Metabolic Engineering of Yarrowia lipolytica for the Production of β-carotene and Zeaxanthin = Yarrowia lipolytica의 대사공학을 통한 베타-카로틴 및 제아잔틴 생산

다국어 초록 (Multilingual Abstract)

Carotenoids are valuable lipophilic compounds with commercial applications in food, animal feed, cosmetics, and nutraceutrical industry. They are found widespread in nature, as they can be naturally synthesized by all photosynthetic organisms, along with some non-photosynthetic organisms as well. However, natural carotenoid extraction is labor-intensive and costly. By comparison, synthetic carotenoids derived from petrochemical precursors are often deemed unsuitable by regulatory agencies for application in drug, food, and cosmetics. Turning to microbial production have long been suggested as an alternative to the both sources by offering better sustainability, safety and economics. In this thesis, multiple strategies were explored to produce β-carotene or zeaxanthin in oleaginous yeast Yarrowia lipolytica. Introduction of carotenoid synthesis enzymes resulted in residual precursors, leading to the exploration of multiple corrective strategies such as pathway flux optimization, redox balancing and cellular compartmentalization.
Chapter 3 outline the engineering strategies that enabled the non-producing Y. lipolytica strain to produce β-carotene. After introduction of heterolgous β-carotene pathway, various genes in upstreme pathway were overexpressed. Lycopene accumulation following the increased carotenoid production level was eliminated using previous reported mutation in the CarRP gene. Two approaches to redirect the central carbon pathway were tested to promote NADPH regeneration and reduce ATP expenditure. Lastly flux to lipid production pathway was increased to facilitate more β-carotene accommodation in lipid droplet (LD), while ensuring enough acetyl-CoA pool for carotenoid production.
In chapter 4, a β-carotene hydroxylase gene was introduced into a β-carotene producing strain from the previous chapter, in order to produce zeaxanthin. The best host-compatible enzyme, PaCrtZ was selected after comparison with various homologs. After finding an enzyme mutant of PaCrtZ from co-evolutionary analysis, various approaches were taken to improve zeaxanthin production. From leucine phototrophy, oxygen and pH control, redox cofactor engineering, several conditions that improved zeaxanthin synthesis were found.
Lastly, chapter 5 describes strategies that may promote accomodation of PaCrtZ mutant enzyme and zeaxanthin in cell. Targeted relocation of the PaCrtZ mutant enzyme to ER, peroxisome and LD was performed and analyzed. ER and peroxisome membrane expansion were explored. Strategy that may shift LD morphology toward small and numerous were demonstrated.

국문 초록 (Abstract)

카로티노이드는 식품, 동물 사료, 화장품 및 건강기능식품 산업에서 상적으로 응용되는 중요한 지용성 화합물이다. 이들은 모든 광합성을 하는 생물체와 일부 비광합성 생물체에 의해 자연적으로 합성될 수 있어 광범위하게 발견된다. 하지만 자연에서 추출하는 방법은 노동 집약적이며 비용이 많이 든다는 단점이 있다. 석유화학 원료에서 유래한 인공 카로티노이드는 약물, 식품 및 화장품에 적용하기 부적합 판정을 받는 경우가 많다. 미생물을 통한 생산은 지속 가능성, 안전성 및 경제성 측면에서 앞선 두 가지 방법에 대한 대안으로 여겨져 왔다. 본 연구에서는 Yarrowia lipolytica하는 유지 효모에서 베타-카로틴 또는 제아잔틴을 과생산하기 위해 경로 흐름 최적화, 산화환원 균형 및 세포 구획화와 같은 여러 전략들을 적용했다.
제 3장에서는 비생산 Y. lipolytica 균주가 베타-카로틴을 생산할 수 있게 적용한 전략을 다루고 있다. 외래 베타-카로틴 경로를 도입한 후 상류 경로의 다양한 유전자들을 과발현했고, 이전에 보고된 CarRP 유전자 돌연변이를 사용하여 라이코펜 축적 문제를 해결했다. NADPH 재생을 촉진하고 ATP 소모를 줄이기 위해 두 가지의 중심 탄소 경로 전환 방법이 제시되었다. 마지막으로 베타-카로틴이 지방 방울 (LD)에 더 잘 적재됨과 동시에 충분한 Acetyl-CoA 풀을 확보하고자 지방 생성 경로의 흐름을 적절히 강화하였다.
제 4장에서는 베타-카로틴 생성 균주에 베타-카로틴 수산화효소를 도입해 제아잔틴을 생산했다. 다양한 동종효소들과 비교 후 가장 적합한 숙주 호환 효소인 PaCrtZ를 선택했고, 공진화 분석을 통해 제아잔틴 생산을 증가시킬 수 있는 PaCrtZ 효소의 돌연변이를 찾았다. 발현양, 류신 영양요구성, 산소 및 pH, 산화환원 보조 인자와 같은 제아잔틴 생산을 개선할 수 있는 조건들을 확인하였다.
마지막으로 제 5장에서는 PaCrtZ 돌연변이 효소와 제아잔틴이 세포 내에서 더 잘 적재될 수 있도록 세포 소기관 개량을 진행하였다. PaCrtZ 돌연변이 효소를 ER, 퍼옥시솜 및 LD로 이동시키는 전력을 시도 및 검토하고, ER과 퍼옥시솜의 막 확장을 통해 제아잔틴 생산량 증대를 시도했다. 또한 LD의 제아잔틴의 수용을 늘리기 위해 작고 많은 수의 LD 형태로 전환할 수 있는 공학적 방법을 제시했다.

목차 (Table of Contents)

• Abstract i
• Contents iv
• List of tables vi
• List of figures i
• Chapter 1. Research backgrounds 1
• 1.1. Properties of carotenoids and their uses in industry 2
• 1.2. Recent development in microbial carotenoid production 6
• 1.3. The scope of this thesis 8
• Chapter 2. Materials and Methods 11
• 2.1. Mediums and culture conditions 12
• 2.2. Strain constructions 13
• 2.3. Plasmid constructions 17
• 2.4. Quantification of carotenoids 50
• 2.5. Fluorescence assay 50
• 2.6. RNA extraction and RT-qPCR 51
• 2.7. Confocal microscopy 52
• 2.8. Bioreactor fermentation 53
• 2.9 Generation of PaCrtZ mutants through SCANEER 53
• 2.10. Molecular dynamics simulation 54
• Chapter 3. Engineering Yarrowia lipolytica for the production of β-carotene by carbon and redox rebalancing 57
• 3.1. Introduction 58
• 3.2.1. Construction of β-carotene synthesis pathway 60
• 3.2.2. Lycopene accumulation removal to obtain high-purity β-carotene 62
• 3.2.3. Central carbon redistribution to enhance NADPH and ATP supply 67
• 3.2.4. Flux adjustment to balance β-carotene storage and acetyl-CoA pool 72
• 3.3. Conclusion 75
• Chapter 4. Introduction of β-carotene hydroxylase and zeaxanthin production enhancement by optimizing the enzyme performance 76
• 4.1. Introduction 77
• 4.2.1. Screening of the chassis-compatible BCH homologs 78
• 4.2.2. Co-evolutionary analysis-based PaCrtZ engineering 83
• 4.2.3. Limiting factor 1: expression level and culture conditions 87
• 4.2.4. Limiting factor 2: Redox partners 92
• 4.3. Conclusion 100
• Chapter 5. Spatial partitioning of β-carotene hydroxylase and zeaxanthin 101
• 5.1. Introduction 102
• 5.2.1. Targeting PaCrtZ(S62A) enzyme to various organelles 104
• 5.2.2. Distribution of PaCrtZ(S62A) across organelles 109
• 5.2.3. Zeaxanthin storage maximized by smaller LDs 112
• 5.3. Conclusion 115
• Chapter 6. Overall Conclusion and further suggestions 116
• 6.1. Overall conclusion 117
• 6.2. Further suggestion 1: Integration of necessary prosthetic groups in the electron transfer chain toward PaCrtZ enzyme 119
• 6.3. Further suggestion 2: Expansion of Zeaxanthin-Derived Carotenoids via Minimal Metabolic Pathway Engineering 120
• References 122
• Abstract in Korean 132