Domesticating the non-model yeast Wickerhamomyces ciferrii as a synthetic biology chassis through development of genetic toolkits and genome engineering strategies
- 주제(키워드) Wickerhamomyces ciferrii , non-model yeast , genetic toolkit development , NHEJ multicopy integration , CRISPR/Cas9 system
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 이평천
- 발행년도 2026
- 학위수여년월 2026. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000036267
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Wickerhamomyces ciferrii is an industrial diploid yeast that naturally produces and secretes tetraacetyl phytosphingosine （TAPS）, an acetylated sphingoid base derivative used in cosmetic and pharmaceutical applications. Despite this industrial potential, rational strain engineering in W. ciferrii has been limited by the lack of validated genetic tools, incomplete genome-level information, and inefficient homologous recombination caused by a strong preference for non-homologous end joining （NHEJ）. This dissertation aimed to establish the genetic and genomic basis for engineering W. ciferrii as a tractable host for sphingolipid bioproduction. First, a modular plasmid toolkit was developed by evaluating episomal replication origins, selectable markers, fluorescent reporters, and endogenous promoters. The Saccharomyces cerevisiae 2µ and CEN6/ARS4 origins supported plasmid maintenance, whereas the Kluyveromyces lactis panARS element did not. Nourseothricin, geneticin, and zeocin resistance markers were validated as practical, and promoter strength was ranked as pTDH3 〉 pPGK1 〉 pPDA1. A positive correlation between codon adaptation index and transcript abundance indicated that codon optimization is an important design factor in this low-GC host. The toolkit was functionally validated by overexpressing a phosphorylation-insensitive ACC1 variant, which increased TAPS production by 2.4-fold. Second, genome-level features of the industrial diploid strain were analyzed to guide downstream engineering. Reference-free k-mer analysis estimated a haploid genome size of approximately 15.6 Mb and revealed substantial heterozygosity of approximately 3.95%. Smudgeplot analysis supported a predominantly diploid allele structure. Assembly-based analyses identified the native 11-bp telomeric repeat sequence and 370 predicted tRNA loci, providing key sequence resources for donor DNA design and tRNA-based sgRNA expression. Third, the strong NHEJ activity of W. ciferrii was used as an engineering advantage rather than suppressed. An NHEJ-mediated multicopy genome integration platform was developed by combining three design features: telomeric repeat sequences added to donor DNA ends to improve donor stability, a truncated URA5 promoter to enrich high-copy integrants, and 5′-phosphorylated donor ends to improve end joining. These elements were integrated into the platform vector pTdmVU5. Using this system, multicopy chromosomal integration of LCB1 and LCB2, which encode the two subunits of serine palmitoyltransferase, increased TAPS production by 2.7-fold relative to the wild-type control. Finally, a CRISPR/Cas9 system was established for targeted genome engineering in W. ciferrii. Cas9 expression was adapted to the host by codon optimization, nuclear localization signal screening, single-copy chromosomal integration, and biallelic target spacer design. sgRNA expression was achieved using an endogenous tRNA-based Pol III promoter, and target spacers were selected using allele-aware analysis of the diploid genome. The resulting system achieved 67–70% editing efficiency at single-copy URA5 and Venus targets and approximately 20% biallelic disruption at the endogenous HIS3 locus. In addition, a proof-of-concept screen for homology-independent insertion of an mRuby2 cassette at a Cas9-directed Venus target identified 9 of 64 transformants （14.1%） as candidates by dual-fluorescence readout, supporting the principle that NHEJ can be programmed for both gene disruption and targeted cassette insertion. Together, this dissertation establishes W. ciferrii as a genetically tractable non-model yeast chassis by connecting genetic part validation, genome-informed design, NHEJ-based multicopy integration, and CRISPR/Cas9-mediated genome editing. The resulting framework provides a foundation for systematic engineering of TAPS biosynthesis and offers a transferable strategy for other NHEJ-dominant non-model yeasts.
more초록/요약
Wickerhamomyces ciferrii는 화장품 및 의약품 분야에 활용되는 아세틸화 스핑고이드 염기 유도체인 tetraacetyl phytosphingosine （TAPS）을 자연적으로 생산하고 분비하는 산업용 이배체 효모이다. 이러한 산업적 잠재력에도 불구하고, W. ciferrii에서의 균주 개량은 검증된 유전자 도구의 부재, 불완전한 게놈 수준 정보, 그리고 비상동 말단 연결 （non-homologous end joining, NHEJ）에 대한 강한 선호로 인한 낮은 상동 재조합 （homologous recombination, HR） 효율로 인해 제한되어 왔다. 본 학위논문은 W. ciferrii를 스핑고지질 생산을 위한, 활용 가능한 숙주로 개량하기 위한 유전학적·게놈학적 기반을 확립하는 것을 목표로 하였다. 첫째, 독립적으로 복제되는 복제원점, 선별 마커, 형광 리포터, 그리고 내인성 프로모터를 평가하여 모듈형 플라스미드 툴킷을 개발하였다. Saccharomyces cerevisiae의 2µ 및 CEN6/ARS4 복제원점은 플라스미드 유지에 적합한 반면, Kluyveromyces lactis의 panARS 복제원점은 그렇지 못하였다. Nourseothricin, geneticin, zeocin 저항성 마커는 실용적이라고 검증되었으며, 프로모터 강도는 pTDH3 〉 pPGK1 〉 pPDA1 순으로 나타났다. 또한 코돈 적응 지수 （codon adaptation index）와 전사체 존재량 사이의 양의 상관관계는 낮은 GC 함량을 가진 이 숙주에서 코돈 최적화가 중요한 설계 요소임을 보여주었다. 개발된 툴킷의 기능성은 인산화 비민감성 ACC1 변이체를 과발현함으로써 검증되었으며, 이를 통해 TAPS 생산량은 2.4배 증가하였다. 둘째, 후속 균주 엔지니어링 설계를 위해 산업용 이배체 균주의 유전체 수준 특성을 분석하였다. Reference-free k-mer 분석을 통해 반수체 유전체 크기는 약 15.6 Mb로 추정되었으며, 약 3.95%의 높은 이형접합도가 확인되었다. Smudgeplot 분석은 해당 균주가 주로 이배체 상동염색체 구조를 가진다는 것을 뒷받침하였다. Assembly 기반 분석을 통해 내인성 11-bp 텔로미어 반복 서열과 370개의 예측 tRNA loci를 확인하였으며, 이들은 각각 donor DNA 디자인과 tRNA 기반 sgRNA 발현을 위한 핵심 서열 자원으로 활용되었다. 셋째, W. ciferrii의 강한 NHEJ 활성을 억제하는 대신 유전공학적 장점으로 활용하였다. 이를 위해 donor DNA 말단에 텔로미어 반복 서열을 추가하여 안정성을 높이고, 고복제수 （high-copy） 삽입체를 선별하기 위해 말단 부분 절단으로 길이가 감소된 URA5 프로모터를 사용하며, 말단 연결의 효율을 향상시키기 위해 5′-인산화된 말단을 적용하는 세 가지 설계 요소를 조합하여 NHEJ 기반 다중 복제 （multicopy） 유전체 삽입 플랫폼을 개발하였다. 이러한 요소들은 pTdmVU5 플랫폼 벡터에 통합되었다. 이 시스템을 이용하여 serine palmitoyltransferase의 두 소단위체를 암호화하는 LCB1과 LCB2를 염색체에 다중복제로 삽입한 결과, 야생형 대조군 대비 TAPS 생산량이 2.7배 증가하였다. 마지막으로, W. ciferrii의 표적 유전체 공학을 위한 CRISPR/Cas9 시스템을 확립하였다. Cas9 발현은 코돈 최적화, 핵 위치 신호 선별, 단일복제 염색체 삽입, 그리고 이배체 유전체를 고려한 spacer 설계를 통해 숙주에 맞게 조정되었다. sgRNA 발현은 내인성 tRNA 기반 Pol III 프로모터를 이용하여 달성하였으며, target spacer는 이배체 유전체 구조를 고려하여 선정하였다. 구축된 시스템은 단일 카피 수 URA5 및 Venus 타겟에서 67–70%의 편집 효율을 보였고, 두 개 카피의 내인성 HIS3 locus에서는 약 20%의 편집 효율을 나타내었다. 또한 Cas9에 의해 생성된 Venus 내부 이중가닥 절단 위치에 mRuby2 발현 카세트를 상동 비의존적으로 삽입하는 개념증명 스크리닝에서 64개 형질전환체 중 9개 （14.1%）가 이중-형광 측정 결과 기반 후보로 확인되었으며, 이는 NHEJ가 유전자 파괴뿐만 아니라 원하는 위치로의 표적 삽입에도 활용될 수 있음을 보여준다. 종합하면, 본 학위논문은 유전적 도구 부품 검증, 유전체 정보 기반 설계, NHEJ 기반 다중 복제 삽입, 그리고 CRISPR/Cas9 매개 유전체 편집을 연결함으로써 W. ciferrii를 유전공학적으로 조작 가능한 비모델 효모 섀시 균주로 확립하였다. 이 연구에서 구축한 공학적 프레임워크는 TAPS 생합성의 체계적 개량을 위한 기반을 제공하며, 나아가 NHEJ 우세성을 보이는 다른 비모델 효모에도 적용 가능한 전략을 제시한다.
more목차
Chapter 1. General introduction 1
1.1. Introduction 2
1.2. Synthetic biology and metabolic engineering 5
1.3. Non-model yeast engineering 9
1.4. W. ciferrii as an industrial host for sphingolipid biosynthesis 14
1.5. Aims of this study 18
Chapter 2. Construction of modular plasmid system and functional characterization of genetic parts in Wickerhamomyces ciferrii 21
2.1. Abstract 22
2.2. Introduction 23
2.3. Materials and Methods 27
2.3.1. Strains, media, and culture conditions 27
2.3.2. Genomic DNA extraction 28
2.3.3. Plasmid construction and sequencing 29
2.3.4. Transformation of W. ciferrii and E. coli 33
2.3.5. Copy number determination by qPCR 34
2.3.6. Fluorescence microscopy 35
2.3.7. Transcriptional gene expression analysis 36
2.3.8. TAPS extraction and quantification 37
2.3.9. Codon adaptation index calculation 38
2.3.10. Statistical analysis 39
2.4. Results and Discussion 47
2.4.1. Screening of episomal replication origins for stable plasmid maintenance 47
2.4.2. Determination of plasmid copy number and sequence integrity 50
2.4.3. Antibiotic susceptibility profiling and evaluation of selectable markers 51
2.4.4. Functional characterization of fluorescent reporter proteins 55
2.4.5. Comparative evaluation of endogenous promoters for heterologous gene expression 58
2.4.6. Relationship between codon adaptation index and transcriptional expression levels 61
2.4.7. Application of the genetic toolkit through ACC1 overexpression for enhanced TAPS biosynthesis 62
Chapter 3. Comprehensive genomic characterization of industrial diploid Wickerhamomyces ciferrii for downstream strain engineering 67
3.1. Abstract 68
3.2. Introduction 69
3.3. Materials and Methods 73
3.3.1. Strain, media, and culture conditions 73
3.3.2. Genomic DNA extraction and whole-genome sequencing 73
3.3.3. K-mer frequency analysis 73
3.3.4. Diploid k-mer spectrum modeling and genome size estimation 74
3.3.5. Allele-pair structure visualization by Smudgeplot analysis 75
3.3.6. Telomeric repeat identification through Tandem Repeats Finder 75
3.3.7. Genome-wide tRNA gene prediction through tRNAscan-SE 76
3.4. Results and Discussion 78
3.4.1. K-mer spectrum analysis and genome size estimation 78
3.4.2. Diploid status confirmation and heterozygosity quantification 81
3.4.3. Allele-pair structure of diploid genome through Smudgeplot 82
3.4.4. Telomeric repeat sequence at contig termini 85
3.4.5. Genome-wide inventory of tRNA genes as a Pol III promoter resource 88
Chapter 4. Non-homologous end joining-mediated multicopy genome integration in Wickerhamomyces ciferrii 91
4.1. Abstract 92
4.2. Introduction 93
4.3. Materials and Methods 97
4.3.1. Strain, media, and culture conditions 97
4.3.2. Genomic DNA extraction 98
4.3.3. Generation of the uracil auxotrophic strain 98
4.3.4. Plasmid construction and sequencing 98
4.3.5. Transformation of W. ciferrii and E. coli 104
4.3.6. Fluorescence measurement 104
4.3.7. Telomeric repeat sequence identification 105
4.3.8. Copy number determination by qPCR 105
4.3.9. Time-course analysis of donor DNA persistence 106
4.3.10. Fluorescence-activated cell sorting （FACS） 107
4.3.11. TAPS extraction and quantification 108
4.3.12. Transcriptional gene expression analysis 108
4.3.13. Statistical analysis 108
4.4. Results and Discussion 119
4.4.1. Intrinsic preference of W. ciferrii for NHEJ-mediated DNA repair 119
4.4.2. Establishment of a uracil auxotrophic mutant and the VU5 dual-function reporter cassette 125
4.4.3. Enhancement of donor DNA integrity and integration efficiency by telomeric end-shielding 129
4.4.4. Enrichment of high-copy integrants by a defective auxotrophic marker strategy 136
4.4.5. Effect of 5′-phosphorylation of donor DNA termini on transformation and integration efficiency 139
4.4.6. Multicopy LCB1/LCB2 integration via platform vector pTdmVU5 for enhanced TAPS production 142
Chapter 5. CRISPR/Cas9-mediated targeted gene knock-out and cassette knock-in through homology-independent repair mechanism in Wickerhamomyces ciferrii 149
5.1. Abstract 150
5.2. Introduction 151
5.3. Materials and Methods 155
5.3.1. Strains, media, and culture conditions 155
5.3.2. Codon optimization of Cas9 155
5.3.3. Nuclear localization signal screening 156
5.3.4. Plasmid construction and sequencing 157
5.3.5. Generation of the Cas9-integrated TC9 strain 158
5.3.6. sgRNA expression vector construction 158
5.3.7. Biallelic target spacer design 159
5.3.8. Transformation of W. ciferrii and E. coli 160
5.3.9. Fluorescence measurement 160
5.3.10. Editing efficiency quantification 160
5.3.11. Homology-independent cassette knock-in 161
5.4. Results and Discussion 164
5.4.1. Codon optimization of Cas9 for the low-GC genome of W. ciferrii 164
5.4.2. Identification of a functional nuclear localization signal through fluorescent protein fusion screening 165
5.4.3. Chromosomal integration of the Cas9 expression cassette via random integration 167
5.4.4. Construction of a tRNA-based sgRNA expression cassette 170
5.4.5. Biallelic target sequence selection in diploid W. ciferrii 173
5.4.6. CRISPR/Cas9-mediated gene disruption at single- and two-copy loci 176
5.4.7. Homology-independent targeted cassette knock-in via CRISPR/Cas9-induced NHEJ repair 179
6. Conclusion 184
7. References 188
ABSTRACT IN KOREAN 203
