Molecular Characterization of Isoprenoid Biosynthesis Gene Cluster and Enhanced Production of Astaxanthin
- Alternative Title
- 이소프레노이드 생합성 유전자군의 분자생물학적 특성과 아스타잔틴 생산량 증대 연구
- Abstract
- Isoprenoids are most important natural products. Recently, each country makes a tremendous effort to isolate and express the isoprenoid biosynthesis genes resulting in the production of isoprenoids in a heterologous host. The objectives of this research were the isolation of new microorganisms producing isoprenoids, the molecular cloning of isoprenoid biosynthesis genes from the isolated organisms in heterologous microorganisms, and the characterization of isoprenoid enzymes overexpressed in Escherichia coli with isoprenoid biosynthesis genes.
In a previous study, we isolated and characterized a marine bacterium, Kocuria gwangalliensis strain SJ2, which produces isoprenoids. The isoprenoid biosynthesis genes has been cloned and this gene composed of eight genes that identified as dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. The dxs gene of 2013bp encodes 670 amino acid residues. The ispC gene of 1305 bp encodes 434 amino acid residues. The ispD gene of 765 bp encodes 254 amino acid residues. The ispE gene of 1014 bp encodes 337 amino acid residues. The ispF gene of 516 bp encodes 171 amino acid residues. The ispG gene of 1146 bp encodies 381 amino acid residues. The ispH gene of 1059 bp encodies 352 amino acid residues. The idi gene of 543 bp encodes 180 amino acid residues.
In order to characterize the enzymatic properties of each gene product of carotenoid biosynthesis genes from K. gwangalliensis strain SJ2, each gene was subcloned into the expression vector, pColdII vector. The result showed that the plasmid containing DXS, IspC, IspD, IspE, IspF, IspG, IspH and IDI genes produced the recombinant proteins to be about 72 kDa, 46 kDa, 26 kDa, 35 kDa, 18 kDa, 40 kDa, 38 kDa and 20 kDa, respectively. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding of enzyme kinetics studies result. The goal of this study was to increase the production of astaxanthin in recombinant E. coli by an engineered non-mevalonate pathway.
We previously reported on the structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis strain BC74171. The carotenoid biosynthesis gene cluster which was involved in astaxanthin production and contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE). The recombinant E. coli harboring the six carotenogenic genes from P. haeundaensis strain BC74171, produced 400 μg/g DCW (dry cell weight) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we cloned dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi in the non-mevalonate pathway from K. gwangalliensis strain SJ2 and co-expressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain, containing both the non-mevalonate pathway and the astaxanthin biosynthesis gene cluster, produced 1100 μg/g DCW of astaxanthin, resulting in approximately three times of the production of astaxanthin.
- Issued Date
- 2015
- Awarded Date
- 2015. 2
- Type
- Dissertation
- Publisher
- 부경대학교 자연과학대학
- Alternative Author(s)
- Tae Hyug Jeong
- Affiliation
- 부경대학교 자연과학대학 미생물학과
- Department
- 대학원 미생물학과
- Advisor
- 김영태
- Table Of Contents
- Abstract I
Contents III
List of Tables VIII
List of Figures IX
List of Abbreviations XIII
Part I. General Introduction of Non-mevalonate Pathway and Isoprenoid Biosynthesis Genes 1
Chapter 1. General Introduction of Nom-mevalonate Pathway and Isoprenoid Biosynthesis Genes 2
1. Introduction 2
2. Isoprenoid compounds 5
3. Gene and enzymes of the non-mevalonate pathway 7
4. Importance of the non-mevalonate pathway 10
5. Importance of the carotenoids 12
6. Objectives of research 16
7. References 17
Part II. Molecular Characterization of Isoprenoid Biosynthesis Gene Cluster and Enhanced Production of Astaxanthin 21
Chapter 2. Cloning And Characterization of Isoprenoids Biosynthesis Genes from The Marine Bacterium, Kocuria gwangalliensis strain SJ2 22
1. Abstract 22
2. Introduction 24
3. Materials and methods 27
3.1 Bacterial strains and growth condition 27
3.2 Genomic DNA extraction and construction of cosmid library 27
3.3 Cloning of isopenoid biosynthesis genes from K. gwangalliensis strain SJ2 28
3.4 Comparative sequence analysis of isoprenoid biosynthesis gene (dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi) from K. gwangalliensis strain SJ2 28
4. Results and discussion 31
5. References 56
Chapter 3. Overexpression and Characterization of Isoprenoid Biosynthesis Genes from The Marine Bacterium, Kocuria gwangalliensis strain SJ2 58
1. Abstract 58
2. Introduction 60
3. Materials and methods 62
3.1 Construction of isoprenoid biosynthesis gene expression vector and overexpression 62
3.2 Extraction and purification of the recombinant DXS, IspC, IspD, IspE, IspF, IspG, IspH and IDI proteins 65
3.3 Western blot analysis of the recombinant DXS, IspC, IspD, IspE, IspF, IspG, IspH and IDI proteins 67
3.4 In vitro enzymatic assay 67
4. Results and discussion 70
5. References 86
Chapter 4. Enhanced production of astaxanthin by metabolically engineered non-mevalonate pathway in Escherichia coli 88
1. Abstract 88
2. Introduction 89
3. Materials and methods 93
3.1 Bacterial strains and growth conditions 93
3.2 Construction of vector system for co-expression in order to increase the production of astaxanthin 93
3.3 Extraction of astaxanthin 94
3.4 Purification and measurement of astaxanthin 97
4. Results and discussion 98
4.1 Vector construction and expression of the isoprenoid biosynthesis genes 98
4.2 Measurement of astaxanthin production 99
4.3 Astaxanthin production by recombinant E. coli growth patterns 101
5. References 106
Part Ⅲ. APPENDIX 109
Chapter 5. Overexpression and Characterization of Lycopene Cyclase (crtY) from Marine Bacterium, Paracoccus haeundaensis strain BC74171 110
1. Abstract 110
2. Introduction 111
3. Materials and methods 113
3.1 Overexpression and purification of lycopene cyclase (crtY) gene 113
3.2 Purification procedures of recombinant CrtY protein 113
3.3 Analysis of the expressed protein by SDS-PAGE and Western blotting 114
3.4 Enzyme assay 115
4. Results and discussion 116
5. References 125
Chapter 6. Cloning and Characterization of D-xylulose Kinase (XK) From Kocuria gwangalliensis strain SJ2 127
1. Abstract 127
2. Introduction 128
3. Materials and methods 129
3.1 Bacterial strains and growth conditions 129
3.2 Genomic DNA extraction and construction of cosmid library 129
3.3 Cloning of D-xylulose kinase gene from K. gwangalliensis strain SJ2 130
3.4 Comparative sequence analysis of D-xylulose kinase gene 130
3.5 D-xylulose kinase overexpression 131
3.6 Refinement of D-xylulose kinase through His-tag affinity chromatography 131
3.7 Identification of D-xylulose kinase through western blot analysis 132
3.8 In vitro enzymatic assay 132
4. Results and discussion 135
4.1 D-Xylulose kinase gene cloning from K. gwangalliensis strain SJ2 135
4.2 Amino acid sequence analysis of D-xylulose kinase 135
4.3 Expression of D-xylulose kinase in E. coli 136
4.4 Purification of the D-xylulose kinase 136
4.5 Enzyme activity of D-xylulose kinase 141
5. References 143
Chapter 7. Cloning and Characterization of Phosphoinositide 3-kinase γ cDNA from Flounder (Paralichthys olivaceus) 145
1. Abstract 145
2. Introduction 147
3. Materials and methods 149
3.1 RNA isolation and construction of the flounder cDNA library 149
3.2 Screening PI3Kγ cDNA and DNA sequencing 149
3.3 Comparative sequence analysis of flounder PI3Kγ 151
3.4 Reverse transcription polymerase chain reaction (RT-PCR) 151
3.5 Expression of flounder PI3Kγ gene in Escherichia coli 152
3.6 Purification of recombinant PI3Kγ proteins 152
3.7 Enzyme activity assay 153
3.8 Protein determination 153
4. Results and discussion 154
4.1 Nucleotide sequences of flounder PI3Kγ 154
4.2 Sequence identity and the phylogenetic tree 154
4.3 Tissue distribution of PI3Kγ 155
4.4 Expression of flounder PI3Kγ in E. coli 160
4.5 Western blot analysis 160
4.6 Purification of the PI3Kγ protein 162
4.7 Enzyme activity of PI3Kγ 162
5. References 167
Korean Summary 170
- Degree
- Doctor
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