Cas9-sgRNA 리보핵산단백질복합체를 이용한 류코노스톡 시트륨 유전체 편집기술 개발 = Development of Genome Editing System in Leuconostoc citreum Using Cas9-sgRNA Ribonucleoprotein Complexes

다국어 초록 (Multilingual Abstract)

Lactic acid bacteria (LAB) have been used as starters or probiotics for foods fermentation and cell factories for bioresource production. Leuconostoc citreum is a gram positive, facultative anaerobic, and hetero-fermentative LAB frequently found in fermented vegetables, and has been used as a starter culture in kimchi fermentation. To improve their probiotic properties, removal of undesired characteristics and addition of new desirable properties by efficient genome engineering tool is necessary for probiotics or industrial use. Recently, the CRISPR-Cas9 system which is bacterial defense mechanism against virus infection has been widely used as a powerful genome editing tools in various cell types such as animal, plant, fungi, and yeast. Delivery of Cas9 by ribonucleoprotein complexes (RNP), in which recombinant Cas9 protein preassembled with sgRNA, are preferable methods due to easy production, low off-target effect, and no requirement of vector system. Although RNP system have been used for various eukaryotes cells, no studies have been applied to LAB due to limitations such as complex cell wall structure, intracellular nucleases, and lack of recombinase. Therefore, the aim of this study is development of efficient genome editing system in L. citreum using Cas9-sgRNA ribonucleoprotein complexes. To achieve the goal, RNP concentration and electroporation conditions were optimized for introducing RNP into L. citreum, and thiolated DNA and RecT recombinase originated from Lactobacillus plantarum WCFS1 were employed to increase the repair efficiency. In addition, to support above experiments, complete genome sequences of L. citreum EFEL2700 was analyzed and species/strain-specific monitoring technique using real-time qPCR was established.
In chapter 2, the complete genome sequence of L. citreum EFEL2700 was analyzed by Illumina and Pacbio sequencing. As results, this strain has 1 chromosome and 4 plasmids (total 1,923,830 bp) with a G + C content of 39.0% and high homology (≥ 99%) in ANI value to the reference strain L. citreum KM 20. The KEGG analysis of metabolic pathways showed a typical hetero-type lactic acid fermentation with biosynthetic route for coenzyme A, and terpenoid backbone including isoflavonoid and riboflavin. No core genes for primary metabolism were present in plasmid 4 and it could be eliminated to create an efficient host for gene transformation.
In chapter 3, species/strain-specific quantitative PCR for Leuconostoc spp. were developed and they were applied for identification and enumeration of starter culture in fermented food. As results, primers, NlacF and NlacR, for detecting L. lactis strains were developed based on pan-genome analysis. PCR analysis against genomic DNA of various LAB exhibited the primer set made a specific binding with genomic DNA of L. lactis species. The qPCR with these primers was proved to enumerate accurate population of L. lactis during kimchi fermentation. In addition, three primer sets for detecting L. mesenteroides DRC1506 were developed based on pan-genome analysis. PCR analysis against genomic DNA of various LAB exhibited the primer set made a specific binding with genomic DNA of L. mesenteroides DRC1506 strains. The qPCR method was proved to count the L. mesenteroides DRC1506 during kimchi fermentation.
In chapter 4, genome editing of L. citreum via Cas9-sgRNA ribonucleoprotein complexes with RecT was conducted. For this, dextransucrase gene was selected as a target DNA and pre-assembled RNP complexes with RecT were introduced to L. citreum by electroporation. As result, dextran-free colonies were successfully obtained on sucrose agar medium, and it was found that single nucleotide deletion occurred resulting in gene knock-out by a frame shift. To increase the mutation efficiency, thiolated donor DNA and RecT recombinase were employed and the efficiency was increased up to 3.9%.
In chapter 5, characteristics of L. citreum Δdsr mutant obtained in the above chapter were analyzed and its fermentation profile was investigated during dongchimi fermentation. As result, the cell growth rate and pH change of the mutant were not significantly different (p < 0·05) compared to the wild type during fermentation. When the mutant strain was inoculated as a starter in dongchimi, the mutant slowly metabolized sucrose resulting in significant reduction (6.5-folds) of dextran production compared to the wild type during fermentation. NMR analysis revealed that the polymer produced by the mutant was alternan having α-(1→3) (39.5%) or α-(1→6)-linkage (60.5%) possibly synthesized by alternansucrase. Meanwhile, viable cell counts and pH change were not significantly different (p < 0·05) compared to the wild type.
In conclusion, for the first time in LAB, dextransucrase gene in L. citreum EFEL2700 was deleted by electroporation of Cas9-sgRNA ribonucleoprotein complexes and the knock-out efficiency was increased up to 3.9% by employing both thiolated donor DNA and RecT recombinase. As a universal genome engineering tool customized for LAB, this RNP complex method can be applicable to other LAB such as Bifidobacterium, Lactobacillus, and Lactococcus beyond the genus barrier. In addition, the DNA-free tools used in this study can be regarded as non-LMO technology, when no exogenous DNA were used.

목차 (Table of Contents)

• CONTENTS
• CONTENTS.............................................................................................. i
• LIST OF TABLES................................................................................ vi
• LIST OF FIGURES.................................................................................. viii
• ABBREVIATIONS................................................................................... xii
• SUMMARY............................................................................................... xiv
• Chapter I. Literature review and research objectives........................... 1
• 1.1 Lactic acid bacteria............................................................................... 2
• 1.2 Leuconostoc citreum............................................................................. 4
• 1.3 Genome sequencing and its application................................................. 7
• 1.3.1 Whole genome sequencing............................................................... 7
• 1.3.2 Species/strain-specific barcode PCR................................................. 9
• 1.4 Genome engineering tools.................................................................... 10
• 1.5 CRISPR-Cas9 system........................................................................... 15
• 1.5.1 CRISPR-Cas9.................................................................................. 15
• 1.5.2 Guide RNA...................................................................................... 20
• 1.6 Ribonucleoprotein................................................................................. 23
• 1.6.1 Delivery of ribonucleoprotein......................................................... 23
• 1.6.2 Difficulities of delivery of RNP...................................................... 26
• 1.7 Recombineering.................................................................................... 28
• 1.7.1 Non-homologous end joining.......................................................... 28
• 1.7.2 Homology-directed repair................................................................ 33
• 1.7.3 Single stranded DNA recombineering.............................................. 34
• 1.7.4 Double stranded DNA recombineering............................................ 36
• 1.8 Recombinase......................................................................................... 38
• 1.9 Applications of CRISPR-Cas systems in lactic acid bacteria................. 40
• 1.10 Research necessity............................................................................... 42
• 1.11 Research objectives and experimental designs.................................... 42
• Chapter II. Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors .................
• 44
• 2.1 Introduction........................................................................................... 45
• 2.2 Materials and methods......................................................................... 47
• 2.2.1 Bacterial culture and DNA extraction............................................. 47
• 2.2.2 Microbial characteristics.................................................................. 47
• 2.2.3 Whole-genome sequencing, assembly, and annotation.................. 47
• 2.2.4 Phylogenetic analysis and ANI comparison................................... 48
• 2.2.5 Plasmid transformation into L. citreum EFEL2700........................ 51
• 2.2.6 Determination of virulence factors and undesirable genes................ 51
• 2.2.7 Determination of antibiotic resistance.............................................. 52
• 2.3 Results................................................................................................... 53
• 2.3.1 Microbial features............................................................................ 53
• 2.3.2 Genomic features.............................................................................. 57
• 2.3.3 Metabolic pathway analysis............................................................. 66
• 2.3.4 Transformation efficiency................................................................ 68
• 2.3.5 Determination of virulence factor and toxin-encoding genes............ 70
• 2.3.6 Antibiotic resistance genes................................................................ 74
• 2.4 Discussion............................................................................................. 79
• 2.5 Summary............................................................................................... 80
• Chapter III. Development of species/strain specific quantitative PCR and its application for identification and enumeration in fermented food .......................................................................
• 81
• 3.1 Introduction........................................................................................... 82
• 3.2 Materials and methods........................................................................... 85
• 3.2.1 Bacterial strains and culture conditions............................................ 85
• 3.2.2 Primer design for PCR...................................................................... 89
• 3.2.3 Kimchi preparation........................................................................... 90
• 3.2.4 Polymerase chain reaction................................................................ 90
• 3.2.5 Quantitative polymerase chain reaction............................................ 92
• 3.2.6 Enumeration of inoculated starters.................................................... 93
• 3.2.7 Microbial and chemical analysis in kimchi fermentation.................. 94
• 3.2.8 Statistical analysis............................................................................. 95
• 3.3 Results................................................................................................... 95
• 3.3.1 L. lactis study results......................................................................... 95
• 3.3.1.1 Design of L. lactis-specific primers............................................... 95
• 3.3.1.2 Polymerase chain reaction for L. lactis.......................................... 100
• 3.3.1.3 Standard curve for qPCR............................................................... 102
• 3.3.1.4 Recovery yield of gDNA from MRS medium and kimchi............. 105
• 3.3.1.5 Growth monitoring in MRS medium............................................. 107
• 3.3.1.6 Growth monitoring in kimchi........................................................ 109
• 3.3.2 L. mesenteroides DRC1506 study results.......................................... 111
• 3.3.2.1 L. mesenteroides DRC1506-specific primers................................ 111
• 3.3.2.2 Multiplex PCR for L. mesenteroides DRC1506............................. 114
• 3.3.2.3 Standard curve for qPCR............................................................... 116
• 3.3.2.4 Recovery yield of gDNA from MRS and kimchi........................... 119
• 3.3.2.5 Growth monitoring in MRS medium............................................. 121
• 3.3.2.6 Growth monitoring of L. mesenteroides DRC1506 during kimchi fermentation................................................................................
• 123
• 3.4 Discussion............................................................................................. 125
• 3.5 Summary...............................................................................................
• 129
• Chapter Ⅳ. Gene editing in Leuconostoc citreum via Cas9-sgRNA ribonucleoprotein complex.......................................................................
• 131
• 4.1 Introduction........................................................................................... 132
• 4.2 Materials and methods........................................................................... 134
• 4.2.1 Bacterial strains, plasmids, and growth conditions........................... 134
• 4.2.2 Design of sgRNA for knock-outs of interest..................................... 134
• 4.2.3 Cas9 protein expression and purification.......................................... 137
• 4.2.4 In vitro transcription of sgRNAs........................................................ 138
• 4.2.5 Assay method for in vitro cleavage reaction...................................... 141
• 4.2.6 RecT expression............................................................................... 141
• 4.2.7 Construction of donor DNA............................................................. 144
• 4.2.8 Preparation of competent cell........................................................... 147
• 4.2.9 RNP electroporation......................................................................... 147
• 4.3 Results................................................................................................... 149
• 4.3.1 Preparation of recombinant Cas9....................................................... 149
• 4.3.2 sgRNA for dextransucrase knock-out................................................ 151
• 4.3.3 Expression of RecT recombinase...................................................... 154
• 4.3.4 RNP nuclease activity assay.............................................................. 158
• 4.3.5 Optimization of competent cell.......................................................... 160
• 4.3.6 Construction of donor DNA............................................................. 165
• 4.3.7 Effect of thiolated donor DNA........................................................... 167
• 4.3.8 Electoporation of RNP complex with thiolated DNA and RecT......... 169
• 4.4 Discussion............................................................................................. 173
• 4.5 Summary............................................................................................... 177
• Chapter Ⅴ. Characteristics of mutated Leuconostoc citreum using Cas9-sgRNA ribonucleoprotein complex and its application ………
• 179
• 5.1 Introduction........................................................................................... 180
• 5.2 Materials and methods........................................................................... 183
• 5.2.1 Bacterial strains, plasmid, and growth condition.............................. 183
• 5.2.2 Cas9 protein expression and purification.......................................... 183
• 5.2.3 In vitro transcription of sgRNA........................................................ 183
• 5.2.4 RNP electroporation......................................................................... 183
• 5.2.5 Mutation confirmation....................................................................... 183
• 5.2.6 Preparation of kimchi (dongchimi-kimchi)...................................... 184
• 5.2.7 Microbial and chemical analysis....................................................... 184
• 5.2.8 Analysis of sugar metabolites............................................................ 185
• 5.2.9 Proton NMR analysis....................................................................... 185
• 5.2.10 Structural analysis of EPS............................................................... 186
• 5.3 Results................................................................................................... 187
• 5.3.1 DNA-free RNP transformation.......................................................... 187
• 5.3.2 Enzyme expression and polysaccharide formation........................... 189
• 5.3.3 Comparison of cell growth............................................................... 192
• 5.3.4 Sequencing analysis......................................................................... 194
• 5.3.5 Color changes during dongchimi fermentation.................................. 196
• 5.3.6 Cell growth and sugar changes during dongchimi fermentation........ 199
• 5.3.7 Structure of exopolysaccharide........................................................ 202
• 5.4 Discussion............................................................................................. 206
• 5.5 Summary............................................................................................... 208
• REFERENCES.......................................................................................... 209
• SUMMARY IN KOREAN....................................................................... 240