Direct conversion of CO₂ to squalene by metabolically-engineered Synechococcus elongatus PCC 7942

다국어 초록 (Multilingual Abstract)

Synechococcus elongatus PCC 7942, a photosynthetic bacterium, has great potentials for value-added chemicals production due to autotrophic growth and CO2 conversion as bio-solar cell factories. Here, S. elongatus PCC 7942 strains were engineered by transforming the overexpressed MEP modular genes with squalene synthase. And also, we investigated to confirm increment contents of SQ accordance to application of cpcB1 protein and ispA, as fusion partner with squalene synthase. Moreover, we analyzed transcriptome to understand the metabolic changes for nitrogen starvation conditions. Thus, we performed RNA-sequencing according to either nitrogen rich or free conditions. The accumulation of squalene in intra cell was confirmed by GC_MS and TEM analysis method. The squalene synthase led to the synthesis of a highest levels (11.98 mg/L/OD730 ± 0.9) reported so far in cyanobacteria. We identified differentially express genes and confirmed physiological response changes for up or down regulation genes among differentially expressed genes (p value < 0.01) during N starvation.
In conclusions, this is the first demonstration of photosynthetic production of squalene from CO2 in engineered S. elongatus PCC 7942 strains. Further, transcriptome result could be useful to metabolically engineer S. elongatus PCC 7942 under nitrogen starvation condition.

Keywords: Synechococcus elongatus PCC7942, Metabolic engineering

목차 (Table of Contents)

• ABSTRACOF THESISV……………………………………VII
• LIST OF FIGURES…………………………………………VIII
• LIST OF TABLES…………………………………………XIV
• Chapter 1. Research background………………………1
• 1.1. Microbial as cell factory……………………………2
• 1.1.1. Technologies for biosystem metabolic engineering…………………………………………………4
• 1.1.2. System metabolic engineering of microorganism for biobased material…………………………………………9
• 1.1.3. Squalene biosynthesis through metabolic engineering………………………………………………13
• 1.2. Prospects of microbial cell factories by Cyanobacteria……………………………………………19
• Chapter 2. Research objective and significance………………………23
• 2.1. Research objective and significance………………………24
• Chapter 3. Photosynthetic production of Squalene from CO2 using engineered Synechococcus enlongatus PCC 7942 ………………………29
• 3.1. Abstract………………………30
• 3.2. Introduction………………………32
• 3.3. Materials and Methods………………………35
• 3.3.1. Medium and culture conditions ………………………35
• 3.3.2. Plasmid construction for squalene production using SyneBrick vector ………………………35
• 3.3.3. Transformation………………………36
• 3.3.4. GC_MS………………………37
• 3.3.5. Electron Microscopy………………………38
• 3.3.6. High resolution microscopy and confocal microscopy ………………………39
• 3.3.7. RNA preparation and qRT PCR………………………40
• 3.3.8. Chlorophyll a measurment………………………41
• 3.3.9. Chemicals and reagents………………………41
• 3.4. Results and Discussion……………………… 42
• 3.4.1. Designing modularized pathways for the production of squalene………………………42
• 3.4.2. Transformation confirmation of constructed vectors as synthetic biology platform………………………46
• 3.4.3. Analysis of Photosynthetic production of squalene from CO2 by GC-MS ………………………50
• 3.4.4. Analysis of SQ in engineered S. elongatus PCC 7942 using Microscopy………………………53
• 3.4.5. Alleviating the cyanobacterial growth inhibition by expressing a terpene synthase in S.elongatus PCC 7942 strain with the optimized MEP pathway only………………………59
• 3.4.6. The cyanobacterial RNA expression level of downstream FPP by expressing squalene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway ………………………60
• 3.5. Conclusion………………………65
• Chapter 4. Improvement of squalene production from CO2 in engineered Synechococcus elgonatus PCC 7942 using a push-and-pull strategy………………………67
• 4.1. Abstract………………………68
• 4.2. Introduction ………………………69
• 4.3. Meterials and Methods………………………73
• 4.3.1. Medium, culture condition………………………73
• 4.3.2. Conjugation………………………73
• 4.3.3.Construction of plasmid to make fusion protein and transformation………………………74
• 4.3.4. Fluometric analysis of squalene contents by using TECAN………………………76
• 4.3.5. Confirmation of SQ production engineered strain with fusion………………………76
• 4.3.6. Scalable culture in tubing bag ………………………77
• 4.4. Results and Discussion………………………78
• 4.4.1. The growth of conjugated strain………………………78
• 4.4.2. Direct use of IspA and cpcB1 as fusion partner for squalene production………………………79
• 4.4.3. Introduction engineered strain with concept of direct fusion and gene copy in cyanobacteria genome for increasing of SQ……………………… 89
• 4.4.4. The relative fluorescence analysis by staining of Nile red………………………94
• 4.4.5. The optimization of IPTG concentraion………………………96
• 4.4.6. Scalable production in bag-type photobioreactor………………………98
• 4.5. Conclusion………………………105
• Appendix I. Transcriptome analysis of Synechococcus elongatus PCC 7942 for nitrogen starvation responses using RNA-seq………………………107
• A.1. Abstract………………………108
• A.2. Introduction………………………109
• A.3. Meterials and Methods………………………112
• A.3.1. Cyanobacterial strain and growth conditions……………………… 112
• A.3.2. RNA preparation and RNA-seq.………………………112
• A.3.3. Data analysis and statistical analysis………………………114
• A.3.4. Measurement of Chlorophyll a and phycocyanin contents using spectrophotometer………………………115
• A.3.5. Calculations for carbon fixation rate using CO2 analysis………………………115
• A.4. Results and Discussion………………………117
• A.4.1. Nitrogen starvation response of S. elongatus PCC 7942………………………117
• A.4.2. Global transcriptomic analysis of S. elongatus PCC 7942 for nitrogen starvation response………………………117
• A.4.3. The classification of differentially expressed gene along module………………………124
• A.4.4. Transcriptomic analysis in specific metabolisms of S. elongatus PCC 7942 for nitrogen starvation response………………………131
• A.4.5. Biological evidences for down-regulated photosynthesis and up-regulated bicarbonate transporter of S. elongatus PCC 7942 for nitrogen starvation response………………………138
• A.5. Conclusion………………………144
• Supplementry table ………………………149
• Appendix II. Abbreviations………………………166
• Summary………………………167
• Reference………………………169
• Abstract in Korean……………199