Improvement Of Ethanol Production By Inducer Addition In Recombinant Escherichia Coli Culture Under Aerobic Conditions

IMPROVEMENT OF ETHANOL PRODUCTION BY INDUCER
ADDITION IN RECOMBINANT Escherichia coli CULTURE
UNDER AEROBIC CONDITIONS

FITHRIANI

POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

DECLARATION OF ORIGINALITY AUTHENTICALY AND
COPYRIGHT*
I declare that the thesis entitled Improvement of Ethanol Production by
Inducer Addition in Recombinant Escherichia coli Culture under Aerobic
Conditions is the result by myself and guidance of the supervisor committee; and
has not been submitted before in any institutions. Secondary source of information
published or unpublished has been acknowledged in text and included in the
reference chapter.
Herewith I bestow the copyright of my paper to Bogor Agricultural
University.

Bogor, March 2016
Fithriani
P051120231

SUMMARY
FITHRIANI. Improvement of Ethanol Production by Inducer Addition in
Recombinant Escherichia coli Culture under Aerobic Conditions. Supervised by
DJUMALI MANGUNWIDJAJA and PRAYOGA SURYADARMA.
Isopropyl-β-D-1-thiogalactopyranoside (IPTG) plays significant role in
initiating expression of recombinant genes in Escherichia coli under the control of
lac-derived promoter. The addition of IPTG is frequently performed in exponential
growth stage by using high concentrations as an effort to fully induce lac-derived
promoter. Although generally results in increasing recombinant genes expression,
metabolic load that occurs in host cells after induction leads to reduce cell growth,
in turn, lowering product yield. The objective of this study was to investigate the
effect of IPTG addition in terms of induction time and IPTG concentration in
recombinant E. coli culture to improve ethanol production under aerobic conditions.
This study was consisted of three experimental stage: effect of pdc and adhB
introduction, role of fdh reaction and formate availability, and effect of IPTG
induction on ethanol production. E. coli strains used in this study were BW25113

and BW25113∆pta. Plasmid pTadhB-pdc harboring ethanol producing genes of pdc
and adhB from Zymomonas mobilis; and pHfdh harboring NADH regeneration
gene of fdh from Mycobacterium vaccae, were used to transform E. coli strains.
Improvement of ethanol production was first investigated by introducing pdc and
adhB genes into E. coli BW25113; and then was further improved by introducing
fdh and ethanologenic Z. mobilis genes into E. coli BW25113∆pta, following 4 g
L-1 formate supplementation into cultivation medium. The effect of induction time
in recombinant E. coli BW25113/pHfdh/pTadhB-pdc culture was investigated by
IPTG addition at 0, 6, 12 and 18 h of cultivation time; while impact of inducer
concentration was investigated by addition of IPTG in the range concentration of
50-2000 µM at an optimum induction time.
Analysis of mRNA and ethanol showed pdc and adhB were successfully
expressed in recombinant strain BW25113/pTadhB-pdc, and led to induced two
fold greater ethanol compared to the parent strain BW25113 (0.2 g L-1 ethanol). The
ethanol production improvement was further observed at 1.10 g L-1 in recombinant
culture of BW25113∆pta/pHfdh/pTadhB-pdc by using 4 g L-1 formate
supplementation. IPTG induction in BW25113∆pta/pHfdh/pTadhB-pdc culture
showed significant higher ethanol production than non-induced culture. Addition
of IPTG at concentration up to 50 µM strongly led to ethanol production
improvement. However, higher IPTG concentrations could not further improve the

production of ethanol. This study demonstrated that induction time at 0 h by using
low concentration of IPTG at 50 µM is effective to improve ethanol production in
recombinant E. coli culture under aerobic conditions.
Keywords: aerobic, Escherichia coli, ethanol production, IPTG induction, NADH
regeneration

RINGKASAN
FITHRIANI. Peningkatan Produksi Ethanol melalui Penambahan Induser pada
Kultur Escherichia coli Rekombinan dalam Kondisi Aerobik. Dibimbing oleh
DJUMALI MANGUNWIDJAJA dan PRAYOGA SURYADARMA
Isopropil-β-D-1-tiogalatopiranosida (IPTG) berperan penting dalam
menginisisasi ekspresi gen rekombinan pada Escherichia coli yang dikontrol oleh
promoter turunan lac. Penambahan IPTG biasanya dilakukan pada fase
eksponensial dengan menggunakan konsentrasi yang tinggi sebagai upaya untuk
menginduksi sepenuhnya promoter turunan lac. Meskipun umumnya dapat
meningkatkan ekspresi gen rekombinan, beban metabolik setelah induksi dapat
menyebabkan penurunan pertumbuhan sel sehingga mengakibatkan penurunan
yield produk. Penelitian ini bertujuan untuk menginvestigasi pengaruh penambahan
IPTG dalam segi waktu induksi dan konsentrasi IPTG pada kultur Escherichia coli
rekombinan untuk meningkatkan produksi ethanol dalam kondisi aerobik.

Penelitian ini terdiri dari tiga tahapan eksperimen: investigasi mengenai
pengaruh introduksi gen pdc dan adhB, pengaruh reaksi fdh dan penambahan
format, serta pengaruh induksi IPTG. Strain E. coli yang digunakan yaitu BW25113
dan BW25113∆pta. Plasmid yang mengandung gen untuk produksi etanol yaitu
piruvat dekarboksilase (pdc) dan alkohol dehidrogenase (adhB) dari Zymomonas
mobilis; dan plasmid pHfdh yang membawa gen fdh untuk regenerasi NADH dari
Mycobacterium vaccae, digunakan untuk mentranformasi strain E. coli.
Peningkatan produksi etanol pertama kali dilakukan dengan mengintroduksikan
gen pdc dan adhB pada E. coli BW25113; kemudian ditingkatkan lebih lanjut
dengan mengintroduksikan gen fdh dan juga gen etanologenik Z. mobilis pada E.
coli BW25113∆pta. Untuk mengivestigasi pengaruh waktu induksi terhadap kultur
E. coli rekombinan, induksi dilakukan melalui penambahan IPTG pada waktu
kultivasi jam ke 0, 6, 12 dan 18. Sedangkan pengaruh konsentrasi induser
diinvestigasi melalui penambahan IPTG dengan rentang konsentrasi 50 -2000 µM
pada waktu induksi yang optimum.
Analisis mRNA dan etanol menunjukkan bahwa gen pdc dan adhB berhasil
terekspresi pada E. coli rekombinan BW25113/pTadhB-pdc, dan dapat
menginduksi produksi ethanol dua kali lipat lebih besar dibandingkan strain induk
BW25113 (0.2 g L-1 etanol). Peningkatan etanol lebih lanjut terobservasi sebesar
1.10 g L-1 pada kultur E. coli rekombinan BW25113∆pta/pHfdh/pTadhB-pdc

melalui penambahan 4 g L-1 format. Induksi IPTG pada kultur
BW25113∆pta/pHfdh/pTadhB-pdc menunjukkan peningkatan etanol yang
signifikan dibandingkan kultur tanpa induksi. Produksi etanol tertinggi sebesar 4.09
g L-1 terobservasi pada waktu induksi jam ke 0. Pemberian konsentrasi IPTG pada
0-50 µM dapat meningkatkan produksi etanol. Namun, konsentrasi IPTG lebih
tinggi tidak dapat meningkatkan etanol lebih lanjut. Penelitian ini menunjukkan
bahwa penambahan IPTG pada waktu induksi jam ke 0 dengan konsentrasi IPTG
minimum sebesar 50 µM efektif untuk meningkatkan produksi etanol pada kultur
E. coli rekombinan dalam kondisi aerobik.
Kata kunci: aerobik, Escherichia coli, induksi IPTG, produksi etanol, regenerasi
NADH

© Copyright IPB, 2016
All Rights Reserved by Law
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IMPROVEMENT OF ETHANOL PRODUCTION BY INDUCER
ADDITION IN RECOMBINANT Escherichia coli CULTURE
UNDER AEROBIC CONDITIONS

FITHRIANI

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

POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

External Thesis Examiner: Dr Ir Mulyorini Rahayuningsih, MSi

PREFACE
Praise and gratitude are addressed to Allah subhanahu wa ta’ala for all His
Grace during the research and study and who has made possible this scientific
writing successfully completed. The study was conducted from November 2014
until December 2015 and entitled Improvement of Ethanol Production by Inducer
Addition in Recombinant Escherichia coli Culture under Aerobic Conditions.
My deepest appreciation is delivered to all those who helped in this scientific
writing process:
1.
Prof Dr Ir Djumali Mangunwidjaja, DEA and Dr Prayoga Suryadarma, STP
MT as supervisors for their enormous enthusiasm in the scientific research,
inspiration, motivation and guidance during study, and research.
2.
Dr Ir Mulyorini Rahayuningsih, MSi as external examiner for her guidance
and advice in the thesis examination and scientific writing.
3.
Prof Dr Ir Suharsono, DEA as Head of Biotechnology Study Program and
moderator in the thesis examination for his guidance and sagacity during
study and examination.
4.

Lecturers and laboratories staffs in Biotechnology Study Program, Research
Center of University; and Agroindustrial Technology Department for their
help in conducting the research.
5.
My parents (The late Icang Sudaryat; and Siti Nuraeni), sisters and brother
(Kartika Dewi Puspa, Uswatun Hasanah, Anugrah Akhirut Tasyrik and Ali
Mursid); and the all family for their support, affection, prayers, and constant
believing me.
6.
The members of Bioprocess Engineering Research Group (Indra Kurniawan
Saputra, Wahyu Suradi Pranata, Resa Denasta Syarief, Budimandra Harahap,
Lianitha Kurniawati, Dian Sukma Riany, Mujtahid Al Fajri, Ricky Susanto
Putera, Nurul Muhibbah, Ari Permana Putra, M. Jiyad Hijran and Mara Anda
Rival) for their help, motivation, and insightful discussions about scientific
matters.
7.
Bioindustry Laboratory Member (Ardhi Novrialdi Ginting and Fatimah
Jumiati Pasaribu) for their motivation and even help to overcome the
difficulties.
8.

Biotechnology 2012 Classmates for their motivation and enthusiasm during
study and research.
9.
My beloved friends (Ratna Sartika, Lestari, Fera Deviyanti Mugni, Imas Siti
Solehah, Astri Gustriana Safe’i, Lia Dewi Sri Yanti, Anita Fatimah, Desti
Taryanti, Niki Nurul Haq, Rima Vera Ningsih and Dini Lestari) for their great
support in study and life at any time; and even lend hand throughout this
research.
Hope the thesis useful.

Bogor, March 2016
Fithriani

TABLE OF CONTENTS
LIST OF TABLES

vi

LIST OF FIGURES

vi

LIST OF APPENDICES

vi

1 INTRODUCTION
Background
Problem Statement
Objective
Significance of Study

1
1
2
3
3

2 LITERATURE REVIEW
Metabolic Engineering of E. coli for Ethanol Production

The fdh Reaction for Regenerating NADH in E. coli Cells
IPTG Induction in Recombinant E. coli Cells

4
4
5
6

3 METHODOLOGY
Place and Time
Bacterial Strains, Plasmids, Media, and Chemicals
Procedure

8
8
8
9

4 RESULT AND DISCUSSION
Effect of pdc and adhB Introduction on Ethanol Production
Role of fdh Reaction and Formate Availability on Ethanol Production
Effect of IPTG-Induction on Ethanol Production in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc Culture

12
12
13

5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation

19
19
19

REFERENCES

19

APPENDIX

23

BIOGRAPHY

29

14

LIST OF TABLES
1 Development of ethanologenic E. coli strains
2 Strains and plasmids used in the study
3 Ethanol production in recombinant E. coli of
BW25113∆pta/pHfdh/pTadhB/pdc after 24 h cultivation
4 The effect of IPTG-induction time on ethanol production, ethanol yield
on glucose, ethanol yield on biomass, and biomass yield on glucose of
E. coli BW25113pta/pHfdh/pTadhB-pdc at 24 h of cultivation
5 Comparison of metabolite yields in ethanologenic E. coli strains
6 Summary of the effect of various IPTG concentrations on ethanol
production, biomass and their yields in recombinant E. coli of
BW25113pta/pHfdh/pTadhB-pdc at 0 h induction after 24 h cultivation

5
9
14

15
17

18

LIST OF FIGURES
1
2
3
4

5

Framework of the study
The stage of study
Relative mRNA expression of pdc and adhB genes in E. coli strains
Time profile of cell growth towards cultivation conditions in the
cultures of E. coli BW25113∆pta/pHfdh/pTadhB-pdc supplemented
with 4 g L-1 formate
Comparison of organic acids accumulation in the cultures of
no IPTG-induction and IPTG-induction at 0 h in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc after 24 h cultivation

3
9
12

15

16

LIST OF APPENDICES
6
7
8
9
10
11

Transformation of E. coli strains
Preculture of E. coli strains
Growth profile of no IPTG-induction culture of E. coli
BW25113∆pta/pHfdh/pTadhB-pdc
Ethanol assay
Glucose assay
Acetaldehyde assay

23
24
25
26
27
29

1 INTRODUCTION
Background
The growing demand of ethanol in replacing fossil fuel has stimulated its
production by fermenting renewable carbon using homoethanologenic biocatalyst,
including Saccharomyces cerevisiae and Zymomonas mobilis. Renewable carbon
such as lignocellulose has been considered as an effective ethanol feedstock due to
the non-competing demands (Hahn-Hagerdal et al. 2006). As lignocellulose
contains significant amount of pentose sugars, their conversion to ethanol are
recalcitrant both for S. cerevisiae and Z. mobilis (Dien et al. 2003). On the other
hand, the most studied and characterized organism Escherichia coli can use all of
sugar constituents of lignocellulose (Dien et al. 2003). With this capability, E. coli
has been considered as an excellent biocatalyst for ethanol production.
E. coli naturally ferments sugars to a mixture of ethanol and organic acids
(Dien et al. 2003). Ethanol is produced from the two step reduction of acetyl-coA
by alcohol dehydrogenase (adhE), oxidizing two NADH to NAD+. This native E.
coli ethanol-producing pathway is insufficient in facilitating high yield due to the
poor efficiency to generate ethanol (Dien et al. 2003). In order to improve ethanol
productivity, metabolic engineered E. coli has been attempted by introducing
pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) genes from Z.
mobilis under the control of lac promoter (Ingram et al. 1987; Ingram and Conway
1988). With further gene deletions and process modification in anaerobic culture,
the ethanol yield could be increased. However, anaerobic fermentation reduces
cellular growth and provokes byproduct accumulation, caused by the slow
regeneration and competition of utilizing NADH through lactate dehydrogenase
(ldh) pathway (Dien et al. 2003). On the other hand, as ethanol is primary metabolite,
its production in microbial cells is made during the exponential phase of growth
whose synthesis is an integral part of the normal growth process (Sanchez and
Demain 2008). That is, a decrement of cellular growth in E. coli cells can be lead
to lower ethanol yield. Therefore, it is essential to improve cell performance to
obtain maximum ethanol production.
For cell performance improvement, it should provide appropriate culture
conditions while maintaining a high level expression of the target product. NADH
has profound role in cellular metabolism as cofactor for redox reaction. The effect
of NADH regeneration on reduced metabolite compounds had been studied
previously (Galkin et al. 1997). Berrios-Rivera et al. (2002) reported that increment
of NADH availability by introducing cofactor-dependent formate dehydrogenase
(fdh) provoked significant change in the final metabolite concentration pattern both
anaerobically and aerobically. Interestingly, an increase in intracellular NADH
under aerobic condition enhanced final cell density and the reduced metabolite
compounds, including ethanol (Berrios-Revira et al. 2002; Suryadarma et al.
2012a). In addition, aerobic culture can represses lactate accumulation (Clark 1989),
while the presence of oxygen triggers acetate formation (Eiteman and Altman 2006).
To suppress acetate accumulation in aerobic growth, Ojima et al. 2012 employed
mutant pta of E. coli under high intracellular NADH. However, applying the
appropriate culture conditions may not result in maximum recombinant product

2
yield. This is limited by non-optimum expression of recombinant genes (Donovan
et al. 1996). For this purpose, it is important to gain an effective expression of
recombinant DNA.
The expression of recombinant genes under control lac-derived promoter
has been utilized isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer
(Donovan et al. 1996). Regardless of the effectiveness of IPTG as gratuitous
inducer, it is expensive and should be consider in low amounts when used in
industrial scale. Commonly, a wide range of IPTG concentration are used without
obvious reason. Interestingly, the induction frequently performed at exponential
stage culture of E. coli due to the higher cell density (Sambrook and Russel 2001).
However, in most cases recombinant DNA expression after induction reduced cell
growth in consequence of metabolic load (Glick 1995; Bentley et al. 2009).
Therefore, it is of interest to investigate the influence that can be achieved on
ethanol production by inducing recombinant E. coli at different of induction times
and IPTG concentrations under aerobic conditions.

Problem Statement
Regulating culture conditions while maintaining a high level expression of
recombinant genes under the control regulatable promoter have been attempted to
enhance the E. coli performance for producing recombinant product (Donovan et
al. 1996), including ethanol. Production of ethanol in E. coli under aerobic
conditions can be achieved by introducing plasmid harboring pdc and adhB (Ingram
et al. 1987), and plasmid harboring fdh following formate supplementation into
cultivation media (Suryadarma et al. 2012a). The reaction of fdh regenerates NADH
from NAD+ by degrading formate to overcome the deficiency of NADH by
respiration system while the mutant pta of E. coli usage and aerobic cultivation
conditions suppressed byproduct accumulations (Ojima et al. 2012; Suryadarma et
al. 2012a). However, expressions of recombinant DNA after induction by IPTG
addition often caused metabolic load and result in the reduction of cell growth
(Bentley et al. 1991; Glick 1995), thereby lowering ethanol yield as target product.
Therefore, strategy for regulating IPTG addition for effective recombinant gene
expression was required to obtain maximal ethanol production. Implementing the
appropriate induction conditions in terms of induction time and IPTG concentration
for effective gene expression of pdc and adhB under the control trc promoter; and
fdh under the control lac promoter is expected to improve ethanol production in
recombinant E. coli culture under aerobic conditions; as can be illustrated in Figure
1.

3

Figure 1 Framework of the study. The illustration showing the strategy for
ethanol production improvement in recombinant E. coli culture by
regulating IPTG induction. (
) up regulated, (
) down
regulated, (1) affected by induction, (2) affected by oxygenation, (3)
affected by mutation, (4) affected by fdh activity. Abbreviations:
PDHc pyruvate dehydrogenase complex, ldh lactate dehydrogenase,
pta phosphotransacetylase, pdc pyruvate decarboxylase, adhB alcohol
dehydrogenase, fdh formate dehydrogenase

Objective
The aim of this study is to investigate the effect of IPTG induction in terms
of induction time and IPTG concentration in recombinant E. coli culture under
aerobic conditions.

Significance of Study
This study will assist to gain the information regarding the strategy of IPTG
addition in recombinant E. coli culture. Thus, assist towards the ethanol production
improvement in recombinant E. coli culture under aerobic conditions.

4

2 LITERATURE REVIEW
Metabolic Engineering of E. coli for Ethanol Production
Current development in industrial ethanol production requires biocatalyst that
produce ethanol in high yield and capable in utilizing lignocellulose as a feed stock
(Dien et al. 2003). Ethanol-producing strains such as S. cerevisiae and Z. mobilis
are limited to growth on hexoses (Hahn-Hagerdal et al. 2006). On the other hand,
E. coli can use all of sugar constituents of lignocellulose. With several advantages
including no requirements for complex growth factor, prior industrial use, its wellcharacterized genetic, and its well-studied physiological regulation (Dien et al.
2003; Atsumi et al. 2008); the facultative anaerobic E. coli is considered as an
effective biocatalyst for ethanol production.
Ethanol is primary metabolite, which is one of the mixed-acid fermentation
end products of E. coli. It is produced in wild type cells of E. coli from the two step
reduction of acetyl-coA by alcohol dehydrogenase (adhE), oxidizing two NADH
to NAD+. Meanwhile, glycolysis generates only one NADH per pyruvate. As a
consequence, the native E. coli pathway for ethanol results in an NADH deficit.
This is overcome by converting one of the acetyl-coA to acetate, resulting in equal
amounts of acetate and ethanol produced by E. coli. Due to the requirement two
NADH per ethanol, the native ethanologenic pathway prohibits E. coli from
conducting S. cerevisiae or Z. mobilis like-homoethanol fermentation, which
generated ethanol as the sole product and only consumes one NADH per ethanol
produced (Dien et al. 2003).
To improve ethanol production in E. coli cells, metabolic engineering by
expressing homoethanol pathway has been developed. The development of
ethanologenic E. coli strain by different researchers is shown in Table 1. It has been
widely reported that metabolically engineered E. coli expressing Z. mobilis genes
encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) under
control lac promoter is efficiently produced ethanol from pyruvate (Ingram and
Conway 1988) because Z. mobilis Km for pdc is quite low compared to pyruvateconsuming reactions (Peterson and Ingram 2008). Chromosomal integration those
genes into E. coli genome under the control pyruvate formate lyase (pfl) promoter
was further conducted to increased genetic stability of the recombinant strain (Ohta
et al. 1991). However, lower gene dosage than plasmid-bearing strain resulted in
low yield of ethanol, with high contaminations by lactate and acetate. Further, the
mutation in competing-pathways including the primary acetate pathway (pta,
phosphotransacetylase; ackA, acetate kinase) and lactate producing-pathway (ldh,
lactate dehydrogenase) were attempted to improve ethanol production in E. coli
(Trinh et al. 2008).

5

Table 1 Development of ethanologenic E. coli strains
Strain

Modified
propertya

Media, substrate, Time Ethanol
and process
(h)
yield
conditions
(g
ethanol
g-1
glucose)

Reference

TC4

Plasmid
pLOI308-2
containing
pdcZm and
adhBZm
Plasmid
pLO1295
containing
pdcZm and
adhBZm
pfl+pfl:(pdc+
adhB+ Cmr)

LB medium,
glucose 50 g L-1,
batch
fermentation

24

0.02

Ingram
and
Conway
1988

LB medium,
glucose 50 g L-1,
batch
fermentation

24

0.08

Ingram
and
Conway
1988

LB medium,
72
glucose 100 g L-1,
batch
fermentaton
LB medium,
72
glucose 80 g L-1,
batch bioreactor
fermentaton

0.13

Ohta et al.
1991

0.48

Trinh et
al. 2008

TC4

KO3

TCS083

a

∆zwf ∆ndh
∆scfA ∆maeB
∆poxB ∆ldhA
∆frdA
∆pta:Kan−,
carrying
plasmid with
pdcZm and
adhBZm

pdcZm, pdc from Z. mobilis; adhBZm, adhB from Z. mobilis; Cmr, chloramphenicol resistance.

The fdh Reaction for Regenerating NADH in E. coli Cells
NADH plays an essential role in cellular metabolism by functioning as a
cofactor for redox reactions, particularly in dehydrogenase-catalyzed reactions
involved in respiration (Chenault et al. 1988). Due to its function, the availability
of this reducing agent determines the metabolic fluxes of many pathways as well as
the transcriptional level of many genes. For example, the allosteric inhibition of
accumulated NADH on citrate synthase (Weitzman 1966), the inhibition of the high
NADH/NAD+ ratio on pyruvate dehydrogenase complex (PDHc) reaction (de
Graef et al. 1999), the induction of adhE expression in E. coli by NADH availability

6
(Leonardo et al. 1993), and the triggering factor of acetate overflow by high
NADH/NAD+ ratio (Vemuri et al. 2006).
As NADH influences the bioacatalysis of cofactor-dependent enzyme system,
deficiency of this reducing agent limits the generation of the reduced metabolites
including ethanol. Therefore, an increment NADH availability is believed to be
useful for improving ethanol production. The most effective strategy to increase
NADH availability utilizes formate dehydrogenase (fdh) to catalyzes NADH
regeneration from NAD+ through degradation of formate into CO2 (Chenault et al.
1988). The advantages of fdh reaction include the use of formate as an inexpensive,
stable, innocuous substrate; and the generation of CO2, which can be easily removed
from the reaction (Chenault et al. 1988).
It has been reported that level of NADH achieved by fdh reaction induces the
production of cofactor-dependent product (Galkin et al. 1997; Berrios-Rivera et al.
2012; Suryadarma et al. 2012a). Since formate is required as a substrate by fdh to
regenerates NADH, its concentration should be maintain as high as possible so that
this substrate does not become limiting for fdh reaction. However, high
concentration of formate may be inhibitory to growth of E. coli due to its toxic
effect (Ostling and Lindgren 1993). Therefore, appropriate concentration of formate
added in E. coli culture is required to facilitate fdh for regenerating NADH.
Previous study reported that different formate concentrations across the range 0−9
g L-1 provided different levels of NADH (Berrios-Rivera et al. 2012), while
concentration at 4−10 g L-1 formate was effective in elongating NADH availability
by fdh reaction in E. coli cells to generate the reduced metabolite (Suryadarma et
al. 2012a).

IPTG Induction in Recombinant E. coli Cells
The lac promoter is widely used as the common system for recombinant
protein production in E. coli and it is one of the well-known regulation mechanism.
Various promoters were constructed from lac-derived regulatory element (Polisky
et al. 1976), including the synthetic trc promoter (Brosius et al. 1985). Both lac and
trc promoters are regulatable promoter systems that provide the ability to activate
the expression of foreign genes by varying an environmental factor such as the
concentration of a particular component in the growth medium (Donovan et al.
1996), whereas a drawback to these promoters is the basal level of transcription
(Donovan et al. 1996; Terpe 2006).
Induction of the lac and trc promoters for expressing foreign genes could be
achieved by adding chemical inducers such as non-hydrolysable lactose analog
isopropyl-β-D-1-thiolgalactopyranoside (IPTG). This inducer binds the lac
repressor, the product of lacI gene, and consequently releases the operator allowing
DNA transcription (Donovan et al. 1996). Since IPTG is metabolic free, the level
of induction remains constant, thereby making IPTG the most utilized in inducing
foreign genes expression for protein production.
Expression of DNA recombinant using IPTG is a common procedure to
improve the yield of target product. IPTG induction frequently performed at
exponential phase because the culture is growing fast and protein translation is
maximal (Sambrook and Russel 2001), while induction throughout the entire

7
growth phase is also possible to induce the expression of foreign genes (Ramirez et
al. 1994). Additionally, IPTG is often added at 1000 µM to fully induce lac-derived
promoter, while a wide range of IPTG concentrations from 0 to 10000 µM can be
used to induce gene expression (Wood and Peretti 1991; Bentley et al. 1991;
Donovan et al. 1996). However, recombinant DNA expression after induction
generally causes a metabolic load on host cell which can result in reduced cellular
growth, cell yields, product expression, and plasmid stability (Bentley et al. 1991;
Glick 1995). In order to balance the decreasing cell yield after induction, and
increasing recombinant expression by the cells; the optimal induction time in the
cultivation for inducing expression as well as the optimal concentration of IPTG
that needs to be used in the process must be examined (Donovan et al. 1996).
d in the

determined.

8

3 METHODOLOGY
Place and Time
This study was conducted at Biotechnology Research Indonesia-The
Netherlands (BIORIN), Research Center of Bio-Resources and Biotechnology; and
Bioindustry Laboratory, Department of Agroindustrial Technology, Bogor
Agricultural University during November 2014 to December 2015.

Bacterial Strains, Plasmids, Media, and Chemicals
E. coli strains and plasmids used in this study are listed in Table 2. E. coli
BW25113 and its pta deficient (BW25113∆pta) (Baba et al. 2006) were obtained
from National BioResource Project of Japan and used as the host strains. The pHfdh
plasmid bearing fdh gene from Mycobacterium vaccae was obtained from previous
study (Ojima et al. 2012). Plasmid bearing adhB and pdc genes from Z. mobilis was
constructed by cloning these genes into pTrc99A vector, denoted as pTadhB-pdc.
These plasmid, pHfdh and/or pTadhB-pdc were further transformed into BW25113
and BW25113∆pta strains.
Luria broth (LB) medium contained 10 g L-1 peptone, 10 g L-1 NaCl and 5 g
-1
L yeast extract was used for preculture medium (Miller 1992). Solutions were
filtered sterilized using a 0.22 µm filter from Sartorius. Stock solutions of
kanamycin (30 mg mL-1, Sigma) were prepared in Milli-Q water, filter sterilized
and stored at -20 °C. Stock solutions of chloramphenicol (34 mg mL-1, Sigma) were
prepared in absolute ethanol, filter sterilized and stored at -20 °C. Stock solutions
of carbenicillin (50 mg mL-1, Sigma) were prepared in Milli-Q water, filter
sterilized and stored at -20 °C. Stock solutions of formate (200 g L-1) were prepared
by dissolving 30.22 g sodium formate (Merck) into 100 mL bidest water, filter
sterilized and stored at 25 °C. IPTG was purchased from Sigma-Aldrich and the
stock solutions were prepared by dissolving 2.38 g into 10 mL Milli-Q water and
filter-sterilized to obtain of 1 M stock solutions and stored at -20 °C.
The cultivation medium was prepared in glucose-enriched LB medium as
described by Suryadarma et al. 2012b. The cultivation medium contained 10 g L-1
peptone, 10 g L-1 NaCl, 5 g L-1 yeast extract, 40 g L-1 glucose, and 20 g L-1 CaCO3.
When necessary, 4 g L-1 formate and IPTG (as described in the procedure section)
were added to the cultivation medium. All medium were adjusted to pH 7 using
aqueous NaOH. Carbenicillin (50 mg L-1), chloramphenicol (34 mg L-1), and
kanamycin (30 mg L-1) were added as appropriate.

9
Table 2 Strains and plasmids used in the study
Name
E. coli strains
BW25113
BW25113∆pta
Plasmids
pHfdh
pTadhB-pdc
a

Descriptiona

Reference

parent strain, lacIQ
[BW25113]∆pta, lacIQ, Knr

Baba et al. 2006
Baba et al. 2006

lacP, Cmr, containing fdh from
M. vaccae
Q
trcP, lacI , Apr, containing pdc
and adhB from Z. mobilis

Ojima et al. 2012
This study

Knr, kanamycin resistance; Cmr, chloramphenicol resistance; Apr, ampicillin resistance.

Procedure
This study was consisted of three experimental stages: effect of pdc and adhB
introduction on ethanol production, role of fdh reaction and formate availability on
ethanol production, and effect of IPTG-induction on ethanol production in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc culture. The stage of this study is presented in
Figure 2.
Improvement of Ethanol Production by Inducer Addition in
Recombinant Escherichia coli Culture under Aerobic Conditions

1. Effect of pdc and adhB Introduction on Ethanol
Production
2. Role of fdh Reaction and Formate Availability on
Ethanol Production
3. Effect of IPTG-Induction on Ethanol Production in
E. coli BW25113∆pta/pHfdh/pTadhB-pdc Culture

Figure 2 The stage of study

10
Effect of pdc and adhB Introduction on Ethanol Production
To investigate the effect of introduction pdc and adhB, plasmid pTadhB-pdc
was introduced into E. coli BW25113 cells by standard genetic method (Sambrook
and Russel 2001) as described in Appendix 1. Then followed by preparing the
preculture for recombinant strain BW25113/pTadhB-pdc and parent strain
BW25113 (Appendix 2). A 5% (v/v) preculture with OD660 nm = 1.0−1.5 was
transferred into 50 mL cultivation medium (in 250 mL baffled flask, Sigma) and
IPTG at concentration 1000 µM was added to recombinant culture. The cultures
then were grown aerobically in an incubator shaker (Ivymen System) at 37 °C under
200 rpm agitation. After 18 h of cultivation, the cultures were harvested and then
analyzed for gene expression of pdc and adhB; and ethanol production.
Role of fdh Reaction and Formate Availability on Ethanol Production
Plasmid pHfdh and pTadhB-pdc were introduced into BW25113∆pta strain.
The recombinant strain of BW25113∆pta/pHfdh/pTadhB-pdc was then precultured.
After OD660 preculture reached 1.0−1.5, a 5% (v/v) of preculture was transferred
into cultivation media. The role of fdh and formate availability were studied by
culturing
recombinant
strain
BW25113∆pta/pHfdh/pTadhB-pdc
with
-1
supplementation of 4 g L formate into cultivation medium (Suryadarma et al.
2012a). For comparison, recombinant E. coli of BW25113∆pta/pHfdh/pTadhB-pdc
without formate supplementation was also cultured. The cultures were incubated at
37 °C and 250 rpm agitation. Ethanol production then was analyzed after 24 h
cultivation.
The Effect of IPTG-Induction on Ethanol Production in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc Culture
The effect of IPTG induction was investigated by analysing the influence of
induction time and IPTG concentration on ethanol production. The influence of
induction time was performed by preparing the preculture of
BW25113∆pta/pHfdh/pTadhB-pdc until OD660 reached 1.0−1.5. A 5% (v/v) of
preculture then was transferred into 50 mL cultivation media (in 250 mL baffled
flask, Sigma) with 4 g L-1 formate supplementation and incubated at 37 °C and 250
rpm agitation for 24 h. The cultures were induced using 500 µM IPTG and added
at 0, 6, 12, and 18 h of cultivation represent different induction phases namely lag,
exponential, end of exponential and stationary (Appendix 3). The induction phases
were examined by monitoring growth profile of non-induced recombinant E. coli
BW25113∆pta/pHfdh/pTadhB-pdc with 4 g L-1 formate addition. During
cultivation, growth profile was analyzed at 6 h interval of cultivation time. Ethanol
and organic acids were measured in the cultures after 24 h cultivation.
The optimal time of induction was used to analyse the effect of IPTG
concentration. In order to investigate the effect of inducer concentration, a 5% (v/v)
of preculture BW25113∆pta/pHfdh/pTadhB-pdc was transferred into 250 mL
baffled flask (Sigma) contained 50 mL cultivation media and supplemented by 4 g
L-1 formate. The cultures then were induced by addition of various IPTG
concentrations in the range 50-2000 µM (Terpe et al. 2006) at 0 h of cultivation,
and incubated at 37 °C and 250 rpm agitation for 24 h. Biomass and ethanol
concentrations were determined in the cultures after 24 h cultivation.

11
Analysis
Growth of E. coli was monitored by optical density measurement at a
wavelength of 660 nm using a spectrophotometer (Hewlett Packard 8453). Samples
were diluted with 1 N HCl to dissolve CaCO3, until the final OD660 value was within
the range 0.2−0.8. Biomass was represented as dry cell weight (DCW), in which 1
OD660 is equivalent to 0.36 g L-1 (Ojima et al. 2012).
Gene expression analysis for determination activity of pdc and adhB was
performed by real time PCR as described previously (Suryadarma et al. 2012b).
For determination of glucose, acetaldehyde and ethanol in the culture, the
samples were heated at 80 °C for 15 min and followed by centrifugation (Hettich
Zentrifugen, Micro 22R) at 10000 rpm for 5 min. The supernatant was filtered
sterilized; and glucose, acetaldehyde, ethanol were determined by enzymatic
method using D-Glucose 716251 Kit, Acetaldehyde 668613Kit and Ethanol 176290
Kit, respectively (Roche, Germany) with slight modification (Appendix 4,5,6) .
Concentration of organic acids (pyruvate, lactate, acetate, and formate) in the
cultures were quantified by HPLC system (Hewlett Packard, 1100 Series) with
mobile phase containing 99% 20 mM Na2HPO4 and 1% acetonitrile. The samples
were prepared by centrifugation at 10000 rpm for 5 min, followed by filtration of
supernatant using 0.22 µm syringe filter (Sartorius). The samples then diluted by
bidest water and injected into the column (Zorbax SB-Aq 883975-914) to separate
the compounds. The analysis were performed at 35 °C, 1.0 mL min-1 flow rate and
detected at 210 nm.
Metabolite yields on glucose basis (Ymetabolite/glucose) were determined by the
formula:
mass of metabolite
Ymetabolite/glucose (g of metabolite/g of glucose) =
mass of glucose
where mass of metabolite and mass of glucose represent the metabolite production
or appeared in the culture; and glucose consumption, respectively. The percent of
theoretical yield of ethanol was calculated by the following equation:
actual yield
x 100%
Theoretical yield of ethanol (%) =
theoretical yield
where
mole of ethanol produced
Actual yield =
mole of glucose consumed
Ethanol yield on biomass basis (Yethanol/biomass) was determined by the formula:
mass of ethanol
Yethanol/biomass (g of ethanol/g of biomass) =
mass of biomass
where mass of ethanol and mass biomass represent the ethanol and biomass
production or appeared in the culture.
The data were represented as averages with standard deviations, obtained
from two or three independent cultures and analysed using curveExpert
professional 2.20 and Microsoft Excel 2013. The statistical analysis were assessed
using t test to estimate the effect of each variable, with all results giving P < 0.05
being considered statistically significant.

12

4 RESULT AND DISCUSSION
Effect of pdc and adhB Introduction on Ethanol Production

Relative mRNA expression (105)

A major challenge in ethanol production by E. coli fermentation is the low
yield of ethanol as end product. Although the engineered of E. coli has been shown
to produce ethanol efficiently (Ingram and Conway 1988), the ethanol yield is
usually held back by the accumulation of byproduct.
In this study, improvement of ethanol production was first investigated by
introducing plasmid pTadhB-pdc carrying the Z. mobilis genes encoding pdc and
adhB under the control trc promoter into competent E. coli BW25113. The trc
promoter was chosen to control those Z. mobilis genes since the level of expression
under control this promoter system is moderately high (Terpe et al. 2006). The
recombinant strain BW25113/pTadhB-pdc and its parent strain BW25113 then
were grown aerobically in order to suppress the generation of lactate (Suryadarma
et al. 2012a) during ethanol production. After 18 h, mRNA expression of pdc and
adhB were determined.
Result presented in Figure 3 shows that the expressions of pdc and adhB were
found in BW25113/pTadhB-pdc, whereas expression of those genes were not
detectable in parent strain. This revealed that ethanologenic pathway from Z.
mobilis were successfully expressed in E. colI cells BW25113/pTadhB-pdc.
Expression of pdc and adhB in recombinant E. coli were expected to divert the
carbon flux from pyruvate into ethanol efficiently and hence can be improved
ethanol production (Ingram et al. 1987). To evaluate the effect of introduction those
recombinant genes on ethanol production, determination of ethanol concentration
in BW25113/pTadhB-pdc and BW25113 cultures were conducted. As expected, the
recombinant E. coli expressing pdc and adhB was observed two fold greater of
ethanol than parent strain (0.2 g L-1 ethanol). This indicates that the expressions of
pdc and adhB induce ethanol production in E. coli BW25113/pTadhB-pdc under
pdc

40

adhB
30

Nd: Not detected

20

10

0

Nd

Nd

BW25113

BW25113/pTadhB-pdc

Figure 3 Relative mRNA expression of pdc and adhB genes
in E. coli strains

13
aerobic condition. This obtained result also supports previous study that an elevated
level of ethanol in recombinant E. coli culture is the results of relatively high level
expression of pdc and adhB (Ingram and Conway 1988). However, although
ethanol production was increased by pdc and adhB introduction; the concentration
that achieved was still low and needs further improvement.

Role of fdh Reaction and Formate Availability on Ethanol Production
During the aerobic catabolism of glucose by E. coli, acetate production has
been associated with a retardation of growth (Eiteman and Altman 2006); while
NADH levels remain relatively low since oxygen is used as terminal electron
acceptor (Causey et al. 2004). These conditions may lead to low production of the
reduced metabolite compounds such as ethanol. Therefore, considerable efforts
have been attempted to minimize acetate production, and increase NADH
availability as a means of improving the production of reduced metabolite (Galkin
et al. 1995; Ojima et al; Suryadarma et al. 2012a). This can be achieved by
regenerating intracellular NADH in a primary acetate-producing pathway
(phosphotransacetylase, pta) mutant of E. coli (Ojima et al. 2012; Suryadarma et
al. 2012a).
In this study, to further improve ethanol production, pHfdh containing fdh
was introduced into E. coli BW25113∆pta/pTadhB-pdc and cultivated with 4 g L-1
formate supplementation. It was reported that fdh reaction contributes in increasing
intracellular NADH (Galkin et al. 1995), while formate concentration at 4 g L-1
was chosen since it is effective to support fdh reaction in E. coli cells (Suryadarma
et al. 2012a). The fdh reaction provides NADH from NAD+ for cofactor-dependent
production system by catalyzing the oxidation of formate into CO2 (Chenault et al.
1988; Galkin et al. 1995). Furthermore, these reports suggest that an increment of
NADH availability may facilitates production of the reduced metabolite compounds
from pyruvate, including ethanol.
Table
3
compares
ethanol
production
of
E.
coli
BW25113∆pta/pHfdh/pTadhB-pdc cultures with formate supplementation and the
control (without formate). In the case of the culture following formate
supplementation, ethanol production was observed 1.10 g L-1 and significantly
higher than that achieved from the control culture. This result indicates that the
addition of formate into culture could be degraded to regenerate NADH (BerriosRivera et al. 2002; Suryadarma et al. 2012a), resulting that the increased NADH
availability induces ethanol production under aerobic condition. Therefore, it
appears that fdh reaction following formate supplementation into recombinant E.
coli culture contributes to ethanol production improvement. The obtained ethanol
concentrations in these recombinant cultures with and without formate
supplementation, however, were the result of basal level transcription the lac and
trc promoters; since no IPTG were added into these cultures. The basal level
transcription of lac-derived promoter could be ocurred because the operator site is
not continously occupied by lac repressor (Donovan et al. 1996). Hence, ethanol
production by E. coli BW25113∆pta/pHfdh/pTadhB-pdc can be further improve in
this study by addition the inducing stimulus IPTG.

14
Table 3 Ethanol production in recombinant E. coli of BW25113∆pta/pTadhB-pdc
after 24 h cultivationa
E. coli strains

Formate added
(g L-1)

Ethanol
(g L-1)b

BW25113pta/pHfdh/pTadhB-pdc

0

0.13a

BW25113pta/ pHfdh/pTadhB-pdc

4

1.10 ± 0.12b

The data were obtained from three independent experiments.; bValues in a column followed by
different letters were statistically significantly different with P < 0.05.
a

Effect of IPTG-Induction on Ethanol Production in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc Culture
IPTG plays an important role in initiating expression of recombinant DNA
under the control of lac-derived promoter. This chemical has been considered as an
effective inducer because it is not metabolized by cells and the level of induction is
remain constant (Donovan et al. 1996). Meanwhile, recombinant DNA expression
after induction generally lead to reduce cellular growth (Glick 1995), and thus
consequence in lowering yield of target product (Donovan et al. 1996). Thus,
appropriate induction condition is required to obtain maximum product yield
(Bentley et al. 1991).
In order to gain the appropriate induction condition for maximum
recombinant protein production, the optimal time of inducer added into the culture
should be determined. The optimal induction time would depend on the response
of strains during the induction phase (Donovan et al. 1996). Generally, induction
of recombinant DNA in E. coli is performed at exponential phase due to E. coli
cells’ optimum physiology (Sambrook and Russel 2001). But later it was reported
that rRNA was degraded upon induction at high specific growth rate (Sanden et al.
2003). Performing induction at stationary phase reduced cell viability (Duan et al.
2000). Besides, early induction may lengthen the lag phase. However, in contrast,
induction throughout the entire growth phase was reported could be possible for
recombinant protein production (Ramirez et al. 1994).
In this study, the effect of induction time in E. coli
BW25113∆pta/pHfdh/pTadhB-pdc was investigated by addition 500 µM IPTG at
0, 6, 12 and 18 h of cultivation time represents growth phase of lag, exponential,
end of exponential and stationary, respectively. The obtained result from those
cultures for ethanol production, ethanol yield on glucose (Yethanol/glucose), ethanol
yield on biomass (Yethanol/biomass) and biomass yield on glucose (Ybiomass/glucose) are
shown in Table 4. In average, ethanol production in the cultures with IPTG addition
at different induction times were found significantly higher than without induction.
These results clearly demonstrate that the addition of IPTG allowed the regulatable
promoter to activate the expression of recombinant genes (Donovan et al. 1996),
including fdh, pdc, and adhB, which in turn induces ethanol production. When a
regulatable promoter is used for foreign expression, the common practice is to
promote high cell growth first without induction, then followed by induction step

15
to obtain high yield product (Sambrook and Russel 2001). In contrast, early
induction (IPTG addition at 0 h) was observed the highest ethanol concentration at
4.09 g L-1 in this study, suggesting IPTG-induction at 0 h of cultivation time
effectively improves ethanol production among the other induction times. To
explain
these
observations,
growth
profiles
of
E.
coli
BW25113∆pta/pHfdh/pTadhB-pdc in those cultures were monitored as shown in
Figure 4. Biomass concentrations after 24 h of cultivation were higher in all IPTGTable 4 The effect of IPTG-induction time on ethanol production, ethanol yield on
glucose, ethanol yield on biomass, and biomass yield on glucose of E. coli
BW25113pta/pHfdh/pTadhB-pdc at 24 h of cultivationa
Time of
IPTGinduction

Ethanol
(g L-1)b

Yethanol/glucose
(g ethanol
g glucose-1)b

Yethanol/biomass
(g ethanol
g biomass-1)b

Ybiomass/glucose
(g biomass
g glucose-1)b

No induction

1.10 ± 0.12a

0.04 ± 0.01a

0.78 ± 0.07a

0.05a

0h

4.09 ± 0.23b

0.14 ± 0.01b

2.17 ± 0.18b

0.06b

6h

3.42 ± 0.31c

0.18 ± 0.02c

1.96 ± 0.23b

0.09 ± 0.01c

12 h

1.95 ± 0.11d

0.10d

1.43 ± 0.01c

0.07c

18 h

2.50 ± 0.31d

0.14 ± 0.02c

1.87 ± 0.33b

0.08c

a

The data were obtained from three independent experiments. Acetaldehyde concentrations are less
than 0.01 g L-1 and their yield on glucose were not calculated. ; bValues in a column followed by
different letters were statistically significantly different with P < 0.05.

No IPTG-induction
IPTG-induction at 0 h
IPTG-induction at 6 h
IPTG-induction at 12 h
IPTG-induction at 18 h

DCW (g L-1)

2

1

0
0

6

12
Cultivation time (h)

18

24

Figure 4 Time profile of cell growth towards cultivation conditions in
the cultures of E. coli BW25113∆pta/pHfdh/pTadhB-pdc
supplemented with 4 g L-1 formate

16
induction cultures than in the control (no IPTG-induction). The highest biomass
production was observed in the culture that induced at 0 h, followed respectively
by IPTG-induction at 6, 12, and 18 h of cultivation time. These observations
indicate that IPTG induction time influenced the growth of
BW25113∆pta/pHfdh/pTadhB-pdc in association with ethanol production as
primary metabolite. This conclusion was further supported by yields calculation
from recombinant cultures (Table 4). The results showed that IPTG-induction at 0
h yielded the highest Yethanol/biomass, while the values for Yethanol/glucose and
Ybiomass/glucose were relatively lower among induction times strategy. This increase
in Yethanol/biomass is consistent with an increased in ethanol production (Table 4),
suggesting that the higher in biomass synthesis contributes to higher ethanol
concentration.
Studies in recombinant genes expression under control of inducible promoter
reported that IPTG addition had growth inhibiting effect in the host cell of E. coli
(Donovan et al. 1996). However, the obtained results here demonstrate that IPTG
addition makes a significant contribution to the growth of recombinant strain. This
phenomena might has been due to the influence of fdh reaction by IPTG-induction.
With the aim of evaluating the influence of fdh in E. coli growth which is correlated
with ethanol production; the accumulation of organic acids at 24 h of cultivation
time were compared in the cultures of no IPTG-induction and IPTG-induction at 0
h (Figure 5). As can be seen, accumulation of pyruvate, lactate, and acetate for
IPTG-induction at 0 h were similar to no IPTG-induction. Meanwhile, the residual
formate concentration in the culture is considerably lower for IPTG-induction at 0
than for no induction. These results suggest that activity of fdh reaction effectively
regenerates NADH from the degradation of formate and assist in ethanol production
improvement when IPTG was added at 0 h of cultivation time. Since the
degradation of formate released the recombinant cells from cytotoxicity (Ojima e