4 482 LITA TRITARNA OK.CDR


Genome-Shuffling-Improved Acid Tolerance and Lactic Acid
Production in for CommercializationLactobacillus plantarum

LITA TRIRATNA , BUDI SAKSONO , LINDA SUKMARINI ,
ASEP SUPARMAN

1 1 1

2

*

AND

1

2

Research Center for Biotechnology, Lembaga Ilmu Pengetahuan Indonesia, Jalan Raya Bogor Km 46,

Cibinong 16911, Indonesia Department of Food Science and Technology, Institut Pertanian Bogor,
Darmaga Campus, Bogor 16680, Indonesia

Lactobacillus plantarum

L. plantarum

Lactobacillus plantarum

genome shuffling Lactobacillus plantarum

shuffling

genome shuffling

genome shuffling L. plantarum

genome shuffling, Lactobacillus plantarum

;

We applied genome shuffling to improve the acid tolerance of , while simultaneously enhancing

lactic acid production. The starting populations were mutant libraries generated by gradual low pH adaptation and ultraviolet

irradiation and then which were subjected to recursive protoplast fusion. A library of shuffled mutants (fusants) with genetic

exchange is achieved by repetition of this process. After three rounds of genome shuffling, we obtained the best performing

fusant that grow better at low pH 4.0 and consume glucose faster than does the wild type. In addition, lactic acid production of

this fusant was 64% higher than that of the wild type. These results demonstrated that the genome shuffling has been

successful in engineering with multiple beneficial improved phenotypes. In the future, this technology is a

promising candidate to accelerate poorly characterized strains for commercialization.

Key words: genome shuffling, , lactic acid, acid tolerance

Teknik telah diaplikasikan untuk memperbaiki toleransi asam dan secara

simultan meningkatkan produksi asam laktatnya. Populasi awal yang digunakan ialah pustaka mutan yang didapat dengan

menurunkan pH medium pertumbuhan secara bertahap dan iradiasi sinar ultra violet. Pustaka mutan kemudian digunakan

dalam proses fusi protoplas secara berulang. Hasil dari pustaka mutan disebut fusan. Paska pengulangan yang ke-3

dari didapatkan fusan unggul yang mampu tumbuh dengan baik pada pH 4.0 dan mengonsumsi gula lebih

cepat dibandingkan dengan galur liar. Produksi asam laktat oleh fusan ini meningkat 64% dibandingkan dengan galur liar.

Hasil ini menunjukkan bahwa teknik telah berhasil merekayasa yang memiliki keunggulan

fenotipe. Di masa mendatang teknik ini mampu menjadi kandidat yang menjanjikan dalam komersialisasi galur mikrob yang

belum dikarakaterisasi dengan baik.

Kata kunci: , asam laktat, toleransi asam

Lactic acid has been widely used in the food,

pharmaceutical and cosmetics industries. It has

recently emerged as an important source for the

production of other chemicals, particularly polylactic

acid for biodegradable plastic (Singh 2006). Most

lactic acid is manufactured by fermentation process as

opposed to chemical synthesis since it is

environmentally friendly (John . 2009).

Fermentation at low pH contributes to the economics of

the lactic acid purification process. Thus, the

commercial goal of improving the growth and lactic

acid production of microorganisms at acid condition

could decrease waste and reduce the cost of production.

Moreover, it also reduces potential contamination

(Porro . 1999; Carlson and Peters 2002; Singh .

2006).
is of commercial interest for lactic

acid production, but the effect of acid stress on the

bacterium is a complex and poorly understood process.

As a result, it is difficult to improve lactic acid

production with rational engineering or direct genetic

manipulation. On the other hand, classical strain-

improvement-methods have succeeded in obtaining

et al.

et al

et al et al

L. plantarum

many industrial strains, but this is a time-consuming

and laborious process because of many repeated rounds

of random mutation and selection methods, especially

for engineering complex phenotypes (Zhang

2002). Thus, an efficient technology named genome

shuffling has been presented as a novel whole-genome

engineering approach for the rapid improvement of

cellular phenotypes. This approach uses recursive

protoplast recombination throughout the entire

genome without the necessity for genome sequence

data or network information (Patnaik 2002;

Stephanopoulos 2002). At present, genome shuffling

has been successfully applied for the improvement acid

tolerance in (Patnaik 2002), acid

and glucose tolerance in (Wang

2 0 0 7 ; Yu 2 0 0 8 ) , d e g r a d a t i o n o f

pentachlorophenol in

(Dai and Copley 2004) and the production of

hydroxycitric acid in (Hida 2007).

However, shuffling of an industrial strain

has not been reported.
Thus, in the present study, we applied genome

shuffling to improve the acid tolerance of

while simultaneously enhancing lactic acid

production.

et al.

et al.

Lactobacillus et al.

L. rhamnosus et al.

e t a l .

Sphingobium chlorophenolicum

Streptomyces et al.

L. plantarum

L. plantarum

*Corresponding author: Phone: +62-21- 8754587,
Fax: +62-21-8754588,  E-mail: saksonobudi@yahoo.com

ISSN 1978-3477, eISSN 2087-8575
Vol 5, No 1, March 2011, p 21-26

I N D O N E S I A

Available online at:
http://www.permi.or.id/journal/index.php/mionline

DOI: 10.5454/mi.5.1.4



MATERIALS AND METHODS

Bacterial Strains and Media.

Adaptation of to Low pH.

Strain Mutagenesis and Mutant Screening.

Protoplast Preparation and Genome Shuffling.

was obtained from Biotechnology Type

Culture Collection (BTCC) Research Center for

Biotechnology-LIPI, Indonesia. The strain was stored

in a deMan Rogosa and Sharpe (MRS) broth with 10%

(w v ) glycerol at -70 °C. The strain was grown in MRS

broth from a 1% inoculum with 18 h incubation at

37 °C. At least two generations of precultures were

required just before the experiments.

Yeast extract fermentation medium (YE) contained

100 g L glucose and 15 g L yeast extract. Regeneration

medium (RM) was MRS agar without Tween 80 and

supplemented with 0.5% BSA, 25 mM MgCl , 25 mM

NaCl , and 0.5 M sucrose (Patnaik 2002).

Culture

adaptation was achieved by modification of the

procedure from Patnaik (2002), through a

continuous and controlled decrease in broth medium

pH 6.2 to pH 5.0 and pH 4.0. For scenarios in which

the imposed pH was not tolerated, and the culture

steady-state could not be maintained, the culture was

replaced with fresh MRS broth of pH lower than

4.0. Adapted wild-type strain in pH 4.0 medium was

then used as the parental, or starter, for genome

shuffling.

The

cells of wild-type strain were grown in

a 10 ml MRS broth at 37 °C for 18 h. Cultures were

centrifuged at 4500 x for 10 min. UV irradiation

was performed by exposing samples of the wild-type

strain in uncovered petri dish directly to UV light at a

distance 20 cm with 120 mJ cm of intensity for 30 s

using UV Crosslinker (Hoefer UVC500, USA). The

mutants were then spread on MRS agar plates

containing 2% (w v ) CaCO and incubated at 37 °C for

48 h. The fast growing colonies, which had bigger

transparent haloes compared to colony diameter,

were picked off and selected as the starter for the

genome shuffling.

Protoplast formation was carried out as described

by Cocconcelli . (1986) with modification.

Strains were cultured at 37 °C for 18 h in MRS broth.

The cells were harvested by centrifugation at 4500

and 4 °C for 10 min, and washed twice with distilled

water. The cells from a 5 mL culture were suspended

in protoplast b L Tris

HCl 1 M pH 6.3, 2.5 mL sucrose 1 M, 2 mL CaCl 50

mM, and 450 L distilled water) containing lysozyme

(2.0 X 10 U mL ) and incubated at 37 °C for 2 h for

protoplast formation.

Lactobacillus

plantarum

et al.

et al.

L. plantarum

g

et al

x g

Lactobacillus

-1

-1 -1

-2

-1

5 -1

2

2

3

2

L. plantarum

uffer (LPB) (50 μ

μ

et al.

et al

The starting populations for genome shuffling were

mutants-library-generated by gradually low pH

adaptation and ultraviolet (UV) irradiation. Fusions

between protoplast preparations were generated by

mixing an approximately equal number of protoplasts

from two populations as described by Patnaik with

slightly modification (Patnaik 2002). The

shuffled mutants, named fusants were then cultivated

in regeneration medium. Each culture was incubated

at 37 C for 6 h and processing continued by spreading

in selection medium of MRS agar plates containing

2% (wv ) CaCO , pH 4.0, and then incubated at 37 C

for 3-4 d. Subsequent colonies were selected randomly

to carry out further selection with small-scale

fermentation analysis in 2 mL YE broth culture at pH

4.0. The cultures were incubated at 37 C for 48 h.

Profiles of both growth and lactic acid production for

each culture were then followed. The value of both

growth and lactic acid production then were analyzed

using SPSS program to identify the fusants that have a

significant change in their growth and lactic acid

production compared to that of control.

The formation of protoplasts, their fusion, and

subsequent regeneration was repeated three times with

the pooled regenerated cells from one fusion being the

inoculum for the subsequent protoplast culture.

Samples from each of the pooled fusion libraries F1-F3

(first/F1, second/F2, third/F3) were saved for later

analysis. Non-shuffled (unshuffled) controls were

prepared by the recursive formation and regeneration

of protoplast without exposure to PEG.

The best performing

selected shuffled strain, F3-33, after three rounds of

genome shuffling (F3) was cultured in 10 mL MRS

broth before being transferred into 100 mL YE medium

at pH 4.0 in shaking flasks (250 mL) at 100 rpm, 37 °C.

The growth, pH, residual glucose, and lactic acid

production were periodically monitored up to 96 h.

Cell growth was monitored

by measuring cell number via the optical density at

600 nm (A ). Supernatants of the samples for assay

were collected by centrifugation. Total lactic acid

for fusant selection was estimated according to the

titration method, and the total sugar concentration

was determined using the phenol-sulphuric acid

method, as described by Dubois . (1956).

Furthermore, total lactic acid of the selected F3 fusant

was then determined by reversed-phase high-

performance liquid chromatography (HPLC)

(Shimadzu LC-6AD, Japan) using a C18 silica column

(HPLC Packed Column UG120 Shiseido, Japan) with

UV detector set at 220 nm. The column was eluted with

20 mM NaH PO (pH 2.55) as a mobile phase at a flow

o

-1 o

o

3

600nm

2 4

Shaking Flask Analysis.

Analytical Methods.

22 TRIRATNA ET AL. Microbiol Indones



rate of 1.0 mL min . All of the measurements were

performed in duplicate from two independent

experiments.

The first population of

the adapted acid-mutant-library was achieved by

slowly decreasing the pH from 6.2 to 4.0 over a period

of 840 h. A stable population of was able

to grow at pH 4.0, a pH that severely inhibits growth of

the wild-type strain. The second population was

enriched for acid tolerance of the wild-type strain using

UV mutant library selected on MRS-agar-plates at

pH 4.0. Ten colonies which had bigger transparent

haloes compared to colony diameter were obtained

(unpublished data).

The resulting new population

(shuffled strains/fusants) represented a larger

combinatorial library of the original genetic diversity.

After the first fusion, 56 colonies were picked off

randomly for further selection in small-scale

fermentation. Selection was done by SPSS program

based on their growth and lactic acid production

values. Fusants with higher and lower lactic acid

production respectively were shown (Fig 1, 2, 3).

First-round shuffling also resulted 29 fusants with

growth-faster compared to that of wild-type

(unpublished data). Among those fusants, 4 fusants, i.e.

F1-2, F1-16, F1-48, and F1-50, showed the value of

relative lactic acid production per A higher than

1.0 (control) and 2 fusants, i.e., F1-6 and F1-30 showed

relative growth-fastest (120%). Relative lactic acid

production is the value of total lactic acid produced

by sample divided by total lactic acid produced by

control, whereas relative growth is the value of cell

density of sample divided by that of control at A .

Two fusants from the selected F1 fusants library, with

higher relative lactic acid production/ A (p < 0.05)

to that of wild type, F1-30 and F1-16 (shown as red bar)

were used as resources for the next fusion (Fig 1a).

Figure 1b showed 4 fusants (F2-3, F2-22, F2-26,

and F2-65) and 10 fusants (F2-64, F2-77, F2-78, F2-79,

F2-88, F2-89, F2-90, F2-91, F2-109 and F2-116) with

lower and higher lactic acid production, respectively.

Second round of shuffling also resulted 87 of fusants

with growth faster compared to that of F1-30 as control

(unpublished data). Among second shuffled library F2,

4 fusants, namely F2-78, F2-88, F2-89, and F2-91 were

selected and used for the next fusion (Fig 1c). These

fusants were selected due to their higher relative lactic

acid production to that of F1-30 as control (p < 0.05).

The result showed 4 fusants (F3-33, F3-118, F3-119,

and F3-176) and 34 fusants with higher and lower

-1

RESULTS

Isolation of Initial Strains.

Genome Shuffling.

L. plantarum

600nm

600nm

600nm

Volume 5, 2011 Microbiol Indones     23

a

b

c

Fig 1 Relative lactic acid production to the control and relative lactic acid
production per A . a, F1 fusants library; b, F2 fusants library; c, F3

fusants library . Wild-type strain (WT), F1-
30 fusant, and F2-89 fusant is used as the control for F1, F2, and F3-
fusants library respectively (For more detail information see result
session of genome shuffling). X-ordinate represents colonies
numbers from each fusant library. Bar represents relative lactic acid
production of fusants, line represents relative lactic acid production
per A . Red bar indicated the fusants were choose for next rounds.

600nm

600nm

Lactobacillus plantarum

Fig 2 Relative growth of F3 fusants library
to F2-89 fusant as a control. X-ordinate represents colonies numbers
from F3 fusants library.

Lactobacillus plantarum

140

120

100

80

60

40

20

0

L
a
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1
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2
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1
3
3

1
3
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1
4
1

1
4
5

1
4
9

1
5
3

F2 fusants

140

120

100

80

60

40

20

0

R
e
la

ti
v
e
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a
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c
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 p
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d
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 (

%
)

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6
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m1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

140

120

100

80

60

40

20

0

R
e
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ti
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a
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7

2
1

2
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2
9

3
3

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4
1

4
5

4
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6
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1
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1
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F
2
-8

9

F3 fusants

1 5 9

1
3

1
7

2
1

2
5

2
9

3
3

3
7

4
1

4
5

4
9

5
3

5
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5

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3

7
7

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1

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1
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1
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3

1
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1
6
1

1
6
5

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6
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1
7
3

1
7
7

F
2
-8

9

F3 fusants

R
e
la

ti
v
e
 g

ro
w

th
 (

%
)

120

100

80

60

40

20

0

, yield., laktat;



relative lactic acid production, respectively (Fig 1c).

Among third shuffled library, F3-33 showed highest

relative lactic acid production (136%) compared to that

of F2-89 as control (p < 0.05).

The relative growth performance to the control of

180 colonies from third shuffled library (F3) which

were picked off randomly. Figure 2 showed 109 and

6 of fusants with growth faster and slower,

respectively. The relative growth of F3 fusants library

was mostly higher compared with that of the F2-89

fusant as control (Fig 2), while relative lactic acid

production of F3 fusants library was mostly lower than

that of control (Fig 1c). The best performing lactic acid

production of fusants in each round (F1-30, F2-89, F3-

33) were compared for their relative lactic acid

production to that of the wild-type (Fig 3). It is shown

that F3-33 fusant in the third round genome shuffling

performed the highest in lactic acid production.

The performance of

F3-33 was further compared with the wild-type strain

in shaking flasks (Fig 4a-c). Consistent with small-

scale fermentation results, the genome shuffled

strain F3-33 produced about a 64.5 % improvement of

lactic acid production (12.0 g L ) over the wild-type

for 48 h (Fig 4a). This lactic acid production increased

up to 48 h, after which no significant improvement was

found. Moreover, the growth characterization and

glucose consumption were consistent with the lactic

acid production. The fusant F-33 grew 53% faster

compared with the wild-type (Fig 4b), and also

consumed glucose faster (Fig 4c).

As lactic acid fermentation at low pH (at or below

the of lactic acid, pH 3.8) can reduce the cost

Shaking Flask Analysis.

DISCUSSION

-1

pKa

24 TRIRATNA ET AL. Microbiol Indones

180

160

140

120

100

80

60

40

20

0

R
e
la

ti
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a
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c
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ti

o
n
 (

%
)

Wt F1-30 F2-89 F3-33

Fusant

a

ab

bc

cd

Fig 3 Relative lactic acid production of the best performing fusants from
each of three rounds of genome shuffling (F1-F3) to the wild type; wt
is wild type strain of . F1-30 is fusant from
colony number 30 of first fusion round, F2-89 is fusant from colony
number 89 of second fusion round, while F3-33 is fusant from colony
number 33 of third fusion round. Bar with different letters are
significantly different by Dunnet test at p<0.05.

Lactobacillus plantarum

a

b

c

Fig 4 Characterization of lactic acid production: a, cell growth; b, and
residual sugar (glucose) of fusant F3-33 compared to that of wild
type (WT) . ,Lactobacillus plantarum

production and decrease waste, acid tolerance

phenotypes meet the performance criteria for industrial

strain commercialization. Thus, the strains tolerant to

acid conditions are desirable industrial phenotypes

(Patnaik 2002). Environmental tolerance of pH is

a complex and yet poorly understood phenomenon,

therefore, therefore we applied genome shuffling

approach to improve lactic acid production of local

. This approach has successfully been

used to improve acid tolerance in

through fusion between a low-pH-adapted mutant

library population and nitrosoguanidine (NTG)

mutant-library-population (Patnaik . 2002), and in

through UV irradiation and NTG

mutagenesis (Wang . 2007). Here, we used an

et al.

L. plantarum

Lactobacillus

et al

L. rhamnosus

et al

Time (h)

Time (h)

A
6
0
0
n
m

Time (h)

14

12

10

8

6

4

2

0

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

F3-33.

2.00

1.80

1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

0.00

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

1.10

1.00

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0-

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

WT;     ,



Volume 5, 2011 Microbiol Indones     25

adapted low-pH-mutant library population and an UV-

irradiation- mutant library population, instead of a

nitrosoguanidine (NTG) mutant library population, as

the starter of genome shuffling. Since genome

shuffling practically mimics the features of natural

evolution through recursive genetic recombination, it

requires a diverse population of mutants with an

improvement of the desired phenotype compared with

the wild-type as a starting point (Yu 2008).

The present study showed more than 50% of fusants

with change in their relative lactic acid production and

growth. The percentage of fusants with different

performing compared to that of their control was

increased to 60% (9% and 51% with their lactic acid

production and growth, respectively), 66% (9% and

57% with their lactic acid production and growth,

respectively) and 85% (21% and 64% with their lactic

acid production and growth, respectively) in first,

second, and third rounds, respectively. The results of

the present study also proved that genome shuffling is

an effective strategy for generating strains which

become more tolerant to the acid and result in the

improvement of lactic acid production. It was shown

that the relative lactic acid production of the best

performing fusants (F1-30, F2-89, and F3-33) from

each of three rounds of genome shuffling were higher

than that of the wild-type (120%, 144%, and 167.8%

respectively) (Fig 3), corresponding with the higher

growth of those fusants compared with the wild-type

(unpublished data). After three rounds of genome

shuffling, we obtained F3-33 fusant which had the

highest lactic acid production and capable of growing

at pH 4.0. In this regard, F3-33 fusant was further

characterized by comparison with the wild-type strain.

In agreement with small-scale fermentation, the

lactic acid production of F3-33 fusant was 64.5%

higher compared with the wild-type strain in shaking

flasks (Fig 4a). After 48 h of growth at pH 4.0, the wild-

type strain had produced 7.74 g L of lactic acid, while

the F3-33 fusant produced 12 g L .

Characterization of F3-33 fusant and the wild-type

in the shaking flask showed the lactic acid production

was closely correlated with the growth and glucose

consumption ( Fig 4b and 4c). The best performing

fusant grew faster, consumed glucose faster, and

produced more lactic acid, and also brought the

medium to a lower pH (unpublished data), as reported

by Patnaik . (2002) and Wang (2007).

However, these previously published results showed

that the fusants produced three-fold more lactic acid

than the wild-type at pH 4.0. The improvement of lactic

acid of our fusant was lower than that of these previous

reports. This result may due to the fact that the starting

et al.

et al et al.

-1

-1

population for each round of fusion had low diversity.

The fusant library F1 and F2 were generated from two

colonies/ fusants of F0 and F1 fusants library,

respectively, while the fusant library F3 was generated

from 4 fusants of F3 fusants library. From F1, F2, and

F3 fusants library, the percentage of fusants which had

changed in their lactic acid production were 9%, 9% and

21%, respectively. These achievements were agreed

with the number of fusants used as resources to generate

fusants library. Thus, we concluded that the low genetic

diversity within a selected population resulted in low

recombination and combinatorial library.

These results, however, demonstrated that we

had been succeeded in applying the genome shuffling

to engineer with multiple beneficial

improved phenotypes including acid tolerance, lactic

acid enhancement, and glucose/substrate consumption

at one time. In the future, it is promising that this

simple technology is a promising candidate to

accelerate poorly characterized strains for commer

cialization.

We would like to thank Yantyati Widyastuti for

providing BTCC strain.

Funding for this project was provided in part by

the Indonesian Ministry of Education and Culture

Program 2009.

L. plantarum

-

Lactobacillus plantarum

ACKNOWLEDGEMENTS

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