Peningkatan Toleransi Etanol Pada Escherichia Coli Rekombinan Melalui Penambahan Glutamat Dalam Kondisi Aerobik

ENHANCING ETHANOL TOLERANCE OF Escherichia coli
RECOMBINANT BY GLUTAMATE ADDITION
UNDER AN AEROBIC CONDITION

INDRA KURNIAWAN SAPUTRA

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

STATEMENT OF THESIS, SOURCE OF INFORMATION AND
COPYRIGHT*
I hereby declare that thesis with titled “Enhancing Ethanol Tolerance of
Escherichia coli Recombinant by Glutamate Addition under an Aerobic
Condition” was my own work under the supervision of an advisory committee and
has not been submitted before to any institution of higher studies. Secondary
source of information published or unpublished have been mentioned clearly in
the text and listed in references at the end of this thesis.
I herewith bestow the copyright of my thesis to Bogor Agricultural
University.

Bogor, May 2016
Indra Kurniawan Saputra
Student ID P051130151

SUMMARY
INDRA KURNIAWAN SAPUTRA. Enhancing Ethanol Tolerance of Escherichia
coli Recombinant by Glutamate Addition under an Aerobic Condition. Supervised
by DJUMALI MANGUNWIDJAJA and PRAYOGA SURYADARMA.
The exploitation of fossil fuel has negative impacts on the availability of
energy and the environment. Therefore, the use of alternative energy, including
ethanol plays an important role in overcoming the problems. Recently, ethanol is
produced through bioconversion of lignocellulose materials, and E. coli is the
potential bacteria for ethanol production by utilizing biomass as substrate. It is
due to its ability to consume pentose and hexose sugars from biomass for ethanol
production. Meanwhile, metabolic engineering of E. coli by insertion of
ethanologenic genes, namely genes encode pyruvate decarboxylase (PDC) and
alcohol dehydrogenaseII (ADH) enhanced ethanol production. In addition,
phosphotransacetylase (PTA) mutation carry on lowering by-product (acetate)
accumulation and introduction of formate dehydrogenase (FDH) to E. coli
improve NADH availability together with piruvate accumulation for ethanol

production under an aerobic condition. This high ethanol accumulation leads the
cell into ethanol stress condition. It can be suppressed by glutamate addition.
Glutamate is supposed to tighten peptidoglycan as crucial component in holding
stress effect. Hence, the objectives of this study are to investigate the effect of
glutamate addition on ethanol tolerance enhancement of ethanologenic E. coli,
and to look into effectiveness of glutamate addition in increasing tolerance of
ethanol under an aerobic cultures condition.
The name of strain E. coli was BW25113∆pta/pHfdh/pTadhB-pdc. First
step in this study was making growth medium, precultivation and cultivation. The
cultivation medium was arranged in glucose-enriched LB medium. Afterwards,
samples were cultivated with agitation 250 rpm for 24 h at 37 0C, subsequently
was added ethanol and glutamate at 6 h. Further step was determination of ethanol
concentration for stress condition. Choosing ethanol concentration that indicated
ethanol stress was based on the lowest significant cell growth which analyzed on
spechtrophotometry at 660 nm and membrane visualization by scanning electron
microscope (SEM). Next step was exploration of glutamate influence in ethanol
stress which looked into selecting an appropriate glutamate concentration, leaking
membrane analysis, glucose consumption, flux carbon, and glutamate
consumption. Leaking membrane analysis was accorded to the release of
metabolites by spectrophotometry at 260 nm (material genetic) and at 280 nm

(protein). Then, glucose consumption was conducted in enzymatic method by
using glucose kit 716251 (Roche). Meanwhile, flux carbon and glutamate
consumption were investigated on organic acids profile by using high
performance liquid chromatography (HPLC). Last step was examination the
effectiveness of glutamate addition. The cultivation was done by increasing
ethanol concentrations progressively from concentrations showed a significant
lower cell growth to the 50 gL-1 ethanol.
The result showed that the 20 gL-1 ethanol concentration gave significant
effect on inhibiting cell growth and caused leakage of cell membrane that viewed
in SEM micrograph. The 20 gL-1 ethanol was as indicator stress in this study and

applied for next cultivation. Whereas, glutamate influence in ethanol stress
resulted that the 2 gL-1 glutamate was known that had improved cell growth
significantly, reduced the release of metabolites (material genetic and protein).
Next the glutamate’s effect on glucose consumption succeeded to improve
glucose uptake in cells ethanol stress, and also efficiency of glucose consumed
achieved revival as same as cell control, it was 0.07 g.g-1. Then flux carbons in
glutamate addition reduce acetate formation as represented of overflow
metabolism. Meanwhile, glutamate was consumed 65% from glutamate addition
total in cultivation media. Hence, it could be reported that glutamate was required

in ethanol stress response. The last result of examination the effectiveness of
glutamate addition indicated that the 2 gL-1 glutamate helped cell E. coli to
increase its tolerance until 40 gL-1 ethanol. For this reason, glutamate addition was
effective to face ethanol stress.
Accordingly, glutamate addition was able to decrease negative impacts of
ethanol stress to the cell. It got better for tolerance on membrane physically,
glucose consumed, and metabolism stability. It also could be recommended as
reference in increasing ethanol production in cell ethanologenic E. coli harboring
pdc-adhB under an aerobic condition.
Keywords : Aerobic, ethanol-tolerant, Escherichia coli, glutamate addition

RINGKASAN
INDRA KURNIAWAN SAPUTRA. Peningkatan Toleransi Etanol pada
Escherichia coli Rekombinan melalui Penambahan Glutamat dalam Kondisi
Aerobik. Dibimbing oleh DJUMALI MANGUNWIDJAJA dan PRAYOGA
SURYADARMA.
Eksploitasi bahan bakar fosil dapat menyebabkan pengaruh negatif
terhadap ketersedian energi dan lingkungan. Maka, penggunaan energi alternatif
termasuk etanol memiliki peranan penting dalam menanggulangi hal tersebut.
Pada masa sekarang, etanol diproduksi melalui biokonversi material lignoselulosa,

dan E. coli merupakan bakteri potensial untuk produksi etanol dengan
memanfaatkan biomassa sebagai substrat. Hal ini disebabkan karena
kemampuannya dalam mengkonsumsi gula pentosa dan heksosa dari biomassa
untuk produksi etanol. Sementara itu, rekayasa metabolisme E. coli melalui
penginsersian gen etanologenik yakni gen yang menyandikan piruvat
dekarboksilase (PDC) dan alkohol dehidrogenaseII (ADH) mampu meningkatkan
produksi etanol. Selain itu, mutasi pada bagian fosfotransasetilase (PTA)
menyebabkan penurunan akumulasi produk samping (asetat) dan penyisipan
format dehidrogenase (FDH) di E. coli meningkatkan ketersedian NADH seiring
dengan akumulasi piruvat untuk produksi etanol dalam kondisi aerobik. Produksi
etanol yang terakumulasi menyebabkan sel mengalami kondisi stres etanol.
Kondisi stres dapat ditekan dengan penambahan glutamat. Glutamat diharapkan
dapat menguatkan peptidoglikan yang merupakan komponen penting dalam
mempertahankan sel dari pengaruh stres. Oleh karena itu, tujuan dari penelitian ini
adalah menerangkan pengaruh penambahan glutamat dalam meningkatkan
toleransi etanol pada E. coli etanologenik dan menguji keefektifan penambahan
glutamat dalam meningkatkan toleransi terhadap etanol kondisi kultur aerobik.
Strain E. coli ini bernama BW25113∆pta/pHfdh/pTadhB-pdc. Tahap awal
penelitian adalah pembuatan media tumbuh, pra-kultivasi, dan kultivasi. Media
kultivasi merupakan media LB kaya glukosa. Setelah itu, sampel dikultivasi

dengan agitasi 250 rpm selama 24 jam pada suhu 37 oC, selanjutnya ditambahkan
etanol dan glutamat pada jam ke-6. Tahap berikutnya adalah penentuan
konsentrasi etanol dalam pembentukan stres. Pemilihan konsentrasi etanol
didasarkan oleh pertumbuhan sel terrendah secara signifikan yang dianalisis
menggunakan metode spektrofotometri pada panjang gelombang 660 nm dan
visualisasi membran melalui pemanfaatan mikroskop pemayar elektron (SEM).
Tahap selanjutnya adalah pemeriksaan pengaruh glutamat dalam stres etanol yang
melihat pada penentuan konsentrasi glutamat yang tepat, analisis kebocoran
membran, konsumsi glukosa, fluks karbon, dan konsumsi glutamat. Analisis
kebocoran membran didasarkan pada keluarnya metabolit sel melalui metode
spektrofotometri 260 nm (material genetik) dan 280 nm (protein). Kemudian
konsumsi glukosa dilakukan berdasarkan metode enzimatis menggunakan kit
glukosa 716251 (Roche). Sementara itu, fluks karbon dan konsumsi glutamat
diamati pada profil asam organik menggunakan kromatografi cair kinerja tinggi
(KCKT). Tahap akhir adalah pengujian keefektifan dari penambahan glutamat.
Kultivasi dilakukan melalui peningkatan konsentrasi etanol secara progresif dari

konsentrasi yang menunjukkan pertumbuhan terendah secara signifikan hingga
konsentrasi etanol 50 gL-1.
Hasil menunjukkan bahwa konsentrasi etanol 20 gL-1 memberikan

pengaruh signifikan dalam menghambat pertumbuhan sel dan menyebabkan
kebocoran membran sel yang terlihat pada hasil SEM. Konsentrasi etanol 20 gL-1
dijadikan sebagai indikator stres dalam penelitian ini dan digunakan dalam
kultivasi berikutnya. Sedangkan pengaruh glutamat dalam stres etanol
menghasilkan bahwa konsentrasi glutamat 2 gL-1 diketahui dapat meningkatkan
pertumbuhan sel secara signifikan, menurunkan metabolit yang keluar (material
genetik dan protein). Selanjutnya, pengaruh glutamat terhadap konsumsi glukosa
sukses dalam meningkatkan konsumsi glukosa kondisi stres etanol, dan juga
efisiensi konsumsi glukosa dapat dikembalikan seperti kontrol yakni 0.07 g.g-1.
Kemudian fluks karbon dalam penambahan glutamat menurunkan pembentukan
asetat yang merupakan representasi dari overflow metabolism. Sementara itu,
glutamat dikonsumsi sebanyak 65% dari total penambahan glutamat di dalam
media kultivasi. Maka, hal ini dapat dilaporkan bahwa glutamat diperlukan dalam
respon terhadap stres etanol. Hasil akhir adalah pengujian keefektifan
penambahan glutamat yang mengindikasikan bahwa konsentrasi glutamat 2 gL-1
dapat membantu sel E. coli dalam meningkatkan toleransinya hingga konsentrasi
etanol 40 gL-1. Oleh karena itu, penambahan glutamat efektif dalam menghadapi
stres etanol.
Dengan demikian, penambahan glutamat mampu menurunkan pengaruh
negatif dari stress etanol. Ketahanan terhadap membran secara fisik dan konsumsi

glukosa serta kestabilan metabolisme juga membaik. Sehingga penambahan
glutamat dapat dijadikan sebagai acuan rekomendasi dalam meningkatkan
produksi etanol pada sel E. coli etanologenik pembawa gen pdc-adhB pada
kondisi aerobik.
Kata kunci : Aerobik, etanol toleran, Escherichia coli, penambahan glutamat

© Copyright 2016 by Bogor Agricultural University
All Rights Reserved
No part or all of this thesis may be excerpted without including or mentioning the
sources. Excerption should only use for education, research, scientific writing,
reporting, critical writing or review an issue; and those are not detrimental to the
interest of Bogor Agricultural University.
No part or all of this thesis may be transmitted and reproduced in any forms or by
any means without permission from Bogor Agricultural University

ENHANCING ETHANOL TOLERANCE OF Escherichia coli
RECOMBINANT BY GLUTAMATE ADDITION
UNDER AN AEROBIC CONDITION

INDRA KURNIAWAN SAPUTRA

Thesis
as partial fulfillment of the requirement to obtain
Master Science degree
in
Biotechnology Study Program

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

Non-committee examiner:

Dr drh Hasim, DEA

PREFACE
All praise and gratitude to Allah subhanahu wa ta’ala for over all The gifts
and The blessing so that this study is successfully completed. Prayers and peace
are always given to Rasulullah SAW. The theme chosen in the study which

carried out during one year (December 2014 – December 2015) is ethanol tolerant
with the title of Enhancing Ethanol Tolerance of Escherichia coli Recombinant by
Glutamate Addition under an Aerobic Condition.
I would like to express my sincere gratitude to my supervisors, Prof. Dr. Ir.
Djumali Mangunwidjaja, DEA and Dr. Prayoga Suryadarma, S.TP, MT who had
patiently directed, motivated, advised, and spent more times during research and
thesis writing. I also would like to acknowledge with much appreciation the noncomittee examiner Dr. drh. Hasim, DEA and the biotechnology program
representative Prof. Dr. Suharsono, DEA, who had provide suggestions and
recommendations for perfection writing. I very appreciate Bakrie Center
Foundation (BCF) which gave scholarship for study and research. Moreover, I
would like to offer special thanks to my family H. Syafruddin, S.Pd (father), Hj.
Hayati (mother), Irwan Wahyudi Saputra (brother), and Iin Nurintan Safitri
(sister) for loves, attentions, prayers, and supports. I also would like to say many
thanks to all my friends in Bioindustrial Laboratory for technical assistance. It has
been a pleaseure to work with you. Besides I gives special thank to Fitri Khasanah
Srihadining Putri, S.Pd for her attention and editing this thesis text.
Hopefully this thesis is useful.

Bogor, May 2016

Indra Kurniawan Saputra

CONTENTS
LIST OF TABLES

vi

LIST OF FIGURES

vi

LIST OF APPENDIXES

vi

1 INTRODUCTION

1

Background
Problem Statement
Research Objectives
Benefits of the Study
Scope of the Study
2 LITERATURE REVIEW
E. coli Recombinant (BW25113∆pta/pHfdh/pTadhB-pdc) in Metabolic
Engineering Scheme
Cell Membrane Sensitivity to Great Amount of Ethanol
Glutamate Forming Peptidoglycan and Uptake in E. coli
3 METHODOLOGY
Research Framework
Place and Time
Bacterial Strain and Plasmids
Growth Medium, Pre-cultivation, Cultivation
Determination of Ethanol Concentration for Stress Condition
Investigation of Glutamate Influence on Ethanol Stress Condition
Finding out the Effectiveness of Glutamate Addition
4 RESULT AND DISCUSSION

1
2
2
3
3
4
4
6
6
9
9
10
10
10
11
12
13
14

Implication of Ethanol Concentration on the Cell Growth and Membrane 14
The Glutamate Effect on Lowering Ethanol Stress Condition
15
The Effectiveness of Glutamate Addition in Decreasing Ethanol Stress 19
5 CONCLUSIONS AND RECOMMENDATIONS
Conclusions
Recommendations

22
22
22

LITERATURES

23

APPENDIXES

27

BIOGRAPHY

30

LIST OF TABLES
1 The E. coli recombinant used in this study

10

2 The release of metabolites (material genetic A260 and protein A280) as
result of leaking membrane in ethanologenic E. coli recombinant
17
3 Cell growth and glucose consumption after 24 h cultivation in E. coli
recombinant under an aerobic condition with ethanol and glutamate test.
17
4 Calculation of product during exposed to ethanol and glutamate addition in
E. coli recombinant under aerobic condition
18

LIST OF FIGURES
1 The gene distruption strategy of E. coli BW25113

5

2 Basic aerobic and anaerobic metabolic pathways of E. coli

5

3 The cell without wall cell fares best in an isotonic environment unless it
has special adaptations that offset the osmotic uptake or loss of water

6

4 Peptidoglycan biosynthesis

7

5 Mechanism of an ABC transporter

8

6 Research framework

9

7 Map of plasmids

11

8 Dry cell weight of E. coli recombinant at 24 h toward different
concentration of ethanol addition at 6 h
14
9 Micrograph of scanning electron microscope E. coli recombinant after 24
h cultivation in LB media and an hour in PBS media
15
10 Dry cell weight of E. coli recombinant at 24 h toward different
concentration of glutamate supplementation in the cultures of 20 gL-1
ethanol addition
16
11 Schematic ilustration showing central carbon metabolism and membrane
of ethanol stress cell with glutamate addition
20
12 Glutamate effectiveness in increasing ethanol concentration to dry cell
weight
21

LIST OF APPENDIXES
1 Procedure of glucose kit (Roche)

27

2 Research database in glucose kit analysis

28

3 Organic acids standard curve in HPLC analysis

29

1

1 INTRODUCTION
Background
Excessive use of fossil fuel cause negative impact on the availability of
energy and the environment. Therefore, increasing enthusiasm is focused toward
energy alternative, including ethanol. Ethanol production continues evolve
resulting in a change of its production, from First-generation produced primarily
food crop substrates such as grains, sugar beet, and oil seeds to Second-generation
produced from non-food biomass. Feedstock from non-food biomass includes
cereal straw, bagasse, forest residues, and purpose-grown energy crops such as
vegetative grasses and short rotation forests (IEA 2008). Non-food biomass
containing lignocelluloses typically compose 35 – 50 % cellulose, 20 – 35 %
hemicellulose, and 10 – 15 % lignin on dry weight basis (Wyman 1994).
However, one of the major technical obstacles to commercialization of
process for converting lignocelluloses to ethanol is strain development. Generally,
microorganisms used for fermentation (e.g. Saccharomyces cerevisiae and
Zymomonas mobilis) cannot catabolism pentose sugars derived from
hemicellulose and growth rate of S. cerevisiae is lower than other ethanol
producer (Jeppsson et al. 2002). Meanwhile, Escherichia coli having fast
generation time is capable of fermenting those sugars (pentose and hexose)
(Ingram et al. 1987). Hence, potency of E. coli had better uses as microbial
factory.
Normally, in regulation of metabolism E. coli result ethanol in anaerobic
condition via acetyl-coA catalyzed by pyruvate formate lyase (PFL) (Peterson and
Ingram 2008). Nevertheless, in the pathway not only produce ethanol but also
acetate as byproducts. Flux carbon tends to increase acetogenesis (Chang et al.
1999), forms anaplerotic pathway that result succinate (Wolfe 2005), decrease
growth of cell (Peng and Shimizu 2003), those all phenomenon metabolism is due
to citric acid cycle (TCA) do not induction properly. Besides, anaerobic condition
generates high lactate acid which converts pyruvate as substrate intermediate
(Chang et al. 1999). Many byproducts and low level of cell growth show that
production ethanol in anaerobic condition is still not effective.
Meanwhile, metabolic engineering is able to improve ethanol production
through insertion ethanologenic genes, namely genes encode pyruvate
decarboxylase (PDC) and alcohol dehydrogenaseII (ADH) (Ingram et al. 1987).
The reason for selection those enzymes are Michaelis-Menten constants (Km) of
PDC that quite low compared with other pyruvate-consuming reactions and
effectively shifting metabolic products to much higher ethanol production. Thus,
inside E. coli harboring pdc-adhB genes forms new pathway that convert pyruvate
to ethanol directly (Peterson and Ingram 2008) and deletion on
phosphotransacetylase (PTA) reduce acetate formation in cultures (Hahm et al.
1994). Furthermore, growth yields depend on oxygen availability because of
energy improvement in E. coli as facultative anaerobic bacterium (Kayser et al.
2005; Steinsiek and Bettenbrock 2012). Oxygen in aerobic condition trigger TCA
cycle that pathway produce energy and down-regulate lactate dehydrogenase
(LDH) forming lactate together with pyruvate oxidase (POXB) forming acetate

2

via other pathways (Ojima et al. 2012). Oxygen as end electron acceptor consume
NADH in respiration system (Causey et al. 2004) involve imbalance
NADH/NAD+ for ethanol production, but to keep stability of NADH/NAD+
integrate formate dehydrogenase (FDH). Besides, introduction of FDH enhance
the accumulation of pyruvate (Ojima et al. 2012). As consequence, aerobic
condition with accumulation of pyruvate will trigger high ethanol production.
Nonetheless, the increasing ethanol production is still limited, since ethanol
stress affects to the cell. As well as S. cerevisiae, ethanol which has molecule
sized 46 dalton diffuses freely across membrane E. coli to equalization of ethanol
concentration between extracellular and intracellular of cell (Alberts et al. 2010).
Ethanol impacts to the cell membrane integrity by changing fatty acid
composition (Dombek and Ingram 1983), decreasing flux proton in membrane
(Cartwright et al. 1986), inactivating cytosolic enzymes and leaking membrane,
then ultimately cell growth retardation (Huffer et al. 2011).
Cell membrane is first barrier to hold ethanol stress. In gram negative
membrane, peptidoglycan is an essential component of the bacterial cell wall that
protects the bacteria from osmotic rupture (Van Heijenoort 2001). So, the
destruction of ethanol stress should be prevented by glutamate addition. Based on
Underwood et al. (2004) that glutamate as osmolyte protective accumulate in
stress cell in aerobic condition, moreover glutamate is also an important part in
synthesis of peptidoglycan (Madigan et al. 2012). Glutamate is non-essential
protein in cell E. coli, produce from α-ketoglutarate which part of TCA cycle
product, key metabolite for urea cycle and co-substrate others amino acid
(glutamine, proline, and arginine) (Nelson and Cox 2004). Thus, in this research
was conducted by glutamate addition in helping cell toward high ethanol stress
under an aerobic condition that cell had been engineered in its metabolic. This
research gives a new way and potency to increase ethanol production in
ethanologenic E. coli harboring pdc-adhB genes.
Problem Statement
Accumulation of ethanol is such toxic for cell that impacts on lowering
growth and membrane integrity until destruction in cell E. coli producing ethanol.
In this study, to help cells face the stress of ethanol by glutamate addition.
Glutamate addition is expected to increase the rate of cell growth and also
maintains membrane stability. It cause that glutamate as osmolyte protective and
as main building block of peptidoglycan.
Research Objectives
The objectives of this research are to investigate the effect of glutamate
addition on ethanol tolerance enhancement of ethanologenic E. coli, and to look
into effectiveness of glutamate addition in increasing tolerance of ethanol under
an aerobic cultures condition.

3

Benefits of the Study
The benefits of this study are enhancing ethanol production in cell
ethanologenic E. coli harboring pdc-adhB genes, encouraging on using strain E.
coli in utilization non-food biomass, and providing recommendation in bioprocess
engineering to overcoming low cell growth because of high ethanol that useful in
industrialization.
Scope of the Study
The scope of this study include (i) culturing ethanologenic E. coli in
different ethanol addition to find out the ethanol stress condition under an aerobic
condition, (ii) glutamate addition to the ethanologenic E. coli culture under an
aerobic condition to get the role of glutamate addition in an ethanol stress
condition, (iii) culturing the ethanologenic E. coli supplemented with appropriate
glutamate concentration in the elevating ethanol addition.

4

2 LITERATURE REVIEW
E. coli Recombinant (BW25113∆pta/pHfdh/pTadhB-pdc) in Metabolic
Engineering Scheme
Metabolic engineering is a field that includes the construction, redirection,
and manipulation of cellular metabolism through alteration of endogenous and/or
heterologous enzyme activities and levels to achieve the biosynthesis or
biocatalysis of desired compounds. The basic tenet of metabolic engineering, the
use of biology as a technology for the conversion of energy, chemicals, and
materials to value-added products, has a long history. Early applications can be
cited, even prior to development of DNA recombinant technology (Smolke 2010).
The most widely bacterium used in DNA recombinant technology is E. coli, a
rod-shaped bacterium about 1 by 2.5 microns in size. This organism is simple in
structure, grows easily in laboratory, and contains very few genes. Another useful
trait of E. coli is the presence of extrachromosomal elements called plasmids.
These small rings of DNA are easily removed from bacteria, modified by adding
or modifying genes, and reinserted into a new bacterial cell where new genes can
be evaluated (Clark & Pazdernik 2010). Furthermore the increased availability of
genome sequences of E. coli provided comprehensive molecular understanding.
As result create a new strain BW25113 from E. coli K-12 which its genome is
available in highly accurate (Hayashi et al. 2006). The strain BW25113 had been
removed lacI, lacZ, araBAD, and rhaBAD for analysis method purpose by red
recombinase system. The red recombinase system came from bacteriophages that
encode their own homologous recombination system. The basic principle is to
replace a chromosomal sequence (Figure 1) with integrated low-copy-number red
recombinase expression plasmid pKD46 that was reported by Datsenko and
Wanner (2000).
According to Baba et al. (2006) the mutation at pta site also used red
recombinase system. It has been known for a long time that the pta gene encode
phosphotransacetylase (PTA) in which produce acetate together with acetate
kinase (ACKA) pathway, generates two ATP molecules per glucose but consumes
no reducing equivalents (NADH). The PTA-deficient mutant did not accumulate
extracellular acetate (Hahm et al. 1994). It gave advantages for ethanol production
during aerobic growth on glucose (Diaz-ricci et al. 1991). To rise ethanol
production had been introduced the ethanologenic genes (pdc-adhB) from Z.
mobilis that encode pyruvate decarboxylase (PDC) and alcohol dehydrogenase
(ADHB) (Ingram et al. 1997). The reason led to Z. mobilis Km PDC is quite low
compared with other pyruvate-consuming reactions and effectively shifting
metabolic products to much higher concentrations of ethanol (Peterson & Ingram
2008). The PDC-ADHB will convert pyruvate to ethanol. It is contrary to wild
type which produces ethanol anaerobic condition via acetyl-coA (Figure 2).
Meanwhile, in aerobic condition E. coli as facultative organism are capable
of modifying its metabolism to accommodate growth. The transition metabolism
is accompanied by alterations in the rate, route, and efficiency of pathways of
electron flow (Figure 2). Under aerobic condition, the energy yield became more
plentiful when alternate acceptors electron (oxygen) are available (Moat et al.

5

2002). However, respiration chain consumed NADH that also used as coenzyme
for alcohol dehydrogenase to produce ethanol. To recover the stability of NADH,
based on Ojima et al. (2012) was introduced formate dehydrogenase (FDH) from
M. vaccae that also enhanced accumulation of pyruvate. Then pyruvate might be
converted by PDC-ADHB to ethanol.

Figure 1 The gene distruption strategy of E. coli BW25113. H1 and H2 refer to
the homology extensions or regions. P1 and P2 refer to priming sites
(Datsenko & Wanner 2000).

Figure 2 Basic aerobic and anaerobic metabolic pathways of E. coli (Moat et al.
2002).

6

Cell Membrane Sensitivity to Great Amount of Ethanol
Ethanol in cell diffuses freely across membrane allowing equalization of
ethanol concentrations between intracellular and extracellular, normally. It is
isosmotic that same to water. However, the high ethanol concentration showed
growth retardation and finally death. In molecular cell, ethanol disrupt membrane
by dissipating proton motive force (Cartwright et al. 1986).The proton motive
force is movement of H+ ions across the membrane through the H+ channel
provided by ATP synthase. In general terms also called chemiosmosis (Reece et
al. 2011). The stability of chemiosmosis are crucial for cell in which effect to
water or ethanol movement where look into both solute concentration and
membrane permeability. The different of them are divided into three type
environment; isotonic, hypotonic, hypertonic. Those environments will bring cell
membrane into normal, lysed, and shriveled respectively (Figure 3). Moreover,
based on Underwood et al. (2004) that ethanol affect on lost of osmolytes that can
change cell environment.

Figure 3 The cell without wall cell fares best in an isotonic environment unless it
has special adaptations that offset the osmotic uptake or loss of water
(Reece et al. 2011).
Glutamate Forming Peptidoglycan and Uptake in E. coli
Glutamate is an important nonessential amino acid involved in protein
synthesis (Glutamate family is proline, glutamine, arginine) and other
fundamental process such as urea cycle, glycolysis, gluconeogenesis, and the
citric acid cycle (Koolman & Roehm 2005). Catabolism of glutamate occurs
mainly by the action of either glutamate dehydrogenase or glutamate
decarboxylase. The first enzyme, among other roles, is important for assimilation
of ammonia to amino acids, while the second is important for resistance to stress
(Berg et al. 2007). Glutamate biosynthesis is resulted from α-ketoglutarate which
part of citric acid cycle and other glutamate family. The glutamate functions as
osmolyte protective in tonicity condition (Underwood et al. 2004) and also as
important molecule of cell wall peptidoglycan structure. It is incorporated into
nucleotide peptidoglycan precursors by addition to UDP-N-acetylmuramoylalanine (UDP-Mur-Nac-L-Ala), a reaction catalyzed by the D-glutamic acidadding enzyme (the murD gene product) (Pratviel-Sosa et al. 1991). The
enzymatic mechanism is by which glutamate to synthesis peptidoglycan, depicted
in Figure 4.

7

Figure 4 Peptidoglycan biosynthesis. The three stages (cytoplasmic, membrane
bound, and wall bound) are separated by the dashed vertical lines.
GlcNAc = N-acetylglucosamine; MurNAc = N-acetylmuramic acid; LR3, for example, meso-diaminopimelic acid. The sites of action of
antimicrobial agents affecting peptidoglycan synthesis are shown. The
structural genes and names of the enzymes are (1, 2) pyrH, UMP
kinase; (3) UDP-N-cetylpyrophosphorylase; (4) murZ, UDP-Nacetylglucosamine
enolpyruvate
transferase;
(5)
UDP-Nacetylglucosamine enol-pyruvate reductase; (6) murC, L-alanine adding
enzyme; (7) murD, D-glutamate adding enzyme; (8) murE, mesodiaminopimelate adding enzyme; (9) murF, D-alanyl:D-alanine adding
enzyme; (10) alanine racemase; (11) ddl, D-alanine:D-alanine ligase;
(12) mraY, UDP-N-acetyl-muramoyl-pentapeptide transferase (first step
in lipid carrier cycle); (13) murG, N-acetyl-glucosaminyltransferase
(final step in lipid carrier cycle); (14) transglycosylases and transpeptidase; (15) membrane-bound pyrophosphatase; (16) membrane-bound
transpeptidase (target of β-lactam antibiotics); (17) D-ala transport
system (Moat et al. 2002).

8

Peptidoglycan synthesis involves a number of cytoplasmic, membrane, and
periplasmic steps. N-acetylglucosamine (GlcNAc) is first coupled with UDP. A
portion of the UDP-GlcNAc is converted into UDP-MurNAc (N-acetylmuramic
acid), and the peptide chain is developed by sequential addition of amino acids.
The growing chain is then coupled with undecaprenyl-phosphate, enabling its
transfer across the cytoplasmic membrane where it is incorporated into the
growing peptidoglycan. At the interface between the growing cell wall and the
cell membrane, transglycosidation reactions polymerize the growing chain and
transpeptidases introduce cross-linking (Moat et al. 2002).
Furthermore E. coli can uptake glutamate from environment via glutamate
ABC transporter that consist of four subunit transporter; GltI (periplasmic binding
protein), GltK (membrane subunit), GltJ (membrane subunit), GltL (ATP binding
subunit) (Schellenberg & Furlong 1977). According to Madigan et al. (2012)
ABC transports system is transport that employ periplasmic binding proteins
along with membrane transporter and ATP-hydrolyzing proteins. A characteristic
property of periplasmic binding proteins is their high substrate affinity. These
proteins can bind their substrate even when they are at extremely low
concentrations; for example, less than 1 micromolar (10-6 M). Once its substrate is
bounce, the periplasmic binding protein interacts with its respective membrane
transporter to transport the substrate into the cell driven by ATP hydrolisis (Figure
5).

Figure 5 Mechanism of an ABC transporter. The periplasmic binding protein has
high affinity or substrate, the membrane-spanning proteins form the
transport channel, and the cytoplasmic ATP-hydrolyzing proteins
supply the energy for the transport event (Madigan et al. 2012).

9

3 METHODOLOGY
Research Framework
The research framework depict in Figure 6. To enhance ethanol tolerance in
E. coli, firstly it was determined of ethanol stress concentration in an aerobic
culture condition. The stress resulted reducing cell growth and breaking of cell
membrane that visualized in scanning microscope electron (SEM) for
understanding the implication high ethanol in E. coli membrane. Ethanol stress
concentration used for next cultivation for explanation glutamate influence in
ethanol stress and glutamate’s effectiveness. Indicator for impact of glutamate was
investigated by looking cell growth, leaking membrane analysis, glucose
consumption, and flux carbon analysis. Meanwhile, to test effectiveness of
glutamate was carried out by increasing ethanol stress concentration until
decreasing cell growth significantly. All of pre-cultivation, cultivation, and cell
growth analysis was conducted according Ojima et al (2012), leaking membrane
analysis based on procedure modified of Lin et al. (2000), glucose consumption
used kit glucose (Roche), and flux carbon analysis was looked into organic acids
by using High Performance Liquid Chromatography (HPLC).

Enhancing ethanol tolerance of E. coli harboring ethanologenic
genes by glutamate addition under an aerobic condition

Effect of ethanol concentration



Cultivation in high ethanol
concentration
Visualization of membrane cell

Glutamate influence on stress
ethanol
 Cultivation in high ethanol
concentration by glutamate
addition
 Leaking membrane analysis
 Glucose consumption
 Flux carbon analysis
 Glutamate consumption

Effectiveness of glutamate
addition


Increasing ethanol
concentration in culture
with cell growth monitoring

Figure 6 Research framework

10

Place and Time
This research was carried out during December 2014 – December 2015 in
Bioprocess Engineering Laboratory, Research Center of Bioresources and
Biotechnology, Bogor Agricultural University and Bioindustry Laboratory,
Department of Agroindustrial Technology, Faculty of Agricultural Technology,
Bogor Agricultural University.
Bacterial Strain and Plasmids
The bacterial strain employed in this study was E. coli BW25113 which
mutated on phosphotransacetylase (pta) genes originated from National
BioResources Project, National Institute of Genetic (NIG), Mishima, Japan (Baba
et al. 2006). This strain was inserted plasmid pHfdh bearing formate
dehydrogenase genes (fdh) (Ojima et al. 2012) and plasmid pTadhB-pdc
harboring ethanologenic genes namely pyruvate decarboxylase (pdc) together
with alcohol dehydrogenase (adhB) genes. The genes were obtained from
Department Chemical Science and Engineering, Osaka University, Japan. Strain
and plasmids are listed in Table 1.
Table 1 The E. coli recombinant used in this study
Name
E. coli strain
BW25113∆pta
Plasmids*
pHfdh

pTadhB-pdc

Description

Reference or Source

pta-deficient mutant from parent
strain BW25113, Kanr

Ojima et al. 2012

fdh gene from Mycobacterium
vaccae, ligated into pHSG399
vector which contains a pMB1
replicon, Camr
Ethanologenic genes (adhB and
pdc) from Z. Mobilis inserted into
pTrc99A, Ampr

Ojima et al. 2012

Department Chemical
Science and Engineering,
Osaka University

*Map of vector plasmids depict in Figure 7

Growth medium, Pre-cultivation, Cultivation
Growth medium used was Lauria Bertani (LB) medium containing 10 g
peptone, 10 g NaCl, and 5 g yeast extract per litre of deionized water. Precultivation was conducted for refresh of cell E. coli from glycerol stock to reach
optimum growth in cultivation. In pre-cultivation LB medium were added
appropriate antibiotics (50 mgL-1 ampicillin, 34 mgL-1 chloramphenicol, 15 mgL-1
kanamycin) along with 1 % (v/v) strain BW25113∆pta/pHfdh/pTadhB-pdc.
Samples were incubated in incubator shaker (Optic Ivymen System) agitation 120
rpm for 12 h at 37 0C. After OD660 reach 1.0 – 1.5, then moved into cultivation
medium.

11

(a)

Figure 7

(b)

Map of plasmids (a) Z. Mobilis ethanologenic genes (adhB-pdc) were
inroduced into pTrc99A named pTadhB-pdc and, (b) M. vaccae
formate dehydrogenase (fdh) was introduced into pHSG399 named
pHfdh.

The cultivation medium was arranged in glucose-enriched LB medium
according to Suryadarma et al. (2012) that consisted of 5% (v/v) inoculums, 40 g
glucose, 4 g formate, 20 g CaCO3 per liter deionized water, antibiotics (50 mgL-1
ampicillin, 34 mgL-1 chloramphenicol, 15 mgL-1 kanamycin) together with 0.5
mM isopropyl thiogalactoside (IPTG) for inducing protein recombinant
expression. Initially, culture medium was adjusted at pH 7 using NaOH then
cultivation based on Ojima et al. (2012) procedures. Supplementation of CaCO3
was meant to prevent pH medium drop during cultivation, while formate addition
was used for keeping redox status of NAD+/NADH. Before cultivation sample
was taken at 0 h for glucose analysis requirement, subsequently added 20 gL-1
ethanol and 2 gL-1 glutamate at 6 h. Samples were incubated (Optic Ivymen
System) with agitation 250 rpm for 24 h at 37 0C. After 24 h, samples were
cultivated and analyzed. Cultivations were repeated 2 – 3 times.
Determination of Ethanol Concentration for Stress Condition
Ethanol concentration indicating ethanol stress was carried out by
cultivation in high ethanol concentration. Concentrations range was 0 – 25 gL-1
ethanol and that significantly reduced cell growth became stress indicator. In
culture condition, ethanol was added at 6 h, cultivated until 24 h under aerobic
condition, and repeated for 3 times. Cell growth were measured by looking into
dry cell weight which analyzed on spectrophotometry wavelength 660 nm. OD660
was multiply with 0.36 as equivalent for 1 OD660 (Ojima et al. 2012). The data
gained were average and standard deviation values from 3 repetitions with the
significance measurement by t-test.
The purpose of ethanol supply in cultures at 6 h was based on growth profile
of E. coli. At 6 h culturing was exponential phase that monitored effect of ethanol
on growth rate easily and assumed that E. coli strain did not produce ethanol

12

anymore at 6 h culturing because of inhibitor regulations. Inhibitor that inhibited
alcohol dehydrogenase (adhB) in ethanologenic E. coli was end product of
enzyme reactions, namely ethanol and NAD+. Ethanol acts as competitive
inhibitor, whereas NAD+ acts as uncompetitive inhibitor (Hoppner and Doelle
1983).
Afterwards, to proved the effect of ethanol toward cell membranes was
conducted through visualization by Scanning Electron Microscope (SEM). SEM
was prepared in 4 steps (fixation, dehydration, ionic spark, visualization) that
procedures described previously (Bennis et al. 2004). Samples had been contacted
with ethanol, subsequently were fixed in 2 % glutaraldehyde for an hour at 4 oC
and in 1 % osmium tetraoxyde for 30 minutes at 4 oC, dehydrated using critical
cell point in liquid CO2 under 95 bar pressure (Quorum K850) then air dried.
Ionic spark employing Cathodic Sprayer Gold (Quorum Q150 R ES) was
sputtered a small amount of gold ion on the samples to avoid charging in the
microscope. Samples were visualized in scanning electron microscope (Zeizz
EVO MA10).
Investigation of Glutamate Influence on Ethanol Stress Condition
Glutamate addition was intended for helping cells steady in ethanol stress
condition. Glutamate concentrations (0 – 6 gL-1) which had an increasing
significant cell growth were used and analyzed further. The sample cultures were
supplemented glutamate and ethanol at 6 h for 24 h cultivation under an aerobic
condition. Furthermore, understanding roles of glutamate in cells were explored
on cell growth, leaking membrane cells, glucose consumption, flux carbon, and
glutamate consumption.
Leaking membrane analysis was carried out to explain the membrane of
cells condition. Procedure which modified based on Lin et al. (2000). Firstly,
preparation of cells were cultivated until 18 h for reaching end phase of
exponential and followed by centrifugation 4800 g for 15 minutes at 25 oC
(Hettich zentrifugen, micro 22R). The pellets were washed twice and put into
phosphate buffer saline (PBS) pH 7. Then, samples were incubated in rotary
shaker (Optic Ivymen System) 250 rpm agitation for an hour at 37 oC with tests
(control, 20 gL-1 ethanol, 20 gL-1 ethanol and 2 gL-1 glutamate). After incubation,
samples were separated from pellet and supernatant by centrifugation 4800 g for
15 minutes (Hettich zentrifugen, micro 22R). Finally, the supernatant was
measured at wavelength 260 nm for genetic material and 280 nm for protein in
spectrophotometer (Hewlett Packard 8453). The data obtained were average and
standard deviation values from 3 repetitions with the significance measurement by
t-test.
Glucose consumption was conducted in enzymatic method by using glucose
kit 716251 (Roche). Samples which cultivated 24 h were inactivated by heating,
degassing, and followed by centrifugation (Hettich Zentrifugen) with 9727 g for 2
minutes at 4 oC. Respectively, the supernatants were filtered (0.22 µm, Sartorius),
diluted in accordance with estimation of glucose remaining, and measured
spectrophotometry according to the supplier’s instructions (Appendix 1). Output
data from glucose kit analysis were glucose remaining, and then data were
processed to get glucose consumptions by reducing total glucose with glucose

13

remaining (Appendix 2). The data gained were average and standard deviation
value from 2 – 3 repetitions with the significance measurement by t-test
To explain regulation of glutamate in metabolism cell was analized flux
carbon with organic acid by using high performance liquid chromatography
(HPLC) (HPLC Hp Hewlett Packard, 1100 series). Materials preparation were
samples diluting in double-distilled water, standards (succinate, lactate, pyruvate,
glutamate, acetate), and mobile phase mixing of 99 % 20 mM NaH2PO4 pH 2 and
1 % acetonitrile. Samples were injected into columned Zorbax Sb-Aq 883975-914
(Agilent), flow rate 1.0 mLmin-1 at 35 oC and detected on 210 nm. Glutamate
consumption was known from glutamate remaining in cultures media that
detected in HPLC by reducing concentration of glutamate addition. The organic
acid standard curve was depicted in Appendix 3. The data gained were average
and standard deviation values from 2 – 3 repetitions with the significance
measurement by t-test. Carbon recovery (Rc) values were calculated according to
following formula:
� =

mol of Carbon in metabolite produced
× 100%
mol of Carbon in glucose consumed

Finding out the Effectiveness of Glutamate Addition

Cells E. coli were cultivated in ethanol stress that supplemented glutamate
under an aerobic condition. The ethanol concentrations were increased
progressively from concentration showed a lower cell growth significantly to 50
gL-1 ethanol. The glutamate concentration had been standardized in previous
experiments. Decreasing significance cell growths were appropriate concentration
that gave effect to the cell. The data gained were average and standard deviation
value from 2 repetitions with the significance measurement by t-test.

14

4 RESULT AND DISCUSSION
Implication of Ethanol Concentration on the Cell Growth and Membrane
At first, the effect of ethanol concentration on the E. coli cell growth was
investigated (Figure 8). The increasing concentration of ethanol addition lowered
cell growth of ethanologenic E. coli. The 20 gL-1 ethanol concentration gave
significant effect on inhibiting cell growth. It was greater than concentration
ethanol of which produced by E. coli strain AH003 with genotype KO11 mutated
at lactate dehydrogenase (ldhA) and pyruvate formate lyase (pflA) as a towering
ethanol producer strain (Forster and Gescher 2014).
This result was also documented by Huffer et al. (2011) which showed that
excessive ethanol reduced cell growth rate in strain E. coli K12 under aerobic
condition. In addition, high ethanol concentration also disrupted membrane
integrity which was due to ethanol toxicity (Dombek and Ingram 1983). Therefore
the membrane cell leaked. The result was confirmed further by SEM micrograph.
E. coli cells being not in stress condition had membrane flat, soft, and normal
(Figure 9a). Otherwise, E. coli which was exposed 20 gL-1 ethanol was visibly
leaking on membrane cells (Figure 9b). It has been reported previously that
leaking cells is due to the alteration of environment osmolarity (Reece et al.
2011). This condition was also broken off peptidoglycan as layer within periplasm
for giving bacteria shapes and protection from stress (Schwechheimer and Kuehn
2015). Meanwhile leaking membranes as consequence of ethanol diffuse easily
trough membrane which disrupts cross-linking of peptidoglycan (Ingram &
Vreeland 1980; Alberts et al. 2010) increased plasmolysis, accumulated of
material genetic and protein in culture. Hence, in this research the 20 gL-1 ethanol
became ethanol stress indicator for next cultivation.

Dry Cell Weight (gL-1)

*: Significance
the 5% level

*

1,5

at

1

0,5

0
0

10

15
Ethanol

Figure 8

20

25

(gL-1)

Dry cell weight of E. coli recombinant at 24 h toward different
concentration of ethanol addition at 6 h.

15

Figure 9

Micrograph of scanning electron microscope E. coli recombinant after
24 h cultivation in LB media and an hour in PBS media of cells (a)
without ethanol addition as control, then (b) with 20 gL-1 ethanol
addition at 6 h and (c) with 20 gL-1 ethanol addition and 2 gL-1
glutamate at 6 h. Arrow represents leaking cells.

The Glutamate Effect on Lowering Ethanol Stress Condition
To avoid the disrupting cell membrane as an effect of ethanol stress, the
different of glutamate concentration was supplemented at 6 h (Figure 10).
Glutamate is abundant amino acid in cytoplasm of E. coli and related organism
under aerobic condition. It also involve in stress response (Feehily and Karatzas
2012) and a crucial molecule in building block of peptidoglycan. The 2 gL-1
glutamate had brought cells in growth comfortably that gave a significance
increasing dry cell weight. The 2 gL-1 glutamate was as an appropriate
concentration on increasing cell growth in the cultures of 20 gL-1 ethanol addition.
To confirm the glutamate influence in leaking cell membranes was
investigated. Naturally, S. cerevisiae as traditional microbial factory produce
ethanol higher than wild type of E. coli. It can be explained from structure of
membrane that afford struggle in high ethanol concentration. S. cerevisiae
membrane have a more complex structure than E. coli. S. cerevisiae membrane
consist of polysaccharide made up three sugars, such as mannose that link to
protein, glucose from β-glycans, and N-acetylglucosamine that present in the wall
linked to chitin (Cabib et al. 2001). Despite in small amount, chitin is main
structure for yeast survival (Shaw et al. 1991). Meanwhile, in E. coli the main
structure for survival is peptidoglycan.

16

*

Dry Cell Weight (gL-1)

2,5

*: Significance at
the 5% level

*
*

2
1,5
1
0,5
0
0

2
4
Glutamate (gL-1)

6

Figure 10 Dry cell weight of E. coli recombinant at 24 h toward different
concentration of glutamate supplementation in the cultures of 20 gL-1
ethanol addition. Glutamate supplementation and ethanol addition was
at 6 h.
Peptidoglycan is a part of E. coli cell wall. Cell wall disintegrated as high
ethanol (Dombek and Ingram 1983). Disintegrated membrane allowed leaking
membrane as consequence that the realease metabolites (material genetic and
protein) from cytoplasm accumulated in culture. Those materials were
investigated by using spectrophotometry on exact wavelength particle. According
to Mulhardt (2007), materials genetic absorb more ultraviolet radiation at 260 nm
and protein at 280 nm for aromatic amino acids. The absorbance 260 nm in cell
ethanol stress described that material genetic was the most abundant than other
tests (Table 2). The result showed that ethanol disrupt membrane integrity in E.
coli recombinant. Interestingly, glutamate addition reduced the release metabolites
(material genetic and protein). The data showed that glutamate reduced material
genetic significantly from at A260 0.91 to 0.76 and average value of protein from
at A280 0.46 to 0.39. These results showed that glutamate prevented the leaking
cell membrane from ethanol stress condition. Then, in membrane imaging showed
although there was broken part, the cell still looked rigid (Figure 9c). Therefore,
the 2 gL-1 glutamate addition can be assumed that kept stability of membrane in
ethanol stress under an aerobic culture.
To get further understanding of the phenomenon, glutamate addition
influenced on glucose consumption was investigated. The glucose consumption is
fundamental thing to know initially on molecular mechanism in ethanol tolerance
(Ma and Liu 2012). Cells in ethanol stress condition consumed glucose lower than
control and glutamate replenishment (Table 3). It described that high ethanol led
to glucose uptake alleviation in cultures under an aerobic condition. Otherwise,
glutamate supplementation succeeded to improve glucose uptake in cells ethanol

17

stress condition. According to Roth et al. (1985), when cells in culture raised
glucose uptake meant that cells had alleviated the effect of stress.
In addition, in ethanol stress resulted biomass (g) per glucose consumed
(g) 0.09 g.g-1 that higher than control and glutamate addition (Table 3). Cells in
ethanol stress needed more glucose to produce energy, not only for metabolism
regulation but also for stress response, such as regulation of heat shock protein
that increase in amount after stresses (Pipper 1995) and required energy (Clark
and Pazdernik 2010). It also enhanced pyruvate production, that represented in
carbon recovery (Rc) value.