylose uptake.

Several studies have indicated that in S. cerevisiae

glucose and xylose appear to share the same transport

facilities and competitively inhibit their mutual transport

[15,16]. Competition with glucose restricts xylose as-

similation, so heterologous expression of a specific xy-

lose transporter could be very useful.

Researchers have tried to identify genes target for im-

proved xylose assimilation. Two genes (GXF1and GSX1)

encoding xylose/glucose transporters from Candida in-

XR: Xylose reductase; XDH : xylitol dehydrogenase; XK : Xylulo-

kinase

Figure 2. The metabolism of xylose in bacteria.

termedia were isolated by Leandro et al. [17], and ex-

pressed in S. cerevisiae. Gsx1 is the first yeast xy-

lose/glucose–H + symporter to be characterized in

Arabidopsis thaliana at the molecular level. Except

GSX1, xylose transporters from Arabidopsis thaliana

J. Y. Yang et al. / Natural Science 1 (2009) 210-215

212

(At5g59250) and Escherichia coli (xylE) were also ex-

pressed in S. ceresiave TMB3120 and failed to support

vigorous growth of the recipient S. cerevisiae strain on

xylose. Even though, the results warrant further investi-

gations for the development of efficient bioethanol pro-

duction processes from lignocellulosic materials.

The presence of three sugar transporters in P. stipitis,

Sut1, Sut2 and Sut3 has been reported. Although all

three transporters have a higher affinity for glucose than

for xylose, the Sut1 transporter has a higher Vmax for

xylose uptake compared to other two Sut transporters

and for hexose transporters [18,19]. Satoshi Katahira [20]

et al. introduced SUT1 into a xylose-assimilating S. cer-

evisiae strain that expresses xylose reductase, xylosede-

hydrogenase and xylulokinase. The results showed that

expression of Sut1 in xylose-assimilating S. cerevisiae

increased both xylose uptake ability and ethanol produc-

tivity during xylose fermentation. Also, the enhancement

of xylose uptake enables to accelerate the ethanol pro-

ductivity during xylose/glucose co-fermentation. How-

ever, there are researchers with different opinions. Gár-

donyi et al. [21,22] concluded that xylose transport in S.

cerevisiae strains has low control over the rate of xylose

utilization, unless the xylose pathway is significantly

improved. In-depth study of the transporters’ mechanism

along with new modeling should continue to drive this

field forward.

2.2. Construction of Recomnant S.

Cerevisiae Strains with

Xylose-Fermenting Ability

2.2.1. Recombinant S. cerevisiae Expressing XR,

XDH, and XK

Researchers have engaged in the development of engi-

neered yeast strains capable of xylose fermentation by

introducing XR and XDH into S

．

cerevisiae. Both of

these enzymes have been isolated and characterized due

to the central role they play in xylose metabolism.

The purified monomeric XR was NADPH-dependent

with an apparent MW of 37 kDa, which was firstly puri-

fied and characterized by Kuhn et al. [23] XDHs also

have been purified and characterized from various xy-

lose-fermenting yeasts.

Kötter et al. [24] first reported the construction of a S.

cerevisiae strain expressing the XR- and XDH-encoding

genes XYL1 and XYL2 derived from the xylose-utilizing

yeast P. stipitis. Walfridsson et al. [25] also genetically

engineered S. cerevisiae to utilize xylose by introducing

the XYL1 and XYL2 genes on either multicopy plas-

mids or by integrating them into the chromosome.

Although these strains can ferment xylose to ethanol,

the excretion of xylitol occurs unless a co-metabolizable

carbon source such as glucose is added. One of the most

important reason is intercellular redox imbalance due to

a different coenzyme specificity of xylose reductase

(with NADPH+) and XDH (with NAD+) [26]. Protein-

engineering of NADPH+-preferring XR and/or NAD+-

dependent XDH is an alternative approach to solve the

problem.

Anderlund [27] constructed four chimeric genes en-

coding fusion proteins of XYL1 and XYL2 with different

orders of the enzymes and different linker lengths. These

genes were expressed in S. cerevisiae. The fusion pro-

teins exhibited both XR and XDH activity when XYL1

was fused downstream of XYL2. The results showed

that the xylitol yield was lower in these strains than in

strains expressing only native XR and XDH monomers,

0.55 and 0.62, respectively, and the ethanol yield was

higher.

By analyzing the amino acid of coenzyme-binding

domain of XDH, Watanabe [28] modified XDH from P.

stipitis by three- and four-site direct mutagenesis. The

triple mutant (D207A/I208R/F209S) and quadruple mu-

tant (D207A/I208R/F209S/N211R) showed more than

4500-fold higher values in kcat/Km with NADP+ than

the wild-type enzyme, reaching values comparable with

kcat/Km with NAD+ of the wild-type enzyme.

In recent years, the research group introduced these

mutated PsXDHs with the PsXR WT to S. cerevisiae and

estimated effect(s) of the functional modiﬁcation(s) of

PsXDH on fermentation of xylose to ethanol in recom-

binant S. cerevisiae [29]. The results showed that re-

combinant yeast strains gave the highest ethanol produc-

tion and the lowest xylitol excretion.

Zeng et al. [30] altered the coenzyme specificity of P.

stipitis XR via rational design based on the 3D structure.

Lys21, the only one amino acid that has hydrogen bind-

ing interaction with NADP+ but not with NAD+ in the

binding pocket, were changed to Ala and Arg respec-

tively. The results showed that the coenzyme depend-

ence of K21A was completely reversed to NADH+.

2.2.2. Recombinant S. cerevisiae Expressing

Xylose Isomerase

Xylose isomerase (XI), encoded by the xylA gene, cata-

lyzes the isomerization of xylose to xylulose in bacteria

and some fungi [31]. xylA has been cloned into S. cere-

visiae from several bacteria. However the XI produced

by the recombinant S. cerevisiae strains was inactive.

Improper protein folding, postranslational modifications,

inter- and intramolecular disulfide bridge formation, and

the internal pH of yeast have been suggested as possible

reasons [32].

In 1996, Walfridsson et al. [33] cloned the Thermus

thermophilus xylA gene encoding xylose (glucose)

isomerase and successfully expressed in S. cerevisiae

under the control of the yeast PGK1 promoter. The re-

combinant xylose isomerase showed the highest activity

at 85 with a specific activity of 1.0 U/mg. It was the ℃

first successful attempt to express the procaryotic gene

xylA for the enzyme XI in the eucaryote S. cerevisiae,

J. Y. Yang et al. / Natural Science 1 (2009) 210-215

213

which could be due to the relatedness between the two

organisms. The recombinant strains could not covert

xylose to ethanol efficiently because the temperature and

pH optimum for the recombinant enzyme are high.

The XylA gene from the anaerobic fungus Piromyces

sp. E2 (ATCC 76762) was functionally expressed in S.

cerevisiae by Marko Kuyper et al. [34]. After prolonged

cultivation on xylose, a mutant strain was obtained that

grew aerobically and anaerobically on xylose. The an-

aerobic ethanol yield was 0.42 g ethanol /g. xylose and

also by-product formation was comparable to that of

glucose-grown anaerobic cultures.

In 2009, Brat et al. [35] cloned and successfully ex-

pressed a highly active new kind of xylose isomerase

from the anaerobic bacterium Clostridium phytofermen-

tans in S. cerevisiae. The recombinant yeast cells with

heterologous expression got the ability to metabolize

D-xylose and to use it as the sole carbon and energy

source. The new enzyme has low sequence similarities to

the XI Thermus thermophilus and Piromyces sp. E2,

which were the only two xylose isomerases previously

functionally expressed in S. cerevisiae. Importantly, the

new enzyme is far less inhibited by xylitol, which ac-

crues as a side-product during xylose fermentation. The

findings provided an excellent starting point for further

improvement of xylose fermentation in industrial yeast

strains.

3. OPTIMIZATION OF DOWNSTREAM

METABOLIC PATHWAYS

3.1. Xylulokinase

Although recombinant strains containing genes cod-

ing for XR and XDH from the xylose-utilizing yeast

P. stipitis have been reported, such strains ferment

xylose to ethanol poorly. One reason for this may be

the low capacity of xylulokinase, the third enzyme in

the xylose pathway [36,37].

Xylulokinase is an enzyme that catalyzes the

chemical reaction: ATP + D-xylulose ADP +

D-xylulose 5-phosphate. In 1989, Ho et al. [38]

cloned the xylulokinase (xks1) gene from S. cere-

visiae and firstly overexpressed in S. cerevisiae.

Toivari et al. [39] also overexpressed the endogenous

gene for xylulokinase (xks1) in S. cerevisiae along

with the P. stipitis genes for XR and XDH. The me-

tabolism of this recombinant yeast was further inves-

tigated in pure xylose bioreactor cultivation at various

oxygen levels. The results clearly indicated that

overexpression of xks1 significantly enhanced the

specific rate of xylose utilization. In addition, the

XK-overexpressing strain can more efficiently con-

vert xylose to ethanol under all aeration conditions

studied. These two studies represented an important

step in efforts to improve xylose metabolism in S.

cerevisiae, as their results strongly indicated that na-

tive XK activity was insufficient for xylose or xylu-

lose fermentation, and overexpression was required to

obtain high ethanol yields.

3.2. Transketolase and Ransaldolase

Transketolase and transaldolase catalyze transfer of

2-C and 3-C molecular fragments respectively, in

each case from a ketose donor to an aldose acceptor.

The two enzymes have been implicated as being

rate-limiting for xylose and xylulose fermentation.

The TKL1 and TAL1 genes encoding transketolase

and transaldolase were overexpressed individually and

together in the S. cerevisiae strain containing XYL and

XYL2. The strain overexpressing TAL1 showed consid-

erably enhanced growth on xylose compared with a

strain containing only XYL1 and XYL2. Overexpression

of only TKL1 did not inﬂuence growth. The results in-

dicated that the transaldolase level in S. cerevisiae is

insufﬁcient for the efﬁcient utilization of pentose phos-

phate pathway metabolites [40]. Bao et al. [41] also

found that the S. cerevisiae strain overexpressing the

TAL1 and TKL1 showed considerably good growth on

the xylose plate.

4. IMPROVEMENT OF TOLERANCE TO

INHIBITORS

The growth of S. cerevisiae and ethanol production were

limited by multiple inhibitors, including furan deriva-

tives, 5-hydroxymethylfurfural (HMF), weak acids, and

phenolic compounds produced during biomass-to-ethanol

processing. The PPP is an important pathway incarbo-

hydrate metabolism, and a lot of previous studies have

shown a correlation between several PPP genes and spe-

cific stresses such as oxidative [42], sorbic acid [43], and

osmotic [44].

To improve production of fuel ethanol from renew-

able raw materials, laccase from the white rot fungus

Trametes versicolor was expressed under control of the

PGK1 promoter in S. cerevisiae to increase its resistance

to phenolic inhibitors in lignocellulose hydrolysates [45].

To identify target genes involved in furfural tolerance,

Gorsich [46] screened a S. cerevisiae gene disruption

library for mutants with growth deficiencies in the pres-

ence of furfural. As a result, more than 62 genes were

found to be associated with sensitivity to furfural. They

also further showed that overexpression of ZWF1 in S.

cerevisiae allowed growth at furfural concentrations that

are normally toxic, which demonstrated a strong rela-

tionship between PPP genes and furfural tolerance.

Adapting strains is also an alternative to improve the

performance of microorganisms. Liu [47] improved bio-

J. Y. Yang et al. / Natural Science 1 (2009) 210-215

214

transformation by newly developed strains adapted to

tolerate the challenges of furfural and HMF in batch

cultures compared with the parental strains. The results

suggest a possible in situ detoxification of the inhibitors

for bioethanol fermentation using improved yeast strains.

Although they have not been tested against inhibitor

complexes such as those in a biomass hydrolysate, the

development and study of such strains provided neces-

sary materials for further studies of the mechanisms of

the stress tolerance at molecular and genomic levels.

5. CONCLUSIONS

The bioconversion of cellulose and hemicellulose to

biofuels and chemicals is being actively researched with

the aim of developing technically and economically vi-

able processes. D-Xylose is the major product of the

hydrolysis of hemicellulose and considerable research

efforts has been focused on the development of xylose-

fermenting recombinant S. cerevisiae. Significant im-

provements in ethanol productivity from xylose have

been achieved through metabolic engineering. However,

there are still unidentified limiting steps in the xylose

metabolism of metabolically engineered S. cerevisiae,

such as lower ethanol yield, more byproducts, the suit-

ability of these recombinant strains and so on. There are

still many tasks that left in the xylose-metabolic engi-

neering. So far the recombinant S. cerevisiae were con-

structed base on the laboratory strains, which are less

complex in genetic background, growth characters, and

physiological characters comparing with the industrial

yeast strains. To get strains easy to be industrialized,

more emphasis should be focused on the reconstruction

of the wild type yeasts. Further improve the expression

and stability of the heterogenous genes in yeasts can be

expected for higher ethanol yield.

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