Enzymatic modification of dough during bread making may

improve bread quality attributes such as texture and taste. To

develop amylolytic Saccharomyces cerevisiae baker’s yeasts

producing and secreting xylanase and phytase, two integrative

cassettes were constructed to allow co-expression of the Aspergillus

awamori glucoamylase gene (GA1) and Debaryomyces castellii

phytase gene (phytDc), and the Debaryomyces occidentalis α-

amylase gene (AMY) and Bacillus subtilis xylanase gene (xynA),

respectively. The recombinant baker’s yeast strain expressing

GA1 and phytDc and the corresponding strain expressing AMY

and xynA were co-cultured to secrete active glucoamylase and

α-amylase, displaying both xylanase and phytase activities that

degraded starch and consequently grew using it as the sole carbon

source. The glucoamylase and α-amylase activities produced

by the co-culture of these two yeast strains increased by 1.5-fold

and 11-fold, respectively, relative to those in their monoculture

with culture medium containing 2% (w/v) raw wheat starch

within 5 days of growth. These new yeasts hydrolyzed 60% of

the raw starch content during the same period.

Keywords: Saccharomyces cerevisiae, amylases, baker’s yeast,

phytase, wheat starch, xylanase

Wheat flour used in bread making contains non-starch poly-

saccharides, such as hemicelluloses, in relatively small amounts

along with starch as the main polysaccharide; it also contains

phytate as the major storage form of phosphate (Monfort et al.,

1997; Lim et al., 2008). Unfortunately, Saccharomyces cerevisiae,

including baker’s yeast and brewer’s yeast, is unable to meta-

bolize starch, non-starch polysaccharides, and phytate. Addition

of exogenous α-amylase and xylanase that modify starch and

non-starch polysaccharides in the dough may delay the rate of

staling and increase the volume and shelf-life of bread (Monfort et

al., 1996). Further, on consumption, bread made with the

addition of glucoamylase generates a melting feeling in the

mouth (Saito et al., 1996). Intake of large amounts of foods

that are rich in phytate may induce mineral deficiencies and

phosphate pollution problems as phytate is an anti-nutritional

factor that forms complexes with proteins and nutritionally

important minerals. Phytase catalyzes the dephosphorylation

of phytate to inositol and inorganic phosphate, thus alleviating

phytate-related symptoms (Han et al., 1999). However, exogenous

addition of these enzymes during bread making may result in

the incidence of allergenic symptoms in bakers (Nieto et al.,

Korean Journal of Microbiology (2020) Vol. 56, No. 2, pp. 170-176 pISSN 0440-2413DOI https://doi.org/10.7845/kjm.2020.0007 eISSN 2383-9902Copyright ⓒ 2020, The Microbiological Society of Korea

Construction of amylolytic yeasts secreting xylanase and phytase to

improve bread quality

Ja-Yeon Lee1, Young-Kum Im2, Jong-Eon Chin3, and Suk Bai2*

1Department of Biological Sciences and Biotechnology Graduate School, Chonnam National University, Gwangju 61186, Republic

of Korea 2Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of

Korea3Department of Radiology, Donggang University, Gwangju 61200, Republic of Korea

빵의 품질향상을 위한 xylanase와 phytase 분비 전분 분해능 효모의 개발

이자연1 ･ 임영금2 ･ 진종언3 ･ 배 석2*1전남대학교 대학원 생물과학･생명기술학과, 2전남대학교 자연과학대학 생물학과, 3동강대학교 방사선과

(Received January 31, 2020; Revised March 7, 2020; Accepted March 9, 2020)

*For correspondence. E-mail: sukbai@chonnam.ac.kr;

Tel.: +82-62-530-3412; Fax: +82-62-530-3409

Amylolytic baker’s yeasts secreting xylanase and phytase ∙ 171

Korean Journal of Microbiology, Vol. 56, No. 2

1999). To resolve this problem, several studies have been

conducted to develop baker’s yeasts that express Aspergillus α-

amylase, endoxylanase, and phytase to reduce or eliminate the

requirement for adding exogenous enzymes (Saito et al., 1996;

Monfort et al., 1997; Nieto et al., 1999; Li et al., 2009). In S.

cerevisiae, the usual way to maintain integrated foreign enzyme

genes is to use reiterated DNA sequences including δ-sequences

and ribosomal DNA as target sites for homologous recombination,

which results in multiple copies of the integrated genes, resulting

in higher expression levels and mitotic stability. Furthermore,

the unnecessary bacterial plasmid DNA sequences containing

the antibiotic resistance marker and the replication origin for

integrating foreign enzyme genes into yeast chromosomes should

be excised before transformation because recombinant yeasts

with bread enter our bodies orally (Lee and Da Silva, 1997;

Park et al., 2014). In this study, we constructed two linearized

δ-integrative vectors harboring the Aspergillus awamori raw

starch-degrading glucoamylase gene (GA1) (Lin et al., 1998)

and Debaryomyces castellii phytase gene (phytDc) (Lim et al.,

2008) or the Debaryomyces occidentalis α-amylase gene (AMY)

(Kang et al., 2003) and xylanase gene (xynA) from the GRAS

organism, Bacillus subtilis. This facilitated the development of

a baker’s yeast with glucoamylase and phytase activities, and a

yeast with α-amylase and xylanase activities. The growth, starch

utilization and enzyme activities of these new yeast strains

were analyzed and the efficacy of raw starch hydrolysis by their

co-culture was compared with that by the monoculture.

A baker’s yeast strain, Saccharomyces cerevisiae ATCC 6037

was used in the transformation experiments. Bacillus subtilis

ATCC 6633 was the source of the xynA gene. Escherichia coli

DH5α was used as the host for plasmid constructions. YIpδ

AURSAδ, YIpδAURDpδ, and YIpδAGSAδ (Kang et al., 2003;

Lim et al., 2008; Kim et al., 2010) served as the backbones

for the double δ-integrative system. All DNA manipulations

and the E. coli transformation were conducted using standard

protocols. Integrative transformation of yeast was conducted

using the method described by Gietz et al. (1992). The xylanase

gene was amplified by PCR using the oligonucleotides 5'-TCA

GGTACCATGTTTAAGTTTAAAAAGAAT-3' and 5'-CGAT

CTAGATTACCACACTGTTACGTTAGAACT-3'. These primers

were designed using the published nucleotide sequences of the

B. subtilis xylanase gene (xynA) (GenBank accession number

M36648). PCR resulted in 0.64 kb amplified DNA fragments

of the whole open reading frame from the genomic DNA of B.

subtilis ATCC 6633, which underwent digestion with KpnI and

XbaI, and was then inserted into the same sites downstream of

the ADC1 promoter (ADC1p) in YIpδAURSAδ lacking the AMY

gene, thereby generating YIpδAURXδ (7.2 kb). Furthermore,

to obtain expression of both the xynA and AMY genes, a 2.7 kb

DNA fragment containing the ADC1p-xynA cassette of YIpδ

AURXδ, which underwent digestion with ApaI and SacI, was

inserted using the same sites in YIpδAGSAδ lacking the GA1

gene to generate YIpδXSAδ (9.6 kb). To construct the δ-

integrative system containing the GA1 and phytDc genes, a 2.1

kb DNA fragment harboring the ADC1p-phytDc cassette was

excised from YIpδAURDpδ and inserted into the XbaI sites in

YIpδAGSAδ lacking the ADC1p-AMY cassette, thus generating

YIpδAGDpδ (10.9 kb) (Fig. 1).

To develop baker’s yeasts producing and secreting xylanase,

YIpδAURXδ was linearized by digesting the δ sequences with

XhoI, after which the fragments (4.4 kb) harboring the ADC1p-

xynA gene cassettes flanked by δ sequences were integrated

into the reiterated δ sequences dispersed throughout the baker’s

yeast’s genome (Lee and Da Silva, 1997; Kang et al., 2003).

The integrative cassette-harboring transformed yeasts were

designated ATCC 6037/YIpδAURXδ (ATCC 6037X). The

yeast transformants formed halos around their colonies on

YPD plates containing xylan whereas the untransformed baker’s

yeast strain displayed no halo (Fig. 2), indicating that the B.

subtilis xylanase gene is expressed under the control of yeast

ADC1 promoter and that xylanase is secreted under the control

of its own lead sequence in baker’s yeast (Ahn et al., 1992).

Then, linearized YIpδXSAδ (6.8 kb) (Fig. 1) was integrated

into the baker’s yeast genome, generating ATCC 6037/YIpδ

XSAδ that expresses both xynA and AMY genes (ATCC 6037XSA)

to develop amylolytic baker’s yeasts producing xylanase (Monfort

et al., 1997). This double δ integration system generates a

smaller unnecessary fragment (2.8 kb) containing an ampicillin

resistance marker, which was eliminated prior to transformation

(Kang et al., 2003). Large halos developed around ATCC

6037XSA secreting xylanase and α-amylase on YPD plates

containing xylan and YPDS3 plate, respectively (Fig. 2). It

is known that these two enzymes act synergistically on the

polysaccharides in wheat flour, resulting in improved bread

172 ∙ Lee et al.

미생물학회지 제56권 제2호

dough by increasing the bread volume and shelf life through a

retarded rate of staling (Monfort et al., 1996; Nieto et al.,

1999). On the other hand, linearized YIpδAGDpδ containing

both ADC1p-GA1 and ADC1p-phytDc gene cassettes (8.1 kb)

was integrated into the baker’s yeast genome, generating ATCC

6037/YIpδAGDpδ (ATCC 6037AGDp) to develop amylolytic

baker’s yeasts that produce phytase. A smaller halo formed

around ATCC 6037AGDp secreting glucoamylase on the YPDS3

plate unlike ATCC 6037XSA secreting α-amylase (Fig. 2). It is

known that baker’s yeasts with high maltose fermentation

Fig. 1. Recombinant plasmids, YIpδXSAδ and YIpδAGDpδ for the expression of xynA and AMY genes, and GA1 and phytDc genes respectively in S.

cerevisiae ATCC 6037. Schematic plasmid maps showing the relative size, restriction sites and locations of the insert DNA. The bacterial plasmid DNA

sequences containing the ampicillin resistance gene were excised by digestion with XhoI (2.8 kb) before transformation.

(A) (B)

Fig. 2. Xylanase and amylolytic activities of S. cerevisiae transformants demonstrated on YPD plate containing 0.15% (w/v) Remazol brilliant blue-xylan (A)

and on YPDS3 plates (B) at 30°C for 2 days. A clear zone around the colony indicates the secretion of xylanase, glucoamylase and α-amylase. A, S. cerevisiae

ATCC 6037; B, ATCC 6037Dp; C, D, ATCC 6037AGDp; 1, 2, ATCC 6037X; 3, 4, ATCC 6037XSA.

Amylolytic baker’s yeasts secreting xylanase and phytase ∙ 173

Korean Journal of Microbiology, Vol. 56, No. 2

ability are preferred in bread making because they utilize the

maltose in wheat flour. Since α-amylase cannot hydrolyze

maltose, the addition of glucoamylase hydrolyzing maltose is

necessary when using inferior maltose-fermenting yeasts with

a better flavor and taste (Lee et al., 2016). Moreover, Saito et al.

(1996) have reported that bread made with the addition of

glucoamylase was superior in terms of a melting feeling in the

mouth.

Yeast cells were cultured in YPD medium [1% (w/v) yeast

extract, 2% (w/v) Bacto-peptone, and 2% (w/v) glucose]. Yeast

transformants were grown on YPD plates containing 1 μg/ml

aureobasidin A (TaKaRa) and were then transferred onto YPDS3

plates [YPD containing 3% (w/v) soluble starch] and incubated

for 4 days at 30°C. Amylolytic clones were detected by the

observation of halos around the colonies. YPD plates containing

0.15% (w/v) 4-O-methyl-D-glucurono-D-xylan-Remazol brilliant

blue R dye (Sigma) or 1% (w/v) oat spelts xylan (Sigma) were

used to detect the xylanase activities secreted by yeast trans-

formants incubated for 2 days at 30°C (Biely et al., 1985). Halo

formation around a colony is indicative of xylanase activity.

Yeast cells were grown in YPS medium [YP containing 2%

(w/v) soluble starch or 2% (w/v) wheat starch (Sigma)] or YPP

medium [YP containing 0.05% (w/v) potassium disulfite to

prevent contamination (Shigechi et al., 2004)] containing 2%

(w/v) raw wheat starch at 30°C for 5 days to assay the glu-

coamylase, α-amylase, xylanase and phytase activities of the

culture supernatants. Residual starch and raw starch were assayed

via the starch-iodine reaction and phenol sulfuric acid method,

respectively (Kim et al., 1988; Shigechi et al., 2004). All

experiments were conducted three to five times independently.

The results are presented as the mean ± standard deviation of

the mean (SD) of three different experiments. Xylanase activity

was assayed using 0.5% (w/v) oat spelts xylan as a substrate at

pH 6.0 and 55°C. The amount of reducing sugar produced from

xylan as a result of the enzyme reaction was determined using

the dinitrosalicylic acid method (Ghang et al., 2007). One unit

of xylanase activity was defined as the amount of enzyme that

liberated 1 μmol of xylose from xylan in 1 min. Phytase activity

was quantified at pH 4.5 and 70°C using acid molybdate reagent.

One unit of phytase activity was defined as the amount of

enzyme that liberated 1 μmol of inorganic phosphate from

sodium phytate in 1 min (Lim et al., 2008). Glucoamylase and

α-amylase activities were quantified at pH 5.5 and 40°C using

the PGO/ODAD assay (Sigma) and the dinitrosalicylic acid

method, respectively. All assays were repeated three times, and

the means were calculated.

Enzyme activities were examined in culture supernatants

from yeast transformants grown in YPS media containing 2%

(w/v) soluble starch. The selected clones exhibiting the highest

activity among transformants were used for subsequent analyses.

As shown in Table 1, the activities of glucoamylase, α-amylase,

xylanase, and phytase were present in the culture supernatant

from the co-culture of ATCC 6037AGDp and ATCC 6037XSA.

The glucoamylase activity produced by ATCC 6037AGDp

co-cultivated with ATCC 6037XSA, similar to ATCC 6037AGSA

secreting both glucoamylase and α-amylase (Kim et al., 2010)

was 2.2-fold higher than that by the monoculture of ATCC

6037AGDp, indicating that the cooperative interaction between

glucoamylase and α-amylase resulted in efficient degradation

of starch to glucose (Kim et al., 1988; Eksteen et al., 2003). In

Table 1. Enzyme activities in the cell-free supernatants of S. cerevisiae and its transformants

Yeast strainsGlucoamylase activitya

(U/ml)

α-Amylase activity

(U/ml)

Xylanase activity

(U/ml)

Phytase activity

(mU/ml)

S. cerevisiae ATCC 6037 NDb ND ND ND

ATCC 6037AGDp (GA1, phyt) 0.15 ± 0.03e ND ND 107 ± 5

ATCC 6037XSA (AMY, xynA) ND 4.6 ± 0.25 0.5 ± 0.03 ND

ATCC 6037AGSAc (GA1, AMY) 0.32 ± 0.04 5.7 ± 0.15 ND ND

ATCC 6037AGDpd (GA1, phyt) + ATCC 6037XSA (AMY, xynA) 0.33 ± 0.03 5.6 ± 0.2 0.47 ± 0.05 102 ± 7

a Yeast cells were grown in YPS media at 30°C for 3 days.b Not detectedc YIpδAGSAδ (Kim et al., 2010)d ATCC 6037AGDp and ATCC 6037XSA were co-cultured at a ratio of 1 : 1 (w/w) e Values are means of results from triplicate experiments and are expressed in enzyme activities present in the culture supernatants.

174 ∙ Lee et al.

미생물학회지 제56권 제2호

addition, the phytase activity produced by these strains was

4-fold higher than that by D. castellii which was the source of

the phytDc gene (25 mU/ml) (Lim et al., 2008). However, the

xylanase activity produced by ATCC 6037XSA was reduced to

one-third of that by B. subtilis (1.6 U/ml). This problem would

be solved if the xynA signal sequence were replaced with the

signal sequence of a yeast gene (Ahn et al., 1992). Time course

analyses of enzyme activities, cell growth, and starch hydrolysis

produced by the co-culture of ATCC 6037AGDp and ATCC

6037XSA using 2% (w/v) wheat starch as the sole carbon

source over 5 days are shown in Fig. 3. Enzymes were growth-

associated and reached the maximal activities after 3 days of

growth. These strains utilized 100% of the starch in the culture

medium containing 2% (w/v) wheat starch during 3 days of

growth. Xylanase and phytase activities were positively correlated

with high starch assimilation and growth rate due to efficient

degradation of starch to glucose by the cooperative interaction

between glucoamylase and α-amylase (Monfort et al., 1996;

Lim et al., 2008; Kim et al., 2010).

Since raw wheat starch below the gelatinization temperature

of starch is used in the fermentation of bread dough, ATCC

6037AGDp, ATCC 6037XSA, and ATCC 6037AGDp + ATCC

6037XSA (co-culture) glucoamylase and α-amylase activities

as well as starch hydrolysis were compared when 2% (w/v) raw

wheat starch was used as the sole carbon source over 5 days. As

shown in Fig. 4, ATCC 6037AGDp secreting GA1-encoded

glucoamylase exhibited low enzyme activity and consumed the

raw starch content to merely 10% of the initial concentration

after 5 days of growth because GA1 hydrolyzes raw starch

slowly (Kim et al., 2011); ATCC 6037XSA secreting AMY-

encoded α-amylase did not hydrolyze raw starch, whereas the

co-culture of ATCC 6037AGDp and ATCC 6037XSA like

ATCC 6037AGSA (data not shown) displayed 1.5-fold higher

glucoamylase activity and 6-fold more hydrolysis of raw starch

compared to ATCC 6037AGDp. Moreover, they showed 11-

fold higher α-amylase activity than that of ATCC 6037XSA

during the same period. These findings indicate that glucoamylase

with a raw starch-binding domain cleaves the non-reducing

ends of raw starch chains and exclusively forms glucose. This

in turn can serve as a substrate for the growth of ATCC

6037XSA as well as ATCC 6037AGDp, thereby increasing the

activity of α-amylase to release new non-reducing ends for

glucoamylase and the synergistic interaction of glucoamylase

and α-amylase resulting in rapid hydrolysis of raw starch that is

critical to increase the rate of fermentation (Sun et al., 2010; Im

et al., 2013). Thus, these new baker’s yeast strains secreting

glucoamylase, α-amylase, xylanase and phytase may be useful

for improving bread quality and taste, increasing bread shelf

life by retarding the process of staling and reducing phytate

content (Monfort et al., 1996; Saito et al., 1996; Lim et al.,

Fig. 3. Growth curve and time courses of wheat starch hydrolysis and extracellular glucoamylase, α-amylase, xylanase, and phytase activities produced by the

co-culture of ATCC 6037AGDp and ATCC 6037XSA. Yeast cells were grown in YP media containing 2% (w/v) wheat starch at 30°C for 5 days. Growth

was measured on different days based on the cell number. The remaining starch values were presented as percentages considering the starch in the

uninoculated medium as 100%. Enzyme activities were measured in culture supernatants. The data (mean ± SD) are from three independent experiments

performed in triplicate.

Amylolytic baker’s yeasts secreting xylanase and phytase ∙ 175

Korean Journal of Microbiology, Vol. 56, No. 2

2008). These yeast strains can also be useful to avoid the

occurrence of allergenic work-related symptoms due to the

exogenous addition of commercial enzymes in bread making

(Nieto et al., 1999). Further studies are in progress to develop

commercial baker’s yeast such as Saf-instant yeast (S. I. Lesaffre)

with improved glucoamylase and α-amylase activities, thereby

improving the xylanase and phytase activities using these

constructed δ-integrative systems.

적 요

밀가루 반죽에 효소를 처리하면 질감과 식감과 같은 빵의

품질을 향상시킬 수 있다. Xylanase와 phytase를 생산 분비하

는 전분 분해능 제빵효모 Saccharomyces cerevisiae 균주들의

개발을 위해 각각 Aspergillus awamori glucoamylase 유전자

(GA1)와 Debaryomyces castellii phytase 유전자(phytDc), 그

리고 Bacillus subtilis xylanase 유전자(xynA)와 Debaryomyces

occidentalis α-amylase 유전자(AMY)를 공동 발현할 수 있는

두 개의 integrative cassettes를 제조하였다. GA1및 phytDc유

전자를 발현하는 재조합 제빵효모 균주와 xynA 및 AMY 유전

자를 발현하는 제빵효모 균주를 동시 배양하면 전분을 가수분

해하고 이를 유일한 탄소원으로 생장할 수 있게 glucoamylase

와 α-amylase를 분비하였고, 더불어 xylanase와 phytase도 분

비하였다. 2% (w/v) 생밀전분이 함유된 배지에서 배양하였을

때 두 균주의 혼합배양에 의한 glucoamylase와 α-amylase 활

성은 단독배양과 비교하여 5일차에 각각 1.5배와 11배 증가하

였다. 이 새로운 균주들은 같은 기간에 생전분 함유량의 60%

를 소비하였다.

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