COPYRIGHTpsasir.upm.edu.my/19442/1/FBSB_2011_11.pdfbertanggungjawab untuk enzim yang terdapat pada tapak jalan utama, contohnya Glikolisis, Kitaran TriKarboksilik, Fermentasi, dan

© COPYRIG

HT UPM

UNIVERSITI PUTRA MALAYSIA

METABOLIC REGULATION ANALYSIS OF RECOMBINANT Lactococcus lactis BASED ON GENE EXPRESSION AND ENZYME ACTIVITY

FARZANEH HEIDARNIA

FBSB 2011 11

© COPYRIG

HT UPM

i

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

© COPYRIG

HT UPM

ii

Dedicated to my parents: Mohammad Ali and Ashraf

For their endless supports

© COPYRIG

HT UPM

iii

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.

© COPYRIG

HT UPM

iv

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).

© COPYRIG

HT UPM

v

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.

© COPYRIG

HT UPM

vi

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.

© COPYRIG

HT UPM

vii

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.

© COPYRIG

HT UPM

viii

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:

© COPYRIG

HT UPM

ix

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:

© COPYRIG

HT UPM

x

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

© COPYRIG

HT UPM

xi

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�

© COPYRIG

HT UPM

xii

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.�