ELUCIDATION OF PHA BIOSYNTHESIS REGULATORY ELEMENTS IN Pseudomonas sp. USM4-55
KAMARIAH HASAN
UNIVERSITI SAINS MALAYSIA
2013
ELUCIDATION OF PHA BIOSYNTHESIS REGULATORY ELEMENTS IN Pseudomonas sp. USM4-55
By
KAMARIAH HASAN
Thesis submitted in fulfillment of the requirement for the degree of Doctor of Philosophy
SEPTEMBER 2013
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ACKNOWLEDGEMENTS
First and foremost, thank you Allah S.W.T for the strength, health and blessing that You give me, keeping me sane at all time while finishing this research and thesis. Syukur Alhamdulillah.
The deepest appreciation to my supervisor, Professor Mohd Nazalan Mohd Najimudin for his patience, motivation and immense knowledge that has guided me throughout my postgraduate studies. His enthusiasm in science has fueled my interest in this field and I cannot imagine having a better advisor and mentor.
I would also like to thank Prof. Mohd Razip Samian and Prof. Sudesh Kumar for their invaluable advices and their kindness in allowing me to use their chemicals and lab apparatus. Not forgetting Dr. Zary Shariman, Dr. Normi Yahaya and Dr.
Mustafa Fadzil, thank you for allowing me access to their lab apparatus. To Dr.
Suriani thank you for the technical support and kind advice.
For lab 414 members, past and present, I will always treasure our camaraderie. Thank you for the joy, laughter and unconditional friendship. This journey would not be this enjoyable without you guys. For my angels, you girls are the greatest! I’m going to miss all the time we spent together. Not forgetting Lab 409 members especially Yoke Ming, thank you for assisting me with GC analysis.
For staff at EM unit, thank you for your help.
Last but not least, my family especially my mother who has been the pillar of strength for me, always backing me up, giving me full support in every possible way. Words cannot express how much it means to me.
I would also like to thank the Ministry of Science and Innovation (MOSTI) for awarding me the National Science Fellowship (NSF) and Universiti Sains Malaysia (USM) for awarding me the USM-RU-PRGS grant.
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TABLE OF CONTENTS
Page
Acknowledgements ii
Table of Contents iii
List of Tables x
List of Figures xi
List of Plates xiii
List of Symbols and Abbreviations xv
Abstrak xvi
Abstract xviii
CHAPTER 1 GENERAL INTRODUCTION
1.1 Research background 1
1.2 Research objectives 4
CHAPTER 2 LITERATURE REVIEW
2.1 Polyhydroxyalkanoates (PHAs) 6
2.1.1 Properties of PHAs 8
2.1.2 Occurrence of PHAs 10
2.1.3 Biodegradation of PHAs 11
2.1.4 Application of PHAs 12
2.2 Biosynthesis of PHAs 13
2.2.1 Biosynthesis of scl-PHAs 13
2.2.2 Biosynthesis of mcl-PHAs 15
2.2.2.1 Fatty acid β-oxidation pathway 15 2.2.2.2 Fatty acid de novo biosynthesis pathway 16
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2.2.2.3 Chain elongation reaction pathway 16
2.3 PHA granules 17
2.3.1 Structure of PHA granules 17
2.3.2 Formation of PHA granules 18
2.3.2.1 Formation of PHA granules in Pseudomonas sp. USM4-55 20
2.4 PHA synthases 22
2.4.1 Classification of PHA synthases 22
2.4.1.1 PHA synthases in pseudomonads (Class II) 24
2.4.2 Organization of PHA synthase genes 25
2.4.3 PHA gene cluster in genus Pseudomonas 27 2.5 Gene regulation in bacteria
2.5.1 The lac operon 2.5.2 The trp operon
2.6 Regulation of PHA production
28 29 31 33 2.6.1 Regulation of PHA production at enzymatic level 33 2.6.2 Regulation of PHA production at transcriptional level 34 2.6.3 Regulation of PHA production in Pseudomonas 34 2.6.3.1 Regulation of phaF and phaI 35 2.6.4 Regulation of PHA production in other bacteria 37 2.6.5 Other regulators for PHA production 39
2.7 Promoters involved in PHA biosynthesis 41
2.8 Transposon
CHAPTER 3 MATERIALS AND METHODS
42
PART Ι
3.1 Identification of genes involved in the over-expression of phaC1::lacZ
v
3.1.1 Introduction 44
3.1.2 General methods 47
3.1.2.1 Bacterial strains and plasmids 3.1.2.2 Growth conditions of bacteria
47 47
3.1.2.3 Spectrophotometry 50
3.2.1.4 Culture media
3.2.1.4.1 Luria Bertani (LB) medium and agar 50 3.2.1.4.2 Mineral Salt medium (MS) and agar 50 3.1.3 Transposon mutagenesis
3.1.3.1 Transformation of pUTmini-Tn5 Sm/Sp into E.coli DH5α- λpir
51
3.1.3.2 Triparental mating 52
3.1.3.3 Selection of mutant strains 52
3.1.4 ß-galactosidase assay 52
3.1.5 DNA walking 54
3.1.5.1 Purification of genomic DNA 56 3.1.5.2 Design of DNA walking primers 56 3.1.5.3 DNA walking first PCR reaction 56 3.1.5.4 DNA walking second PCR reaction 58 3.1.5.5 DNA walking third PCR reaction 58 3.1.6 Mapping of transposon insertion
3.1.6.1 PCR purification 59
3.1.6.2 DNA sequence analysis 59
PART ΙΙ
3.2. Construction of Pseudomonas sp. USM4-55 secB and lon mutant strains
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3.2.1 Introduction 61
3.2.2 Bacterial strains, media and culturing condition 61
3.2.3 General molecular biology methods 64
3.2.3.1 Polymerase Chain Reaction (PCR) 64 3.2.3.2 Digestion of PCR fragments 67 3.2.4 Construction of secB mutant using broad host range vector system 67 3.2.4.1 Construction of secB gene replacement vector 67 3.2.4.2 Transformation of vector pKEM302-secB into hosts 69 3.2.4.3 Screening of mutant strains 69 3.2.5 Construction of lon mutant using suicide vector system 70 3.2.5.1 Construction of gene replacement vectors 70 3.2.5.2 Introduction of pGR-lon into hosts 72 3.2.5.3 Screening of mutant strains 72 3.2.6 Confirmation of gene disruption in mutant strains 73 3.2.6.1 PCR of DNA fragments from mutant and wild type
strains 73
3.2.6.2 DNA Sequencing of PCR fragments 74 3.2.6.3 Testing of mutant strains on MS medium with X-Gal 74 PART ΙΙΙ
3.3 Characterization of secB and lon mutant strains
3.3.1 Introduction 77
3.3.2 General methods
3.3.2.1 Bacterial strains and plasmids 77
3.3.2.2 Spectrophotometry 77
3.3.2.3 Culture Media 77
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3.3.3 Bacterial population growth study 78
3.3.4 Gene expression level analysis 79
3.3.4.1 RNA extraction 79
3.3.4.2 DNase treatment for RNA samples 3.3.4.3 RNA analysis on agarose gel
3.3.4.4 Reverse transcription-quantitative real-time PCR (qRT- PCR)
79 79 80
3.3.4.5 Determination of housekeeping gene (rrn) expression in all
samples 80
3.3.4.6 Standard curves for qRT-PCR assay 82
3.3.5 Gas Chromatography (GC) analysis 82
3.3.5.1 PHA polymer extraction 82
3.3.5.2 Determination of PHA monomer and composition 83 3.3.5.2.1 Harvesting of cell cultures 83 3.3.5.2.2 Methanolysis 83 3.3.5.2.3 Gas Chromatography analysis 84 3.3.6 Transmission Electron Microscopy (TEM)
3.3.6.1 Preparation of cells 86
3.3.6.2 Samples preparation for TEM 86 3.3.6.3 Cross section with ultramicrotome 87 3.3.6.4 Staining of samples and viewing with TEM 88
CHAPTER 4 RESULTS PART Ι
4.1 Identification of genes involved in the over-expression of phaC::lacZ 89
4.1.1 Isolation of mutant strains 89
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4.1.2 Quantitative analysis of ß-Galactosidase activity in the mutant
strains 93
4.1.3 Transposon-Insertion Mapping 95
4.1.3.1 DNA walking 95
4.1.4 Sequencing and BLAST analysis 104
4.1.4.1 Pseudomonas sp. USM LZC1-EB1 104 4.1.4.2 Pseudomonas sp. USM LZC1-EB2 107 4.1.4.3 Pseudomonas sp. USM LZC1-EB6 107 4.1.4.4 Pseudomonas sp. USM LZC1-EB7 108 4.1.4.5 Pseudomonas sp. USM LZC1-EB9 108 4.1.4.6 Pseudomonas sp. USM LZC1-NB 109 PART ΙΙ
4.2 Construction of Pseudomonas sp. USM4-55 secB and lon mutant strains 110 4.2.1. Construction of gene replacement vector pKEM302-secB 111 4.2.2 Construction of gene replacement vector using a non-replicative
plasmid pDM4 114
4.2.3 Generation of disrupted mutant strain via homologous
recombination 116
4.2.4 Disruption of the chromosomal secB gene 118 4.2.5 Disruption of chromosomal lon gene 120 4.2.6 Confirmation of genes disruption by PCR 122 4.2.7 Comparison of lacZ expression between mutants and parental
strains 124
PART ΙΙΙ
4.3 Characterization of secB and lon mutant strains 127
4.3.1 Bacterial growth study 127
4.3.2 Quantitative real-time PCR (qRT-PCR) 130
ix 4.3.2.2 Real-time PCR
4.3.2.3 Determination of PCR efficiencies of 16S rRNA, phaC1 and phaC2
130 131
4.3.2.4 Relative quantification of phaC1 and phaC2 mRNA
transcripts 132
4.3.2.5 Relative quantification of phaC1 and phaC2 mRNA
transcripts in cells grown on PFAD. 134
4.3.2.6 Relative quantification of phaC1 and phaC2 mRNA
transcripts in cells grown in glucose. 136 4.3.3 PHA accumulation in Pseudomonas sp. USM4-55 and its mutant
strains 138
4.3.3.1 Preparation of copolymer standard: Determination of PHA
polymer composition 138
4.3.3.2 Comparison of PHA production by Pseudomonas sp.
USM4-55 and its mutant strains. 141
4.3.4 Observation of Pseudomonas sp. USM4-55, Pseudomonas sp.
SM100 and Pseudomonas sp. LM100 PHA granules 144
CHAPTER 5 DISCUSSION
5.1 Identification of genes involved in phaC regulation by mini-Tn5
mediated insertional mutagenesis 146
5.2 SecB chaperone as a regulator in PHA biosynthesis 155 5.3 Mutation in Lon protease effect PHA biosynthesis 159 5.4 SecB chaperone and Lon protease share substrates 162
CHAPTER 6 CONCLUSION 164
CHAPTER 7 BIBLIOGRAPHY 167
CHAPTER 8 APPENDICES
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LIST OF TABLES
Page Table 2.1 Comparison of physical properties of various mcl-PHAs 9
Table 2.2 Classification of PHA synthases 26
Table 2.3 Genetic organization of PHA synthase genes encoding various
classes of enzymes 26
Table 2.4 Regulatory proteins involved in PHA metabolism 40 Table 3.1 Bacterial strains and vectors used in Part Ι 49
Table 3.2 Primers for DNA walking 57
Table 3.3 Bacterial strain and vectors used in Part ΙΙ 62 Table 3.4 Primer sequences for gene replacement vectors 66
Table 3.5 Components of PCR mixture 66
Table 3.6 Cycling parameter for PCR 66
Table 3.7 The qRT-PCR primers for amplification of 16S rDNA, phaC1 and phaC2
81
Table 3.8 Parameters used in gas chromatography analysis 85 Table 4.1 BLASTX analysis performed on each sequence to map the
mini-Tn5 insertion 105
Table 4.2 Monomer compositions of pure PHA polymer extracted from
Pseudomonas sp. USM4-55 141
Table 4.3 Compositions of the PHA content detected in Pseudomonas sp.
USM4-55 and its mutant strains 142
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LIST OF FIGURES
Page
Figure 1.1 Outline and flow of thesis 5
Figure 2.1 Chemical structure of PHA 7
Figure 2.2 Major metabolic pathways that supply hydroxyalkanoate
monomers for PHA biosynthesis 14
Figure 2.3 PHA granule formation models 19
Figure 2.4 Predicted granule formation model in Pseudomonas sp.
USM4-55 21
Figure 2.5
Figure 2.6 Figure 2.7
(A) Negative control of the lac operon (B) Positive control of the lac operon Negative control of the trp operon
Hypothetical model proposed for the PHA synthesis regulation
30 30 32 36
Figure 3.1 Experimental approaches for Tn5 mutagenesis and
identification of genes involved in phaC regulation 45
Figure 3.2 Organization of pha genes cluster 48
Figure 3.3 Plasmid map of pUTmini-Tn5 Sm/Sp 48
Figure 3.4 General strategy of DNA Walking ACPTM PCR Technology 55 Figure 3.5 Experimental approaches for construction gene replacement
vector for secB mutant 68
Figure 3.6 Experimental approaches for construction of replacement
vectors for lon mutant 71
Figure 3.7 Experimental approaches for study in Part ΙΙΙ 76 Figure 4.1 Bar graph showing the values of ß-galactosidase assay 94 Figure 4.2 Sequence of mini-Tn5 and design of TSP primers 96
Figure 4.3 Vector map of pKEM200 112
Figure 4.4 Vector map of pKEM202-secB 113
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Figure 4.5 Vector map for pKEM302-secB 115
Figure 4.6 Vector map for pGR-lon 117
Figure 4.7 Construction of secB mutant strain in Pseudomonas sp.
USM4-55 and its derivatives by homologous recombination 119 Figure 4.8 Homologous recombination events that take place in the
chromosome of Pseudomonas sp. USM4-55 and its derivatives using suicide vector
121
Figure 4.9 Growth profile of Pseudomonas sp. USM4-55, Pseudomonas sp. SM100 and Pseudomonas sp. LM100 grown in MS medium supplemented with PFAD
128
Figure 4.10 Growth profile of Pseudomonas sp. USM4-55, Pseudomonas sp. SM100 and Pseudomonas sp. LM100 grown in MS medium supplemented with glucose
128
Figure 4.11 Quantification of phaC1 gene expression in mutant strains relative to Pseudomonas sp. USM4-55 grown in MS medium supplemented with PFAD
135
Figure 4.12 Quantification of phaC2 gene expression in mutant strains relative to Pseudomonas sp. USM4-55 grown in MS medium supplemented with PFAD
135
Figure 4.13 Quantification of phaC1 gene expression in mutant strains relative to Pseudomonas sp. USM4-55 grown in MS medium supplemented with glucose
137
Figure 4.14 Quantification of phaC2 gene expression in mutant strains relative to Pseudomonas sp. USM4-55 grown in MS medium supplemented with glucose
137
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LIST OF PLATES
Page Plate 4.1 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-EB1 90 Plate 4.2 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-EB2 90 Plate 4.3 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-EB6 91 Plate 4.4 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-EB7 91 Plate 4.5 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-EB9 92 Plate 4.6 Comparison blue colonies formation between Pseudomonas sp.
USM LZC1 and Pseudomonas sp. USM LZC1-NB 92 Plate 4.7 Agarose gel electrophoresis showing PCR fragments amplified
from DNA walking process on Pseudomonas sp. USM LZC1- EB1
98
Plate 4.8 Agarose gel electrophoresis showing PCR fragments amplified from DNA walking process on Pseudomonas sp. USM LZC1- EB2
99
Plate 4.9 Agarose gel electrophoresis showing PCR fragments amplified from DNA walking process on Pseudomonas sp. USM LZC1- EB6
100
Plate 4.10 Agarose gel electrophoresis showing PCR fragments amplified from DNA walking process on Pseudomonas sp. USM LZC1- EB7
101
Plate 4.11 Agarose gel electrophoresis showing PCR fragments amplified from DNA walking process on Pseudomonas sp. USM LZC1- EB9
103
Plate 4.12 Agarose gel electrophoresis showing PCR fragments amplified from DNA walking process on Pseudomonas sp. USM LZC1- NB
103
Plate 4.13 Agarose gel electrophoresis showing fragments used in the
cloning of gmR into pKEM100 112
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Plate 4.14 Gel electrophoresis showing the fragments used in the
construction of plasmid pKEM202-secB 113
Plate 4.15 Gel electrophoresis showing the fragments used in the
construction of plasmid pKEM302-secB 115
Plate 4.16 Agarose gel electrophoresis showing fragments involved in the
construction of pGR-lon 117
Plate 4.17 DNA gel electrophoresis result showing amplification of secB
gene using external primers 123
Plate 4.18 DNA gel electrophoresis result showing amplification of lon
gene using external primers 123
Plate 4.19 Comparison of blue colonies formation between Pseudomonas
sp. USM LZC1 (left) and Pseudomonas sp. SM101 (right) 125 Plate 4.20 Comparison of blue colonies formation between Pseudomonas
sp. USM LZC2 (left) and Pseudomonas sp. SM102 (right) 125 Plate 4.21 Comparison of blue colonies formation between Pseudomonas
sp. USM LZC1 (left) and Pseudomonas sp. LM101 (right) 126 Plate 4.22 Comparison of blue colonies formation between Pseudomonas
sp. USM LZC2 (left) and Pseudomonas sp. LM102 (right) 126 Plate 4.23 PHA polymer extracted from Pseudomonas sp. USM4-55 140 Plate 4.24 TEM images of all strains grown in MS supplemented with
PFAD 145
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LIST OF SYMBOLS AND ABBREVIATIONS
β beta
BLAST Basic Local Alignment Search Tool
bp Base pair
CT Threshold cycle
dNTP Deoxyribonucleic acid et al. et alia (and others)
ext external
gmR Gentamycin resistant gene
hrs hours
kDa Kilo dalton
kmR Kanamycin resistant gene
min minutes
mRNA Messenger ribonucleic acid nm
PFAD
Nanometer
Palm fatty acid distillate
rpm Revolution per minute
σ sigma
sec seconds
smR Streptomycin resistant gene
sp. species
Tn5 transposon
U Unit
w/v Weight per volume
v/v Volume per volume
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KAJIAN ELEMEN PENGAWALATUR BIOSINTESIS PHA DALAM Pseudomonas sp. USM4-55
ABSTRAK
PHA sintase ialah enzim penting dalam penghasilan bioplastik oleh bakteria.
Untuk mengenalpasti pengawalatur bagi gen PHA sintase, perpustakaan mutan selitan bagi Pseudomonas sp. USM4-55 menggunakan mini-Tn5 telah dijana. Binaan cantuman transkripsi mengandungi gen lacZ di dalam gen phaC1 and phaC2 dari Pseudomonas sp. USM4-55 telah digunakan dalam kajian ini. Lima mutan yang menunjukkan peningkatan pada ekspresi phaC1 telah berjaya dipencilkan manakala tiada mutan phaC2 dihasilkan. Selitan Tn5-mini ditemui pada gen yang mengkodkan protein berikut; anti-sigma faktor K, subunit protein translocase (SecB), protease bergantung ATP (Lon) dan PhaF (sejenis protein berkaitan granul PHA). Dengan menyelitkan gen gmR, secB dan lon telah mengalami proses ‘knockout’ di dalam jenis liar Pseudomonas sp. USM4-55 dan terbitannya yang mengandungi cantuman transkripsi gen lacZ di dalam gen phaC1 dan phaC2 (Pseudomonas sp. USM LZC1 dan Pseudomonas sp. USM LZC2). Mutan SecB¯ dan Lon¯ dalam Pseudomonas sp.
USM LZC1 mempamerkan peningkatan ekspresi lacZ seperti mutan asal, menunjukkan gen ini memainkan peranan dalam pengawalaturan phaC1. Untuk mengkaji kadar ekspresi gen, PCR transkripsi berbalik kuantitatif-masa benar (qRT- PCR) telah dijalankan untuk mutan SecB¯ dan Lon¯ dalam Pseudomonas sp. USM4- 55. Tahap ekspresi gen phaC untuk strain ini meningkat pada fasa log manakala ia menurun pada fasa malar apabila dibandingkan dengan jenis liar. Melalui kajian fisiologi, strain SecB¯ menunjukkan peningkatan penghasilan PHA sebanyak 50%
hingga 75% apabila dikultur di dalam medium tertakrif bersama ‘Palm Fatty Acid Distillate’ (PFAD) dan glukosa sebagai sumber karbon. Walaubagaimanapun, strain
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Lon¯ yang dikultur di dalam sumber karbon yang sama menunjukkan penurunan penghasilan PHA. Mutan Lon¯ juga menunjukkan pengurangan bilangan sel pada fasa malar dalam medium tertakrif. Hasil kajian ini menunjukkan bahawa Lon memainkan peranan penting dalam pertumbuhan bakteria dan penghasilan PHA dalam Pseudomonas sp. USM4-55. Kesimpulannya, didapati SecB dan Lon protease terlibat dalam pengawalaturan sintesis PHA dalam Pseudomonas sp. USM4-55 melalui cara yang berbeza dan peranan sebenar mereka sebagai pengawalatur memerlukan kajian lanjut.
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ELUCIDATION OF PHA BIOSYNTHESIS REGULATORY ELEMENTS IN Pseudomonas sp. USM4-55
ABSTRACT
PHA synthases are the key enzymes for bioplastic production in bacteria. To determine regulators of PHA synthase genes, a library of insertional mutants of Pseudomonas sp. USM4-55 by mini-Tn5 was generated. Transcriptional fusion constructs containing the lacZ within phaC1 and phaC2 genes of Pseudomonas sp.
USM4-55 were used in this study. Five phaC1 over-expressed mutants were successfully isolated and none for phaC2. The mini-Tn5 insertions were found to be located on genes encoding these proteins: anti-sigma factor K, a protein translocase subunit (SecB), an ATP dependent protease (Lon) and PhaF (PHA granule associated protein). By inserting a gmR gene, the secB and lon genes were knocked out in the wild type Pseudomonas sp. USM 4-55 as well as its derivatives which contained a transcriptionally-fused lacZ gene within the phaC1 and phaC2 genes (Pseudomonas sp. USM LZC1 and Pseudomonas sp. USM LZC2, respectively). The SecB¯ and Lon¯ mutants in Pseudomonas sp. USM LZC1 showed an over-expression of lacZ as similarly shown by the original mutants indicating that these genes indeed play a role in phaC1 regulation. To study gene expression, real time qRT-PCR was performed in the SecB¯ and Lon¯ mutants of Pseudomonas sp. USM4-55. The phaC expression levels of these mutant strains were up regulated at log phase but were down regulated at stationary phase compared to the wild type. In physiological studies, the SecB¯
strain increased PHA production by 50% and 75% when grown in defined medium supplemented with palm fatty acid distillate (PFAD) and glucose, respectively, as a carbon source relative to the wild type. However, the Lon¯ strain grown in these carbon sources showed reduced PHA production. The Lon¯ mutant also exhibited a
xix
reduced cell density at stationary phase in the minimal medium. This result implied that Lon played an essential role in bacterial growth and PHA accumulation in Pseudomonas sp. USM4-55. In conclusion, it was shown that the SecB and the Lon protease are involved in the regulation of PHA synthesis in Pseudomonas sp. USM4- 55 in different ways and their exact regulatory role is yet to be elucidated.
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CHAPTER 1: GENERAL INTRODUCTION 1.1 Research background
Plastic is one of the great inventions that play an important part in our lives due to its many attractive properties including stability and resistance to degradation. Since the mass production of plastics in the 1950s, people have been depending on it without realizing the adverse effects it has on the environment.
While plastics are undeniably very useful in our modern lifestyle, the uncontrolled usage of this non-biodegradable polymer will contribute to various problems including exhausted landfill areas and environmental pollution. Plastics also pose a considerable threat by choking and starving wildlife. As a result, millions of pounds were spend for yearly clean-up operation organized in many countries (Barnes et al., 2009). The growing awareness on the threat of this non- degradable polymer has prompted scientists to find its replacement. The increase in the human population to more than 7 billion recently further enhanced the need for renewable and biodegradable substances as a substitute to the non- biodegradable materials to sustain our future.
The emergence of biodegradable plastics was seen as a potential solution to the problem. Among the materials that caught attention are polyhydroxyalkanoate (PHA), polylactic acid (PLA) and polybutylene succinate (PBS), a synthetic bioplastic. These polymers draw interest due to their ability to be degraded under natural environment and are good candidates to replace the conventional plastics.
Among these environmentally friendly plastics, PHA is the best candidate because this biopolymer is produced by bacteria which can feed on renewable substrate which is an alternative energy source to the depleting fossil resources.
2
The biggest obstacle in replacing conventional plastic with PHA is the high cost of large scale production which resulted in soaring cost of degradable plastics, therefore hindering the global use of this biopolymer. To overcome the problem, many studies have been conducted to understand the behavior of PHA producing bacteria and how they regulate the production and degradation of this bioplastic in the environment. Hopefully the findings will help improve the properties, increase the production and eventually reduced the cost.
The lack of understanding on the regulation of PHA produced by bacteria is one of the obstacles that hampered the research in optimizing PHA production.
Few regulators such as PhaF, PhaR and PhaP that might play roles in PHA production were previously uncovered. However, other proteins that are not directly link to PHA metabolic pathway might also play important roles in regulating PHA production. Therefore, in this study, we aim to uncover more of these regulators.
One of the most studied PHA producing bacteria is from the genus Pseudomonas. These bacteria produce mainly mcl-PHAs (medium chain length PHAs) and some produce both mcl-PHAs and scl-PHAs (short chain length PHAs). One of the few bacteria that can produce both types of PHAs was isolated in a soil sample taken from an oil palm plantation in Felda Tasek Chini, Pahang (Few, 2001). This bacterium was named as Pseudomonas sp. USM4-55 and can accumulate a blend of PHB (poly-3-hydroxybutyrate, scl-PHA) and mcl-PHA homopolymer, and a small amount of random copolymer [P(3HB-co-3HA)]
(Sudesh et al., 2004). Several researches were performed to study the synthases in this bacterium. Mcl-PHA operon (phaC1, phaZ and phaC2) was cloned and
3
characterized (Baharuddin, 2002) while scl-PHA operon (phbC, phbA and phbB) was also cloned and characterized (Tan et al., 2010). The expression of phaC1 and phaC2 genes were also studied in E. coli (Balqis, 2007). Quantitative reverse transcription real-time PCR (qRT-PCR) have revealed that these two synthases are transcribed independently of each other and are controlled by their own promoter (Tan, 2010).
Previously, a reporter system to measure the expression of each phaC1 and phaC2 by integrating a promoterless lacZ in the middle of each gene was constructed. The two resulting strains were named as Pseudomonas sp. USM LZC1 and Pseudomonas sp. USM LZC2 which contained the phaC1::lacZ and phaC2::lacZ, respectively (Kamariah, 2007). These reporter gene infused strains provide the best candidates to study phaC gene regulation by looking at the expression of lacZ. Apart from acting as a reporter, the integration of lacZ has also disrupted the PHA synthase genes. Thus, these mutant strains are excellent candidates to study the role of individual phaC genes.
In this research, the regulation of phaC1 and phaC2 genes in Pseudomonas sp. USM4-55 was investigated. This study focused on finding new regulators that might help in the regulation of the PHA synthase genes utilizing a reporter gene, lacZ as an indicator. In the first stage, random mutagenesis was employed using mini-Tn5 which disrupted certain genes and affects the expression of phaC::lacZ transcriptional fusion. The mutant strains which showed altered expression of lacZ gene were subjected to β-galactosidase assay to verify the LacZ activity.
Genomic DNA from the mutant strains were then subjected to DNA walking process, DNA sequencing and BLAST analysis to identify the genes involved in
4
altering the phaC expression. The second stage of the research is the construction of gene insertion mutants in Pseudomonas sp. USM4-55, Pseudomonas sp. USM LZC1 and Pseudomonas sp. USM LZC2. The genes identified in the first part of the study were mutated in the wild type strain and its derivatives through homologous recombination process utilizing suicide plasmids.
To characterize the wild type and mutant strains, growth population study was carried out by plotting growth curves of bacteria cultured in minimal medium. Gene expression level study was performed utilizing quantitative real- time PCR (qRT-PCR) method to compare the phaC1 and phaC2 gene expression.
Finally, the wild type and mutant strains grown in minimal medium supplemented with either PFAD or glucose as a carbon source were subjected to gas chromatography (GC) analysis to compare PHA production. Formation of PHA granules was also observed utilizing transmission electron microscopy (TEM). The approach that was taken is outlined in Figure 1.1.
1.2 Research objectives
This research was done to fulfill the following objectives:
a) To determine regulators involved in the expression of phaC1 and phaC2 (transcriptionally fused to the reporter gene lacZ) via transposon mutagenesis.
b) To perform targeted disruption of genes uncovered by the transposon random mutagenesis in Pseudomonas sp. USM4-55 and its derivatives.
c) To evaluate the effect of mutations on the phaC genes expression via qRT-PCR.
d) To assess PHA accumulation in wild type and mutants by performing GC analysis.
5
Figure 1.1 Outline and flow of thesis. Experimental approach to identify genes that play roles in the regulation of phaC and characterization of the mutant strains.
PART Ι
Identification of genes involved in regulation of Pseudomonas sp. USM4- 55 phaC1 via mini-Tn5 mutagenesis
Mini-Tn5 mutagenesis of Pseudomonas sp. USM LZC1 and Pseudomonas sp.
USM LZC2 to screen altered expression of phaC through reporter gene lacZ
Identification of genes involved in phaC regulation via DNA Walking, sequencing and BLAST analysis
PART ΙΙ
Construction of secB and lon mutants in Pseudomonas sp. USM4-55 and its derivatives
Construction of gene replacement vectors to mutate genes that play roles in phaC regulation
Site directed mutagenesis in Pseudomonas sp. USM 4-55 and its mutant derivatives Pseudomonas sp. USM LZC1 and Pseudomonas sp. USM LZC2
PART ΙΙΙ
Study of gene expression level and PHA accumulation in secB and lon mutants
Growth population studies to compare growth rates of wild type and mutant strains
Relative quantification of gene expression level through qRT-PCR between wild type and mutant strains
Comparison of PHA accumulation (GC analysis) and granule formation (TEM)
6
CHAPTER 2: LITERATURE REVIEW 2.1 Polyhydroxyalkanoates (PHAs)
Polyhydroxyalkanoates or PHAs are high molecular weight polymers synthesized by microorganisms as storage compounds during imbalanced conditions such as an excess in carbon source and limitation of other growth essential nutrients such as nitrogen or phosphorus (Anderson & Dawes, 1990).
PHAs, accumulated by bacteria up to 90% of their dry cell weight, are deposited as amorphous water insoluble granules in the cytoplasm. They play a role as a sink for carbon and reducing equivalents (Madison & Huisman, 1999; Sudesh et al., 2000). PHAs gained interest due to their biodegradable and biocompatible properties and hence are the best candidates to replace conventional plastics.
When carbon starvation occurs, PHAs are mobilized (degraded) by PHA depolymerases (Jendrossek, 2001). Accumulation of PHAs also increases bacteria survival under stress conditions such as UV irradiation, salinity, thermal and oxidative stress, desiccation and osmotic shock (Castro-Sowinski et al., 2010).
More than 150 different polyhydroxyalkanoic acids with different properties were discovered in more than 300 different microorganisms isolated from various sources including aerobic and anaerobic habitats (Kim do et al., 2007). The most common chemical structure of PHA is shown in Figure 2.1. The side chain length and its functional group influence crystallinity, melting point and glass transition temperature of the polymer (Eggink et al., 1995). The discovery of PHAs has prompted the need to study their structure, physical state and biological properties. The metabolic pathways involved in their biosynthesis and gene regulation are also of interest to many researchers.
7 H H
O C OH H C C
R H O n
n = 1 R= hydrogen poly (3-hydroxypropionate) methyl poly (3-hydroxybutyrate) ethyl poly (3-hydroxyvalerate) propyl poly (3-hydroxyhexanoate) pentyl poly (3-hydroxyoctanoate) nonyl poly (3-hydroxydodecanoate) n = 2 R= hydrogen poly (4-hydroxybutyrate) n = 3 R= hydrogen poly (5-hydroxyvalerate)
Figure 2.1 Chemical structure of PHA. Adapted from (Zinn et al., 2001) and (Ojumu et al., 2004). All monomers have one chiral center (*) in the R position.
*
8 2.1.1 Properties of PHAs
PHAs are composed of 3-hydroxy fatty acid monomers which form linear, head to tail polyester. PHAs can exist as homopolymers, copolymers or a blend of both depending on the producers and growth substrates provided (Peoples &
Sinskey, 1989). Once extracted from the cell, PHAs show crystalline and thermoplastic properties similar to polypropylenes or petrochemical-based plastic.
PHAs can be divided into a few classes according to the size of the monomers. PHAs containing up to 5 carbons are classified as short chain length PHAs (scl-PHAs) while PHAs with 6 to 14 carbons are classified as medium chain length PHAs (mcl-PHAs). Scl-PHAs have properties similar to conventional plastic albeit relatively stiff and brittle while mcl-PHAs are elastomers and sticky (Madison & Huisman, 1999). PHA copolymers, which composed primarily of HB with additional chain monomers such as HV, HH or HO, are more flexible and tough (Rai et al., 2011). Homopolymers of scl-PHAs such as poly(3-hydroxybutyric acid) (PHB) have Tm values of 180°C and Tg
values of 4°C. Copolymers of mcl-PHAs have Tm values of between 40 to 60°C and Tg values between -50 and -25°C. Table 2.1 demonstrates the properties of a variety of mcl-PHAs produced by pseudomonads.
Other than biodegradable, PHAs are biocompatible because their breakdown products are 3-hydroxyacids which are commonly found in animals.
PHAs also exhibit piezoelectricity property which help stimulates bone growth and aid in wound healing. These characteristic of PHAs can be very useful in many medical applications for example implants, gauzes, suture filaments and as matrix material for slow release of drugs (Zinn et al., 2001).
9
Table 2.1 Comparison of physical properties of various mcl-PHAs (adapted from Rai et al., 2011).
Organism Carbon source MW
(kDa) Mn
(kDa) PDI Tg
(°C) Tm (°C)
P. oleovorans
n-hexane n-heptane n-octane n-nonane n-decane 1-octene
333 308 178 240 225 242
182 160 99 131 113 101
1.8 1.9 1.8 1.8 2.0 2.4
-25.8 -30.8 -36.5 -39.7 -38.4 -36.6
NO 38.9 58.5 47.8 47.6 NO
P. putida GPO1
Octanoate 100%
Octanoate 75%
Octanoate 50%
Octanoate 25%
286 253 290 278
118 113 156 118
2.4 2.2 1.9 2.4
-33.1 -39.5 -44.6 -47.4
58.1 44.5 39.9 NO P. putida
IPT046 Glucose and
fructose 223 88 2.5 -39.7 56
P. putida styrene 76.5 25.2 3.0 -41.7 38.1
P. aeruginosa
octanoic decanoic tetradecanoic
316 251 255
191 121 148
1.7 2.1 1.7
ND ND ND
ND ND ND
P. sp 61-3 glucose 176 40 4.4 -43 ND
P. putida Oleic acid vegetable
135 168
40 65
2.8 2.7
-43.5 -52
ND ND P. oleovorans Tallow free
fatty acid 134 68 2.0 ND ND
P. resinovorans Tallow free fatty acid Tallow
157
192
79
90
2.0
2.1
ND
ND
ND
P. citronellis Tallow free ND
fatty acid 125 55 2.3 ND ND
P. putida Tallow free
fatty acid 141 71 2.0 ND ND
P. oleovorans octanoate 339 160 2.1 ND ND
MW = weight average molecular; Mn = Number average molecular weight; PDI = polydispersity index; Tg = Glass transition temperature; Tm = melting temperature; ND = not determined; NO = not observed.
10 2.1.2 Occurrence of PHAs
PHA granules were first observed in bacterial cells under the light microscopy as refractile bodies by Beijerinck in 1888 (reported in Chowdhurry, 1963). However, a French scientist Maurice Lemoigne was the first to chemically identify intracellular PHB granules in Bacillus megaterium (Braunegg et al., 1998). Since then, various microorganisms such as archae and photosynthetic bacteria were found to accumulate PHB. For almost 50 years, PHB was the only known PHA until Wallen & Rohwedder (1974) discovered 3-hydroxyvalerate (3HV) and 3-hydroxyhexanoate (3HHx) in the chloroform extract of activated sludge.
PHA with longer side chains, 3-hydroxyheptanoate (3HHp) monomer was identified in B. megaterium (Findlay & White, 1983). Following this discovery, 3-hydroxyoctanoate (3HO) with 3HHx was discovered in P. putida GPo1 (De Smet et al., 1983). Ten years later, a Pseudomonas strain GP4BH1 which accumulates a blend of PHB and mcl-PHA from various carbon sources was successfully isolated (Steinbüchel & Wiese, 1992). Another strain, Pseudomonas sp. 61-3 which produced a blend of PHB homopolymer and a random copolymer, P(3HB-co-3HA) consisting 3HA units of 4-12 carbon atoms was also isolated (Matsusaki et al., 1998).
After the discovery of PHA production by bacteria, the research has shifted towards cloning and characterization of genes involved in PHA production. By the end of the 1980s the genes coding for enzymes in PHA production from Cupriavidus necator (formerly known as Ralstonia eutropha) were successfully cloned and expressed in E. coli (Peoples & Sinskey, 1989).
11 2.1.3 Biodegradation of PHAs
Unlike conventional plastics that can remain in the environment for decades, biodegradable plastics are easily degraded by a variety of bacteria within months. Degradation of PHAs occur in the absence of carbon and energy sources upon exposure to soil, compost or marine sediments (Macrae & Wilkinson, 1958). Biodegradation is dependent on factors such as microbial activities, composition of the polymer, temperature, moisture, pH and molecular weight (Boopathy, 2000). Bacteria colonize the surface of PHA polymers and degrade PHAs into carbon dioxide and water under aerobic environment and methane is produced under anaerobic environment. PHA degradation by bacteria was achieved by using their own secreted PHA hydrolases and PHA depolymerases which were found attached to the PHA granule surface (Gao et al., 2001).
The types of monomer units were found to affect degradation. PHA copolymers containing the 4-hydroxybutyrate (4HB) monomers degrades more rapidly than homopolymer PHB or P(3HB-co-3HV) copolymers (Doi et al., 1989). In C. necator, R-3-hydroxybutyric acid is the sole product of PHB hydrolysis but a mixture of dimers and monomers of the acid can be found in hydrolysis products of other organisms (Braunegg et al., 1998). PHAs can be degraded within few months in anaerobic sewage or few years in seawater (Madison & Huisman, 1999). Lee (1996) showed that P(HB-co-HV) was absolutely degraded after 6, 75 and 350 weeks in anaerobic sewage, soil and seawater respectively (Lee, 1996). Apart from the environmental conditions, other factors such as composition, crystallinity, additive and surface area of PHAs can significantly affect the degradation rates.
12 2.1.4 Applications of PHAs
Currently, various types of PHAs are being studied for different applications ranging from medical to production of everyday use products due to their special properties of biocompatibility and biodegradability. PHAs can be used to manufacture bottles and fibres for biodegradable packaging. The molten PHA can be applied on papers and cardboards to form a water resistant layer (Hocking & Marchessault, 1994). Other than that, PHAs can also be used as a medium for slow discharge of drugs, hormones, herbicides, insecticides, flavours and fragrances. PHAs are also considered as sources for the synthesis of raw materials for paint production and enantiomerically pure chemicals (Steinbüchel
& Fuchtenbusch, 1998).
Due to its high production cost, PHAs are currently more attractive for use in the medical field. The applications include implants like heart valves, stents, osteosynthetic materials and bone scaffold (Hazer & Steinbüchel, 2007). In tissue engineering, the cells are grown in vitro on biodegradable polymers to reconstruct tissues for implantation purposes (Zinn et al., 2001). Yao and co-workers (2008) used phasins (PhaP) of C. necator as a tag by fusing it to the PHA nanoparticles and attached them to cell specific ligands. Flourescence microscopic examination showed that the PhaP-nanoparticles were taken up by the correct tissue, demonstrating the success of targeted drug delivery (Yao et al., 2008). In agriculture, insecticides are incorporated into PHA granules and sown along with crops. By doing so, the release of insecticides into the environment can be controlled since PHA degrading bacteria can slowly degrade the PHA granules.
However its application is still under study (Philip et al., 2007).
13 2.2 Biosynthesis of PHAs
PHAs can be divided into two different classes based on the number of carbon atoms and their compositions. The first class of PHA is scl-PHAs which is represented by a classic PHB producer, C. necator (Peoples & Sinskey, 1989).
The second class of PHA is mcl-PHAs, which is represented by pseudomonads (Rehm, 2007). Figure 2.2 summarizes the various major metabolic pathways involved in PHA biosynthesis.
2.2.1 Biosynthesis of scl-PHAs
Biosynthesis of PHB in C. necator is through pathway I when sugar is provided as the carbon source (Peoples & Sinskey, 1989). As shown in Figure 2.2, this pathway involved three enzymes, namely ß-ketothiolase, acetoacetyl- Coenzyme A (CoA) reductase and PHA synthase encoded by phaA, phaB and phaC respectively. The first step involves condensation of two acetyl-CoA to form acetoacetyl-CoA catalyzed by the enzyme ß-ketothiolase (PhaA). This is followed by a reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA by the NADPH-dependent acetoacetyl-CoA reductase (PhaB). The last step is catalyzes by the enzyme PHA synthase (PhaC) and it involves polymerization of (R)-3- hydroxybutyryl-CoA monomer into PHB with a release of a free CoA molecule.
During normal growth condition, β-ketothiolase is inhibited by free CoA molecules coming out of the Krebs cycle. However, during imbalance nutrient condition, the entry of acetyl-CoA into the Krebs cycle is restricted. The excess acetyl-CoA is channelled into PHB biosynthesis and resulted in the accumulation of PHAs (Verlinden et al., 2007).
14
PATHWAY I PATHWAY II
(Sugars) (Fatty acids)
Acetyl-CoA Acyl-CoA
Acetoacetyl-CoA (PhaB) 3-ketoacyl-CoA Enoyl-CoA
(R)-3-Hydroxybutyryl-CoA
PHA GRANULES
4-,5-,6-Hydroxyalkanoyl-CoAs
Other pathways
Related carbon sources
Acetyl-CoA
(sugars)
Figure 2.2: Major metabolic pathways that supply hydroxyalkanoate monomers for PHA biosynthesis. PhaA, β-ketothiolase; PhaB, NADPH- dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3- hydroxyacyl-ACP-CoA transferase; PhaJ (R)-enoyl-CoA hydratase; FabG, 3- ketoacyl-CoA reductase (adapted from Sudesh et al., 2000).
(S)-3-Hydroxyacyl-CoA
(R)-3-Hydroxyacyl-CoA
PhaC
(R)-3-Hydroxyacyl-ACP
3-Ketoacyl-ACP Enoyl-ACP
Acyl-ACP Malonyl-ACP
Malonyl-CoA
PATHWAY III (Fatty acid de novo
Biosynthesis) PhaG (Fatty acid degradation)
β-Oxidation
PhaA
PhaB
PhaC
Krebs cycle
FabG PhaJ
15 2.2.2 Biosynthesis of mcl-PHAs
Pseudomonads belongs to the rRNA-homology-group I can synthesize mcl- PHAs from various alkanes, alkanols or alkanoates (Lageveen et al., 1988). These bacteria can also synthesize PHA from unrelated carbon sources such as sugars which produce precursors that do not exhibit similar structure to the monomers (Anderson & Dawes, 1990). Biosynthesis of mcl-PHAs requires an additional enzyme to redirect 3-hydroxyacyl-CoA thioesters towards PHA synthase. From studies on P. putida KT2442, three different pathways were found to be involved in the synthesis of the 3-hydroxyal-CoA thioesters. These are fatty acid β- oxidation cycle, fatty acid de novo biosynthesis pathway and fatty acid chain elongation reaction. These pathways provide the intermediates for the production of mcl-PHAs.
2.2.2.1 Fatty acid β-oxidation cycle
When fatty acids are provided as the carbon sources, Pathway II which involves the fatty acid β-oxidation cycle generates substrates, 3-hydroxyacyl CoA that can be polymerised by the PHA synthase of pseudomonads such as P. putida.
The length of monomers produced via this pathway is similar to the substrates or shortened by 2, 4 or 6 carbon atoms (Lageveen et al., 1988). The intermediates of fatty acid β-oxidation cycle includes enoyl-CoA, 3-ketoacyl-CoA and (S)-3- hydroxyacyl-CoA which is used directly in mcl-PHA synthesis. In this pathway, fatty acids are activated by the acyl-CoA synthetase complex (FadD) forming the corresponding acyl-CoA thioesters. This molecule then enters the β-oxidation cycle where it is shortened by the removal of 2 carbon atoms in the form of acetyl
16
CoA. The shortened acyl-CoA is then reduced to trans-2, 3-enoyl-CoA catalyze by acyl-CoA dehydrogenase (FadE).
2.2.2.2 Fatty acid de novo biosynthesis pathway
The fatty acid de novo pathway is the most important pathway that produces monomers of 3-hydroxyacyl CoA for PHA synthesis as shown in pathway III in Figure 2.2. Simple carbon sources such as glucose, ethanol and acetate can be used as the starting materials (Huijberts et al., 1994). P. putida and P. aeruginosa produce copolyesters with 3-hydroxydecanoic acid as the main constituent and other 3HAs (3-hydroxyalkanoates) as minor constituent from glucose via this pathway (Steinbüchel & Fuchtenbusch, 1998). The (R)-3-hydroxyacyl intermediates from the fatty acid biosynthetic pathway are transferred from their acyl carrier protein (ACP) to CoA form by acyl-ACP-CoA transacylase (PhaG) (Rehm et al., 1998). This enzyme is the key connection between fatty acid synthesis and PHA biosynthesis and was identified in P. putida.
2.2.2.3 Chain elongation reaction pathway
The chain elongation pathway takes place during all types of PHA biosynthesis. In this pathway, acetyl-CoA molecules are condensed to 3- hydroxyacyl-CoA. Ketoacyl-CoA was formed by addition of acyl-CoA to acetyl- CoA. Ketoacyl-CoA was then converted to (R)-3-OH-acyl-CoA by the reaction of ketoacyl-CoA reductase (Hoffmann & Rehm, 2004). PHAs accumulated from sodium octanoate are generated via chain elongation reaction catalyzed by thiolase (Huijberts et al., 1994).
17 2.3 PHA Granules
PHA granules are found as insoluble spherical inclusions in the cytoplasm with hydrophobic PHA polyester as the core. Embedded proteins such as PHA synthases, phasins, depolymerising enzymes and regulatory proteins are present on the lipid monolayer surface (Grage et al., 2009). The size and molecular mass of PHA granules vary between organisms depending on carbon source provided, types of PHA synthase and the biosynthesis pathway involved. The sizes of the PHA molecules are about 50 to 1000 kDa with diameters of between 100 to 500 nm (Steinbüchel et al., 1995). These granules consist of about 103 to 104 monomers (Luengo et al., 2003). The number of granules per cell depends on the bacteria. For example, C. necator accumulates 8 to 12 granules per cell while P.
oleovorans generates only one or two large granules per cell (Klinke et al., 2000).
2.3.1 Structure of PHA granules
An electron microscopy study has shown that the core of PHA granules is surrounded by a 4 nm boundary layer, comprising a phospholipid monolayer (Mayer & Hoppert, 1997). Wide angle X-ray scattering showed that PHA granules are amorphous and treatments that removed lipid components can initiate crystallization (Kawaguchi & Doi, 1992). Analysis of PHA granules by atomic force microscopy (AFM) revealed an extra network layer with globular areas that incorporate structural proteins such as phasins (PhaP) (Dennis et al., 2008). The AFM also showed porin-like structures in the surrounding membrane and these were suggested to provide a portal to the polymer core for PHA metabolism and depolymerisation to occur (Dennis et al., 2003).
18 2.3.2 Formation of PHA granules
Two models for the PHA granule formation was described: the micelle model and the budding model as shown in Figure 2.3. Both models consider the location of the PHA synthase and phasin proteins on the surface of the granule.
The micelle model supported PHA granule formation in vitro and without membranes. This model assumes that once the first PHA chains have been synthesized, the polymer molecules aggregate to form nascent small granules by hydrophobic interaction. PHA synthase binds to the surface of PHA granules and becomes insoluble while phasin and other PHA specific proteins (PhaZ and PhaR) are also attached to the growing surface of the granules (Tian et al., 2005).
The budding model presumes that PHA synthase is linked to the cytoplasmic membrane. At the early stage, granules are close to the inner cell membrane and are not distributed in the cytoplasm (Tian et al., 2005). Electron microscopy studies showing membrane-like material surrounding PHA granules provided evidence for this model (Lundgren et al., 1964). In vivo studies using GFP-labeled PHA synthase from P. aeruginosa also supported the budding model by localizing granule formation close to the cytoplasmic membrane at the cell poles (Peters & Rehm, 2005). The budding model as shown in P. aeruginosa, involves four steps. First, PHA synthase was attached to the inner surface of the cytoplasmic membrane. This was followed by the formation of oligomers that bind to the polymerase in the inner surface of the cytoplasmic membrane. Then, polymer elongation takes place between the phospholipid monolayer of the membrane. This environment is hydrophobic. Finally, granule associated proteins (phasins) direct the formation of the budding granules (Rehm, 2006).
19 Soluble PHA synthase
Amphipathic PHA synthase
Figure 2.3 PHA granule formation models. (I) Micelle model representing in vitro formation in the absence of membranes and (II) Budding model representing granule formation at the cytoplasmic membrane (adapted from Tian et al., 2005).
I II Intracellular
Lipid membrane
Extracellular
PhaP Hydroxyacyl-CoA
PHA granule PHA granule
20
2.3.2.1 Formation of PHA granules in Pseudomonas sp. USM4-55
Pseudomonas sp. USM4-55 is capable of producing a blend of PHB homopolymer and copolymer of mcl-PHAs that contains a small amount of PHB monomers. The copolymers are inseparable by solvent fractionation. The PHA granules of Pseudomonas sp. USM4-55 have been isolated and studied in detail (Sudesh et al., 2004). In this bacterium, PHB and mcl-PHA exist in the same cell but are separated in different granules. The micelle and budding models cannot be used to explain why PHB chain and mcl-PHA growing from different PHA synthases are segregated into separate granules. Therefore, a different mechanism was adopted to describe the granule formation event in this bacterium (Sudesh et al., 2004). Two mechanisms were proposed for the granule formation in Pseudomonas sp. USM4-55 as shown in Figure 2.4. In the first mechanism, two types of PHAs are segregated into different granules due to phasin proteins that can interact directly with the hydrophobic PHA chains (Sudesh et al., 2004). The presence of mcl-PHA in a polymer chain of mostly 3HB monomers resulted in unstable granule (Sudesh et al., 2002). Therefore segregation of PHB and mcl- PHA into separate granules is essential.
The second mechanism demonstrated that the formation of the PHA synthase complex is due to the observation of a lag phase in the polymerization reaction. Enzymes with the same substrate specificity will form a complex with each other and thus each of PHA synthase enzymes will generate one granule (Sudesh et al., 2004). Mechanism II offers a straightforward description for the separation of PHB and mcl-PHA into different granules in Pseudomonas sp.
USM4-55.
21
Figure 2.4 Predicted granule formation model in Pseudomonas sp. USM4-55 (adapted from Sudesh et al., 2004). White spheres represent scl-PHA synthase while black spheres represent mcl-PHA synthase.
A B
Bacterial cell
Observed phenomenon in Pseudomonas sp. USM4-55
Mechanism II
PHA synthase
PHA synthase complexes
MCL-PHA SCL-PHA SCL-PHA
MCL-PHA
PHA synthase Mechanism I
Existing model for PHA granule formation
22 2.4 PHA synthases
PHA synthase is the key enzyme of PHA biosynthesis and determines the type of PHA synthesized. This enzyme catalyzes the polymerization of hydroxyacyl-CoA to PHA and free CoA. PHA synthase enzyme shows a wide range of substrate specificity and hence a variety of monomers can be polymerised.
PHA synthases from a variety of bacteria were cloned and the alignment of the primary structures showed an overall identity of 21-88% with 8 conserved amino acid residues (Amara & Rehm, 2003). All PHA synthases share a conserved cysteine at the catalytic active site to which the growing PHA is covalently bonded. These enzymes are mainly composed of variable loops (49.7%) and α-helical (39.9%) secondary structures, while 10.4% are predicted as β-sheet secondary structures (Cuff & Barton, 2000). PHA synthases are attached to the surfaces of PHA granules in PHA accumulating cells as shown by immunogold labelling in electron microscopy and in vivo analysis using PhaC- GFP fusion (Mayer et al., 1996; McCool & Cannon, 1999).
2.4.1 Classification of PHA synthases
PHA synthases can be divided into four classes based on their primary structure, substrate specificity and subunit composition as shown in Table 2.2(A).
Class I PHA synthases consist of one type of subunit (PhaC) with sizes of 61 kDa to 73 kDa. Synthases in this class prefer to utilize CoA thiosters of various (R)-3- hydroxy fatty acids comprising 3 to 5 carbon atoms (Qi & Rehm, 2001). The PHA synthase in C. necator is an example of a Class I synthase. However, in vivo
23
studies of C. necator PHA synthase in recombinant E. coli showed that this enzyme also accepts mcl 3-hydroxy fatty acid-CoA thioesters as substrate (Antonio et al., 2000).
Class II PHA synthases also comprise of one type of subunit which utilizes CoA thioesters of various (R)-3-hydroxy fatty acids comprising 6 to 14 carbon atoms (Amara & Rehm, 2003). Class II synthases synthesize smaller PHAs with sizes up to 500 kDa while Class I synthases synthesize PHA bigger than 500 kDa (Rehm & Steinbüchel, 1999). The PHA synthases in P. putida is an example of Class II synthases.
Class III PHA synthases represent enzymes with two different subunits of PhaC and PhaE with a size of 40 kDa each. These synthases prefer CoA thioester of (R)-3-hydroxy fatty acids of 3 to 5 carbon atoms (Liebergesell et al., 1992).
The PHA synthase in Allochromatium vinosum is an example of the Class III synthase. PhaC subunit from A. vinosum exhibit low (21% to 28%) but significant sequence similarity with Class I and Class II synthases and shared highly conserved amino acid positions, albeit smaller than the other synthases. However, PhaE does not exhibit any significant homology with other synthases. Aside from A. vinosum, PHA synthase from phototrophic purple sulphur bacteria and cyanobacteria are also classified as Class III PHA synthases (Steinbüchel & Hein, 2001).
Class IV PHA synthase is represented by B. megaterium. This synthase resembles Class III synthases but PhaE is substituted by PhaR. PhaR has the size of 20 kDa. B. megaterium required both PhaC and PhaR for activity in vivo and in vitro (McCool & Cannon, 2001).
24
There are other synthases that do not belong in any class. Thiocapsa pfennigii synthase possesses two different subunits with strong similarity (85%) to PhaC from Class III synthases. This PHA synthase is characterized by broad substrate specificity comprising CoA thioesters of 3 to 14 carbon atoms. This synthase catalyzes the formation of copolyester PHB and P(3HHX) (Rehm, 2003).
2.4.1.1 PHA synthases in pseudomonads (Class II)
Class II PHA synthases (PhaC1 and PhaC2) are primarily found in pseudomonads and they prefer mcl-3HAs containing 6 to 14 carbons as substrates (Huisman et al., 1991). The variation between phaC1 and phaC2 sequences may signify the structural and functional differences (Zhang et al., 2001). A study on phaC1 and phaC2 knockout mutant strains in P. mendocina revealed that PhaC1 is the major enzyme for PHA synthesis while PhaC2 contribute to the PHA accumulation only when cells are cultivated in gluconate (Hein et al., 2002).
PhaC2 could not replace PhaC1 very effectively and resulted in a reduced ability of phaC1 mutant strain to form PHA from gluconate and no PHA was accumulated from fatty acids. Disruption of phaC2 did not show a serious effect.
The same phenomenon was also demonstrated in Pseudomonas sp. USM4-55 and this lead to the conclusion that PhaC1 is the major PHA synthase in pseudomonads (Kamariah, 2007).
Few pseudomonads exhibit broader substrate specificity as shown by Pseudomonas sp. 61-3 and Pseudomonas sp. USM4-55 (Abe et al., 1994; Sudesh et al., 2004). These strains are able to polymerize both scl-PHAs and mcl-PHAs.
