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UNIVERSITI PUTRA MALAYSIA
METABOLIC REGULATION ANALYSIS OF RECOMBINANT Lactococcus lactis BASED ON GENE EXPRESSION AND ENZYME ACTIVITY
FARZANEH HEIDARNIA
FBSB 2011 11
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METABOLIC REGULATION ANALYSIS OF RECOMBINANT Lactococcus lactis BASED ON GENE EXPRESSION AND ENZYME ACTIVITY
By
FARZANEH HEIDARNIA
Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of Requirement for the Degree of Master of Science
July 2011
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Dedicated to my parents: Mohammad Ali and Ashraf
For their endless supports
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Abstract of thesis presented to the Senate of the Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
METABOLIC REGULATION ANALYSIS OF RECOMBINANT Lactococcus lactis BASED ON GENE EXPRESSION AND ENZYME ACTIVITY
By
FARZANEH HEIDARNIA
July 2011
Chairman: Nor’Aini Abdul Rahman, PhD
Faculty: Biotechnology and Biomolecular Sciences
Lactic acid bacteria (LAB) are industrially important microorganisms that are widely
used in industrial food fermentations for dairy production. However, there is a
growing interest in their application in genetic modification and biotechnology
processes. Lactococcus lactis is a non-pathogenic bacterium whose genome has been
completely sequenced and its metabolic pathways are well studied. These reasons
make L. lactis an attractive target for those approaches including creating live vector
vaccine.
The present study was conducted to evaluate the effect of aerolysin on the metabolic
regulation and fermentation characteristics of the recombinant L. lactis. Both L.
lactis NZ9000 and Recombinant L. lactis carrying D1 of aerolysin gene (Lac-D1ae)
were cultivated in M17 medium supplemented with 0.5% (w/v) glucose incubated at
30°C with an agitation of 150 rpm. Chloramphenichol (7.5 µg/mL) was added to
maintain the plasmid. Samples for gene expression and enzyme activity assays were
taken during late-exponential growth phase.
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The alteration of expression of 10 genes (glk, pfk, pyk, ackA, mdh, ldh, pgi, zwf, gnd)
responsible for enzymes at the main metabolic pathways i.e. glycolysis, Triarboxylic
Acid (TCA) cycle, fermentation and pentose phosphate pathway (PP pathway) were
examined by using semi-quantitative RT-PCR and Real-Time PCR. The activity of
these enzymes including glucokinase (GLK), phosphofructokinase (PFK), pyruvate
kinase (PYK), acetate kinase (ACK), Malate dehydrogenase (MDH), Lactate
dehydrogenase (LDH), Phosphoglucose isomerase (PGI), Glucose-6-phosphate
dehydrogenase (G6PDH) and 6-Phosphogluconate dehydrogenase (6PGDH) were
also measured to understand the metabolic regulation in Lac-D1ae.
According to the fermentation results obtained, cell growth, lactate production rate
and acetate production rate in the Lac-D1ae were 0.77 g/L, 0.82 mg/L/h and 0.16
mg/L/h, respectively. The values were slightly lower compared to the parental strain
(0.8 g/L, 0.84 mg/L/h and 0.18 mg/L/h). Glucose consumption rate also showed a
considerable decrease in the recombinant strain (3.48 g/L/h) in comparison with
L.lactis NZ9000 (4.27 g/L/h). HPLC results showed the production of lactate (8.20
g/L) and acetate (1.58 g/L) were reduced in Lac-D1ae in contrast with parental strain
(8.35 g/L and 1.83 g/L).
In conclusion, the fermentation characteristics of the recombinant L. lactis showed
that the presence of aerolysin gene has no inhibitory effect on the growth.
Furthermore, integrating methods based on gene expression and enzyme activities
showed up-regulation of glycolysis and TCA cycle and down-regulation of Pentose
Phosphate pathway in the recombinant L. lactis carrying aerolysin gene (Lac-D1ae).
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
ANALISIS PENGAWALAN METABOLIK REKOMBINAN Lactococcus
lactis BERDASARKAN KEPADA EKSPRESI GEN DAN AKTIVITI ENZIM
Oleh
FARZANEH HEIDARNIA
Julai 2011
Pengerusi: Nor ‘Aini Abdul Rahman, PhD
Fakulti: Bioteknologi dan Sains Biomolekul
Bakteria asid laktik (LAB) merupakan mikroorganisma yang penting dalam industri
dimana ia digunakan secara meluas dalam industri pemakanan untuk produk tenusu.
Disamping itu pada masa kini aplikasi modifikasi gen dan proses bioteknologi
terhadap bakteria ini semakin meningkat. Lactococcus lactis merupakan bakteria
bukan patogen dimana genomnya telah dijujukkan sepenuhnya serta tapak jalannya
telah banyak dikaji. Oleh itu L.lactis menjadi target yang menarik melalui
pendekatan tersebut seperti menghasilkan vektor vaksin hidup.
Kajian ini dijalankan untuk menilai kesan aerolysin terhadap pengawalan metabolik
dan kriteria fermentasi rekombinan L.lactis. Kedua-dua L.lactis NZ9000 (strain asal)
and rekombinan L.lactis pembawa gen aerolysin D1 (Lac-D1ae) telah ditumbuhkan
dalam medium M17 yang dibekalkan dengan glukosa 0.5% (w/v) dan diinkubasi
pada 30°C dengan goncangan pada 150 rpm. Chloramphenicol (7.5 ug/mL) telah
ditambah untuk mengekalkan plasmid. Sampel untuk ekspresi gen dan asai aktiviti
enzim telah diambil semasa akhir fasa eksponen.
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Perubahan ekspresi 10 gen (glk, pfk, pyk, ackA, mdh, ldh, pgi, zwf, gnd) yang
bertanggungjawab untuk enzim yang terdapat pada tapak jalan utama, contohnya
Glikolisis, Kitaran TriKarboksilik, Fermentasi, dan Tapak Jalan Pentos Posfat telah
ditentukan menggunakan semi-kuantitatif RT-PCR dan Real-Time PCR. Aktiviti
untuk enzim-enzim tersebut termasuk glucokinase (GLK), phosphofructokinase
(PFK), pyruvate kinase (PYK), acetate kinase (ACK), Malate dehydrogenase
(MDH), Lactate dehydrogenase (LDH), Phosphoglucose isomerase (PGI), Glucose-
6-phosphate dehydrogenase (G6PDH) and 6-Phosphogluconate dehydrogenase
(6PGDH) juga diukur untuk memahami pengawalan metabolik dalam Lac-D1ae.
Berdasarkan kepada keputusan fermentasi pertumbuhan sel, kadar penghasilan asid
laktik, dan asid asetik oleh Lac-D1ae masing-masing adalah 0.17 g/L, 0.82 mg/L/h
dan 0.16 mg/L/h. Nilai-nilai tersebut adalah kurang sedikit berbanding dengan strain
asal (0.8 g/L, 0.84 mg/L/h dan 0.18 mg/L/h). Kadar penggunaan glukosa
menunjukkan pengurangan yang ketara pada strain rekombinan (3.48 g/L/h)
berbanding dengan strain asal L.lactis NZ9000 (4.27 g/L/h). Keputusan HPLC
menunjukkan penghasilan asid laktik (8.20 g/L) dan asid asetik (1.58 g/L) adalah
kurang dalam Lac-D1ae dibandingkan dengan strain asal (8.35 g/L dan 1.83 g/L).
Secara umumnya kriteria fermentasi Lac-D1ae menunjukkan kehadiran aerolysin
tidak merencatkan pertumbuhan strain tersebut.
Sebagai kesimpulan, kriteria fermentasi rekombinan L.lactis menunjukkan kehadiran
gen aerolysin tidak merencatkan pertumbuhan L.lactis. Melalui kaedah integrasi
berasaskan ekspresi gen dan aktiviti enzim, pengawalan metabolik rekombinan
L.lactis pembawa gen aerolysin (Lac-D1ae) menunjukkan peningkatan pengawalan
Glikolisis dan Kitaran TCA dan penurunan pengawalan tapak jalan Pentos Posfat.
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ACKNOWLEDGEMENTS
Firstly, my praise to Allah for giving me the strength and wisdom to fulfill this
challenging task. I would like to express my sincere appreciation and gratitude to my
supervisor Dr. Nor’Aini Abdul Rahman under whose guidance and supervision
had provided me the opportunity and easily conducive environment to complete this
study. Her invaluable constructive criticisms and continuous supports had built in me
the confidence to undertake the laboratory investigations with patience and optimism
throughout the course of my study.
Special thanks go to the members of my supervisory committee Prof. Dr. Raha
Abdul Rahim, and Assoc. Prof. Dr. Rosfarizan Mohamad for their suggestions
and useful deliberative discussion to make this study more comprehensive and
meaningful.
I would like to thank all staff at the Department of Bioprocess Technology,
Department of Cell and Molecular Biology, Faculty of Biotechnology and
Biomolecular Sciences for their numerous help and support during my study.
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I certify that a Thesis Examination Committee has met on ……… to conduct the final examination of Farzaneh Heidarnia on her thesis entitled “Metabolic Regulation Analysis of Recombinant Lactococcus lactis, Based on Gene Expression and Enzyme Activity” in accordance with the Universities and University Colleges Act 1971 and Construction of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the degree of Master of Science. Members of the Thesis Examination Committee were as follows: …., PhD ……….. (position) Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) …, PhD …….. (position) Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) …, PhD ……….. (position) Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) …, PhD ……………….(position) Faculty of …… University …… (External Examiner)
----------------------------------------------- BUJANG BIN KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra MalaysiaDate:
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The thesis was submitted to the senate of Universiti Putra Malaysia has been accepted as the fulfillment requirement for the degree of Master of Science. The members of Supervisor Committee were as follows: Nor’Aini Abdul Rahman, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Raha Abdul Rahim, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Rosfarizan Mohamad, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
_______________________________ HASANAH MOHD GHAZALII, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:
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DECLARATION
I declare that the thesis is my original work except for the quotation and citation which have been duly acknowledged. I also declare that it has not been previously or currently submitted for any other degree at Universiti Putra Malaysia or at any other institution.
------------------------------------ FARZANEH HEIDARNIA
Date: 18 July 2011
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TABLE OF CONTENTS
Page
DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL viii DECLARATION x LIST OF TABLES x iii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xvi
1. INTRODUCTION 1�
2. LITERATURE REVIEW 5�2.1 Lactic Acid Bacteria 5�
2.1.1 Lactococcus lactis 6�2.1.2 Biotechnology and Genetic Engineering of L. lactis 7�2.1.3 L. Lactis as a Live Vaccine Vector 8�2.1.4 Gene cloning and effects of exogenous gene 9�2.1.5 Metabolic pathway 11�
2.2 Aeromonads 16�2.2.1 Aeromonas hydrophila 17�2.2.2 Aerolysin 21�2.3.3 Structure of Aerolysin 22�
2.3 Gene Expression 24�2.3.1 Northern Blots 25�2.3.2 Ribonuclease Protection Assays 27�2.3.3 DNA Microarray Technology 28�2.3.4 Serial Analysis of Gene Expression 30�2.3.5 RT-PCR 32�2.3.6 Real-Time RT-PCR 33�2.3.7 Real-Time PCR vs. Conventional RT-PCR 35�
2.4 Enzymes 36�2.4.1 Factors Affecting Enzyme Activity 36�
3. METABOLIC REGULATION ANALYSIS OF RECOMBINANT L. LACTIS BASED ON GENE EXPRESSION AND ENZYME ACTIVITIES 39�
3.1 Introduction 39�3.2� Material and Methods 40�
3.2.1 General Experimental Design 40�3.2.1 Bacterial strain and culture condition 42�3.2.2 Verification of the recombinant pNZ8048D1Aer plasmid 43�3.2.3 Inocula Preparation 43�3.2.4 Shake-flask fermentation 44�
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3.2.5 Cell Concentration 44�3.2.6 Sugar Concentration 44�3.2.6 RNA Extraction 45�3.2.7 Primer design 46�3.2.8 Semi-quantitative RT-PCR 46�3.2.9 Real-Time PCR 49�3.2.10 Enzymatic Assays 52�3.2.12 Measurement of organic acids concentration by HPLC 55�
3.3 Results 55�3.3.1 Fermentation characteristics 55�3.3.2 Verification of the recombinant plasmid, pNZ8084D1Aer 61�3.3.3 Optimization of PCR conditions 62�3.3.4 Semi-quantitative RT-PCR 63�3.3.5 Quantitative RT-PCR 65�3.3.6 Real-Time PCR 67�3.3.7 Enzyme activity measurement 72�
3.4 Discussion 74�3.4.1 Glycolytic pathway and PP pathway 74�3.4.2 TCA cycle 77�3.4.3 Fermentation pathway 78�
4. CONCLUSIONS AND RECOMMENDATIONS 80�4.1 Conclusions 80�4.2 Recommendations 81�
REFERENCES 83�APPENDIX 99�BIODATA OF STUDENT ERROR! BOOKMARK NOT DEFINED.�