Engineering Komagataella phaffii via adaptive laboratory evolution for improved methanol tolerance and utilization in β-carotene production = 적응 진화를 이용한 Komagataella phaffii 균주의 메탄올 내성 및 활용 향상에 따른 베타-카로틴 생산성 증가에 관한 연구

국문 초록 (Abstract)

당류 기반 원료의 한계를 극복하고 지속 가능한 생산을 달성하기 위해, 단일 탄소 화합물을 고부가가치 물질로 전환하는 것은 미생물 세포공장 개발에서 중요한 목표로 부상하고 있다. 이들 가운데 메탄올은 수용성이며 다양한 탄소원으로부터 합성될 수 있다는 점에서 매력적인 기질이며, 메틸영양성 효모인 코마가타엘라 파피이는 메탄올 기반 생물공정을 위해 적합한 섀시이다. 그러나 메탄올 대사는 포름알데하이드의 독성을 비롯한 상당한 생리학적 부담을 유발하며, 이는 사용 가능한 메탄올 농도를 제한하고 생산성을 저해한다. 기존의 합리적 엔지니어링은 메탄올 대사 경로의 일부 반응 단계를 개선하는 과정에 기여했으나, 전역적인 조절 네트워크와 에피스타시스에 대한 불완전한 이해로 인해 설계 가능한 공간이 제한되며, 고농도 메탄올 조건에서의 강인한 내성 및 높은 생산성은 아직 충분히 달성되지 못했다. 이러한 측면에서, 실험실 적응진화는 사전에 정의된 설계 가설을 넘어 유리한 돌연변이 조합을 발굴할 수 있는 선택압 기반의 상보적 전략을 제공한다.
본 연구에서는 서로 다른 환경 조건에서 실험실 적응진화를 수행하여 메탄올 내성과 활용이 향상된 코마가타엘라 파피이 균주들을 구축하였다. 제 2장에서는 메탄올 스트레스의 영향을 평가하기 위한 기준 조건으로 영양이 풍부한 배지에서 실험실 적응진화를 수행하였다. 진화한 분리주는 모균주에서는 성장이 억제되던 고농도 메탄올에서 증식할 수 있었고, 유전체 재분석을 통해 포름알데하이드의 독성을 완화하는 것으로 보이는 변이를 찾을 수 있었다. 베타-카로틴 생산 경로를 도입했을 때 진화된 균주는 모균주보다 더 높은 역가를 보여, 실험실 적응진화가 코마가타엘라 파피이에서 메탄올 내성과 활용을 향상시키는 데에 활용될 수 있음을 입증하였다.
제 3장에서는 영양이 제한된 조건에서의 적응 양상을 규명하기 위해 최소 메탄올 배지에서 실험실 적응진화를 수행하였다. 유전체 재분석과 전사체 분석을 통해 메탄올 대사와 연관된 여러 위치에서의 변이와 더불어 경로 수준의 발현 변화가 관찰되었다. 베타-카로틴 생산 경로를 도입하여 평가한 결과, 진화된 균주는 모균주에 비해 더 높은 역가와 수율을 나타냈으며, 이를 통해 최소 배지에서의 진화가 메탄올 활용을 지지하는 보다 심층적인 대사 재구성을 유도할 수 있음을 시사하였다.
제 4장에서는 메탄올 대사의 이화 경로를 제한하고자 포름산염 탈수소효소를 결실시킨 배경 균주를 구축하고, 보다 효율적인 탄소 동화 경로를 선호하도록 최소 메탄올 배지에서 실험실 적응진화를 진행하였다. 실험실 적응진화를 통해 얻어진 분리주는 환원성 글라이신 경로 관련 유전자들의 발현 증가와 함께 단일 탄소 대사 및 보조인자 대사의 광범위한 전사체 수준 재조직화를 보여주었다. 베타-카로틴 생산 조건 하에서 이러한 변화는 추가적인 생산성 향상으로 이어졌으며, 경로 수준의 제약이 코마가타엘라 파피이에서 서로 다른 적응 경로를 향해 진화를 유도할 수 있음을 보여준다.
종합하면, 본 연구는 실험실 적응진화를 통해 코마가타엘라 파피이 균주에서 메탄올 내성과 활용을 향상시킬 수 있음을 증가된 베타-카로틴 생산을 통해 보여주었으며, 적응 경로가 진화 환경과 부여된 대사적 제약에 의해 강하게 규정된다는 점을 제시하였다. 본 연구에서 제시한 접근은 메틸영양성 코마가타엘라 파피이를 기반으로 하는 메탄올 기반 생물공정과 같은 미생물 세포공장 구축을 위한 섀시 균주 개발의 기반 전략을 제공한다.

다국어 초록 (Multilingual Abstract)

To overcome the limitations of sugar-based feedstocks and achieve sustainable production, the conversion of one-carbon compounds into value-added products has emerged as an important goal in the development of microbial cell factories. Among these substrates, methanol is particularly attractive because it is water-miscible and can be synthesized from diverse sources, while native methylotrophs such as Komagataella phaffii provide a suitable chassis for methanol-based bioprocesses. However, methanol metabolism imposes substantial physiological burdens, most notably formaldehyde toxicity, which restricts the usable methanol concentration and thereby limits productivity. Conventional rational engineering has improved specific steps in the methanol utilization pathway, but incomplete knowledge of global regulatory networks and epistatic interactions constrains the accessible design space, and robust tolerance and high productivity at elevated methanol concentrations have yet to be fully achieved. In this respect, adaptive laboratory evolution (ALE) provides a complementary, selection-driven strategy that can uncover beneficial combinations of mutations beyond predefined design hypotheses.
In this thesis, ALE was employed to obtain K. phaffii strains with improved methanol tolerance and utilization under distinct environmental conditions. In Chapter 2, ALE was performed in a nutrient-rich medium as a baseline condition to assess the impact of methanol stress. The evolved isolate was able to grow at high methanol concentrations that inhibited the parental strain, and genome resequencing identified a mutation that appeared to alleviate formaldehyde toxicity. When a β-carotene production pathway was introduced, the evolved strain achieved a higher titer than the background strain, demonstrating that ALE can be used to enhance methanol tolerance and utilization.
In Chapter 3, ALE was carried out in a minimal methanol medium to examine how K. phaffii adapts under nutrient-limited conditions. Genome resequencing and transcriptomic profiling revealed mutations in loci associated with methanol metabolism together with pathway-level changes in gene expression. When evaluated with an engineered β-carotene pathway, the evolved strain displayed higher titers and yields than the parental strain, suggesting that evolution in minimal medium can drive deeper metabolic remodeling to support methanol utilization.
In Chapter 4, the dissimilatory branch of methanol metabolism was constrained by deleting formate dehydrogenase, and the resulting background was subjected to ALE in minimal methanol medium to favor more carbon-efficient assimilation routes. The ALE-derived isolates showed increased expression of genes in the reductive glycine pathway and broad reorganization of one-carbon and cofactor metabolism at the transcriptomic level. Under β-carotene producing conditions, these changes further improved production performance, illustrating how pathway-level constraints can steer evolution toward distinct trajectories.
In conclusion, this thesis demonstrated that adaptive laboratory evolution can be used to enhance methanol tolerance and utilization in K. phaffii, as evidenced by improved β-carotene production, and that adaptive trajectories are strongly shaped by the evolutionary environment and by imposed metabolic constraints. This work provides a robust framework for developing ALE-derived chassis strains for microbial cell factories, particularly for methanol-based bioprocesses using K. phaffii as a methylotrophic host.

목차 (Table of Contents)

• Abstract i
• List of Tables vii
• List of Figures viii
• Chapter 1. Introduction 1
• 1.1. Microbial cell factories for sustainable C1 compound bioconversion 2
• 1.1.1. Methanol as a potential feedstock among C1 compounds 3
• 1.1.2. Constructing synthetic methylotrophy and engineering native methylotrophs 4
• 1.2. Native eukaryotic methylotroph Komagataella phaffii as a promising host 6
• 1.2.1. Rational engineering of methanol metabolism in K. phaffii 8
• 1.2.2. Adaptive laboratory evolution as a promising approach for improving methanol metabolism 9
• 1.3. Scope of this thesis 11
• Chapter 2. Adaptive laboratory evolution of Komagataella phaffii in rich methanol medium for improved methanol tolerance and utilization 14
• 2.1. Abstract 15
• 2.2. Introduction 16
• 2.3. Materials and Methods 18
• 2.3.1. Strain cultivation and adaptive laboratory evolution 18
• 2.3.2. Specific growth rate measurements 18
• 2.3.3. Genome resequencing 19
• 2.3.4. Mutant strain cloning 22
• 2.3.5. Formaldehyde concentration measurement 25
• 2.3.6. Culture in minimal medium 25
• 2.3.7. β-Carotene biosynthesis and measurement 26
• 2.4. Results and Discussion 27
• 2.4.1. Long-term ALE in YPM to improve methanol utilization 27
• 2.4.2. Identification of beneficial mutations for enhanced growth on methanol medium 33
• 2.4.3. Evolved strain enables growth on minimal methanol medium 44
• 2.4.4. Enhanced production of β-carotene using methanol by an evolved PM7a strain 49
• 2.5. Conclusion 55
• Chapter 3. Phenotypic and transcriptomic remodeling of Komagataella phaffii via adaptive laboratory evolution in minimal methanol medium 56
• 3.1. Abstract 57
• 3.2. Introduction 58
• 3.3. Materials and Methods 60
• 3.3.1. Primers, strains, plasmids, sgRNA sequences and reagents 60
• 3.3.2. Construction of mutant strains 64
• 3.3.3. Strain cultivation and adaptive laboratory evolution 64
• 3.3.4. Specific growth rate measurements 65
• 3.3.5. Genome resequencing 65
• 3.3.6. RNA extraction and RNA-seq analysis 66
• 3.3.7. β-Carotene biosynthesis and measurement 67
• 3.4. Results and Discussion 68
• 3.4.1. ALE of a histidine-prototrophic GS115 under buffered minimal methanol conditions 68
• 3.4.2. Genome resequencing and functional analysis of ALE-derived mutations 73
• 3.4.3. Transcriptomic profiling of the ALE isolate PB7c under methanol adaptation 85
• 3.4.4. Evaluation of β-carotene production from methanol in the evolved PB7c strain 95
• 3.5. Conclusion 98
• Chapter 4. Constraint-guided adaptive laboratory evolution of Komagataella phaffii in minimal methanol medium 99
• 4.1. Abstract 100
• 4.2. Introduction 101
• 4.3. Materials and Methods 103
• 4.3.1. Primers, strains, plasmids, sgRNA sequences and reagents 103
• 4.3.2. Construction of deletion strains 106
• 4.3.3. Strain cultivation and adaptive laboratory evolution 106
• 4.3.4. Specific growth rate measurements 107
• 4.3.5. Genome resequencing 107
• 4.3.6. RNA extraction and RNA-seq analysis 107
• 4.3.7. β-Carotene biosynthesis and measurement 109
• 4.4. Results and Discussions 110
• 4.4.1. Construction of the fdh-deletion background and ALE under minimal medium conditions 110
• 4.4.2. Mutational profiling of ALE isolates by genome resequencing 115
• 4.4.3. Transcriptomic profiling of constraint-guided ALE isolate Pf5c under methanol adaptation 128
• 4.4.4. Evaluation of β-carotene production from methanol in the evolved Pf5c strain 143
• 4.5. Conclusion 146
• Chapter 5. Overall conclusion and further suggestions 147
• 5.1. Engineering Komagataella phaffii via adaptive laboratory evolution for improved methanol tolerance and utilization in β-carotene production 148
• 5.2. Further suggestion 1: Functional dissection of adaptive mutations and epistasis 151
• 5.3. Further suggestion 2: Multi-omics characterization of ALE-derived strains 153
• Appendix A1. Lipid extract derived from newly isolated Rhodotorula toruloides LAB-07 for cosmetic applications 154
• A1.1. Abstract 155
• A1.2. Introduction 156
• A1.3. Materials and Methods 158
• A1.3.1. Isolation and identification of microorganisms 158
• A1.3.2. Genomic DNA library construction and sequencing 160
• A1.3.3. Genome assembly and validation 160
• A1.3.4. RNA library construction and sequencing 161
• A1.3.5. Genome annotation 161
• A1.3.6. Flask-scale culture and lipid content analysis 162
• A1.3.7. Identification of fatty acid synthesis pathway and structural analysis of desaturase 163
• A1.3.8. Lipid extraction 164
• A1.3.9. Assay of R. toruloides lipid extract on human keratinocytes 164
• A1.3.10. DPPH radical scavenging assay 165
• A1.4. Results and Discussions 167
• A1.4.1. Identification of isolated pink yeast based on 26S rRNA and ITS sequencing 167
• A1.4.2. De novo genome assembly and genome annotation 170
• A1.4.3. Lipid production and analysis based on docking simulation 174
• A1.4.4. Analysis of the effect on skin cells and antioxidant activity of R. toruloides lipid extract 179
• A1.5. Conclusion 182
• References 183
• Abstract in Korean 198