Biotechnological application of polyunsaturated fatty acid biosynthesis in the protist Thraustochytrium aureum : Thraustochytrium aureum 균주의 다중불포화지방산 생합성 연구의 생물학적 응용

국문 초록 (Abstract)

다중불포화지방산은 특히 망막과 중추신경계의 중요한 구성성분이다. 특히 prostaglandine, leukotrienes 등과 관련된 조절물질의 생합성에 반드시 필요한 전구체이며, 또한 생합성 기작의 전사단계에서 regulator 로 작용하게 된다. 다중불포화지방산은 linoleic acid (LA, C18:2 n6)으로부터 시작하는 ω6 family와 α-linolenic acid (ALA, C18:3 n3)으로 시작되는 ω3 family로 구성되어 있으며, 이들은 탄소원자의 첨가(elongation)와 이중결합 첨가(desaturation) 등의 기작을 하는 각각의 다른 효소들의 연차적인 작용을 통하여 합성된다. 다중불포화지방산 생합성 균주이며 원생미생물인 Thraustochytrium aureum 은 22개 탄소를 가진 다중불포화지방산, 특히 ω6-docosapentaenoic acid (ω6-DPA, C22:5 n6)와 docosahexaenoic acid (DHA, C22:6 n3) 등의 생산능이 우수한 균주로 평가 받고 있다. 따라서, 본 연구에서는 다중불포화지방산 생합성 연구에 관련된 유전자 탐색에 T. aureum 균주를 선택하였다.
본 연구에 사용된 해양원생생물로부터 유전체 추출방법을 최적화하였으며, 추출된 유전체로부터 직접 PCR 기법을 이용하여 1320 bp 핵산을 코딩하고 있는 439 아미노산의 Δ5-desaturase와 1548 bp 핵산을 코딩하고 있는 515 아미노산의 Δ4-desaturase을 확보하였으며, 이 두 유전자의 N-말단에 cytochrome b5 도메인과 3개의 histidine box 가 잘 보존되어 있음을 확인하였다. 동정된 유전자는 메탄자화 균주인 P. pastoris내로 형질전환하여 세포외 기질로 공급된 di-home-γ-linolenic acid (DGLA, C20:3 n6) 에서 arachidonic acid (ARA, C20:4 n6) 로 그리고 adrenic acid (ADA, C22:4 n6) 은 ω6-docosapentanoic acid (ω6-DPA, C22:5 n6)로의 전환을 확인하여 P. pastoris에서 Δ4- and Δ5- desaturase 유전자의 존재와 활성을 확인하였다.
또한, 본 연구에서는 T. aureum 으로부터 확보된 5-fatty acid elongase (TauELO) 가 C20 PUFAs을 기질로 사용하여 C22 PUFAs로 전환시키는 elongation 활성을 확인하였다. TauELO 유전자는 273 아미노산을 코팅하는 825 bp 의 nucleotide ORF로 구성되고, 다른 PUFA elongase와 높은 유사성을 공유하였다. TauELO 유전자를 P. pastoris 균주내 발현을 통해 세포외 기질로 ARA (C20:4 n6)와 EPA (C20:5 n3)을 20% ADA (C22:4 n6)와 28% ω3-DPA (C22:5 n3)로 각각 전환하였다. 추가적으로, TauELO는 γ-linolenic acid (GLA, C18:3 n6)을 기질로 DGLA (C20:3 n6)을 생합성하는 6-elongation 특성을 가지는 것을 확인하였다. PCR error 에 의해 165 amino acid가 methionine 에서 threonine 으로 대체된 TauELO-mut 는 ADA (C22:4 n6) 와 ω3-DPA (C22:5 n3)를 각각 31% 와 34%을 생산하였으며, native TauELO보다 elongase 활성이 증가하였다. 또한, TauELO는 linoleic acid (LA, C18:2 n6)을 α-linolenic acid (ALA, C18:3 n3)로 eicosadienoic acid (EDA, C20:2 n6)을 eicosatrienoic acid (ETrA, C20:3 n3)와 같은 C18 PUFA 전환시키는 Δ9-elongation 활성을 가지는 것을 확인하였으며, oleic acid (OA, C18:1 n9)을 eicosenoic acid (C20:1 n9)로 전환시키는 monounsaturated elongation 활성이 낮지만 존재하고 있음을 확인하였다. 결론적으로 TauELO 효소는 5-, 6- 그리고 9-elongation 활성을 가진 다기능성 elongase 로써 C22와 C20 PUFAs의 생합성 특성을 증명하였다. TauELO elongase 는 unrooted phylogram 내에서는 one-step PUFA-elongase 그룹에 포함되지만, 기능적으로는 C22와 C20 PUFAs의 생합성하기 때문에 Multi-step PUFA-elongase 그룹에 포함되어야 한다.
본 연구에서 확보된 다중불포화지방산 생합성 유전자 TauELO, TauDES4 및 TauDES5를 메탄자화균주인 P. pastoris에 co-expression 하여 세포외 기질 GLA (C18:3 n6)와 SDA (C18:4 n3)로부터 탄소수 22개인 불포화지방산인 ω6-DPA (C22:5 n6)와 DHA (C22:6 n3)로 생합성이 가능함을 확인하였다. 3개의 유전자를 co-expression 하기 위해 TauELO, TauDES4 및 TauDES5 gene 을 각각 yeast expression vector인 pPIC3.5, pPIC6A 과 pPICZA에 클로닝하였다. 본 연구에서는 ω6-DPA (C22:5 n6), DHA (C22:6 n3)와 같은 C22 PUFAs가 multi-functional elongase와 5, 4-desaturase 효소들의 연속적인 기작을 통해 각각의 외부기질인 GLA (C18:3 n6)와 SDA (C18:4 n3)로부터 합성됨을 확인하였다. 종합적으로 볼때, 각각의 GLA (C18:3 n6)와 SDA (C18:4 n3)는 TauELO의 Δ6-elongation 활성에 의해 93.3% DGLA (C20:3 n6)와 98.3% ETA (C20:4 n3) 전환되었으며, 최종으로 TauDES4 에 의해 51.0% ω6-DPA (C22:5 n6)와 78.9% DHA (C22:6 n3)로 전환됨을 확인하였다. 이로써 T. aureum 의 elongase는 다기능 활성(Δ9-, Δ6- 과 Δ5-)을 가지고 있음을 재차 확인하였다.
이들 결과는 PUFAs을 실질적인 생산에 적합한 개량 균주의 개발에 기초자료로 제공될 수 있다.

다국어 초록 (Multilingual Abstract)

Polyunsaturated fatty acids (PUFAs) are important constituents of membranes particularly found in the retina and central nervous system. PUFAs serve as precursors for a number of biologically active molecules including eicosanoids, pheromone, growth regulators and hormones, and also act as a regulator molecule of synthetic pathway at transcription level. PUFAs are synthesized through alternating series of chain elongation and desaturation by the each step of different enzyme mechanisms. The synthesis of the ω6 family begins with linoleic acid (LA, C18:2 n6) and the reaction of the ω3 family starts from α-linolenic acid (ALA, C18:3 n3). In microorganism-based PUFAs biosynthesis, the protist Thraustochytrium aureum with high contents of C22 PUFAs, especially ω6-docosapentaenoic acid (ω6-DPA, C22:5 n6) and docosahexaenoic acid (DHA, C22:6 n3), is well evaluated for their potential as a promising candidate in the practical production PUFAs-rich oil. Therefore, T. aureum were chosen and exploited for the PUFAs biosynthesis and related genes.
In the first study, we attempted to optimize a method of total nucleic acid extraction for metabolic engineering of this marine protist as a preliminary experiment. Using the extracted nucleic acid and degenerated primers for direct PCR, we isolated Δ4- and Δ5-desaturase genes that contained each 1548 bp and 1320 bp and encoded 515 and 439 amino acids contained a predicted N-terminal cytochrome b5-like domain as well as three conserved histidine boxes. Their genes exhibited an expected function, when expressed in the methylotrophic P. pastoris in the presence of appropriate exogenous substrate, as an evidence for Δ5- and Δ4-desaturase activity with conversion of di-home-γ-linolenic acid (DGLA, C20:3 n6) to arachidonic acid (ARA, C20:4 n6) and adrenic acid (ADA, C22:4 n6) to ω6-docosapentanoic acid (ω6-DPA, C22:5 n6).
In the second study, the isolation and characterization of 5-fatty acid elongase from T. aureum was demonstrated to be associated with the synthesis of not only the C22 PUFAs, but also the 20 carbon atoms. The TauELO gene contains 825 bp nucleotide ORF, which encodes protein of 274 amino acids, and it shares high similarity with other PUFA elongases. Expression of the TauELO gene in Pichia pastoris resulted in the production of ADA (C22:4 n6) and ω3-DPA (C22:5 n3) in cells that were provided with ARA (C20:4 n6) and EPA (C20:5 n3), respectively. Approximately, 20% ADA (C22:4 n6) and 28% ω3-DPA (C22:5 n3) of the converted products were obtained in the given substrate. The TauELO-mut (The 165 amino acid threonine was substituted instead of methionine by PCR error) was converted into approximately 31% and 34% of the products ADA (C22:4 n6) and ω3-DPA (C22:5 n3) and is serves to enhance the elongase activity, relative to the native TauELO. Further, the substrate specificity result suggests that the TauELO was also involved in the elongation of γ-linolenic acid (GLA, C18:3 n6) to DGLA (C20:3 n6). In addition, TauELO is able to convert the Δ9-elongation of the C18 PUFAs, such as LA (C18:2 n6) and ALA (C18:3 n3) to eicosadienoic acid (EDA, C20:2 n6) and eicosatrienoic acid (ETrA, C20:3 n3), respectively and also able to synthesis the low level of monounsaturated activity of oleic acid (OA, C18:1 n9) to eicosenoic acid (C20:1 n9). The TauELO protein was confirmed to be a novel multifunctional activity of 5-elongation with 6-, 9- and a low level of monounsaturated elongation activities. This newly found multifunction was attested in the process of production of the C22 and C20 PUFAs. Thus it is evidenced that the TauELO encodes an enzyme having 5-, 6- and 9-elongation activities. The elongase TauELO falls into one-step PUFA-elongase group of unrooted phylogram, but functionally should be fall into Multi-step PUFA-elongase group, because TauELO can synthesize C20 and C22 PUFAs in multi-steps.
In the last study, we constructed and evaluated the co-expression of genes related to the PUFAs synthesis, such as TauELO, TauDES4 and TauDES5 derived from T. aureum. To accomplish co-expression, the TauELO, TauDES4 and TauDES5 gene were subcloned into a yeast expression vector, pPIC3.5, pPIC6A and pPICZA, respectively. We found that C22 PUFAs such as ω6-DPA (C22:5 n6) and DHA (C22:6 n3) were synthesized from GLA (C18:3 n6) and SDA (C18:4 n3), respectively, as exogenous substrates via a series of reactions catalyzed by a multi-functional elongase and 5, 4-desaturase enzymes. Taken together, these finding indicate that ω6-DPA (C22:5 n6) and DHA (C22:6 n3) were produced as follows; GLA (C18:3 n6) and SDA (C18:4 n3) were elongated to 93.3% DGLA (C20:3 n6) and 98.3% ETA (C20:4 n3) by TauELO. Finally, the Δ5-elongated products were converted to 51.0% ω6-DPA (C22:5 n6) and 78.9% DHA (C22:6 n3), repectively by TauDES4. In addition, we confirmed that the TauELO enzyme was involved in multiple reactions leading to the production of PUFAs and that the TauELO, TauDES5, and TauDES4 were capable of functioning together to produce ω6-DPA (C22:5 n6) and DHA (C22:6 n3) using GLA (C18:3 n6) and SDA (C18:4 n3).
These results and information could provide a basis for the construction of engineered stained suitable for the practical production of PUFAs.

목차 (Table of Contents)

• CHAPTER I. INTRODUCTION … 13
• 1.1. Polyunsaturated Fatty Acids … 13
• 1.1.1. Overview … 13
• 1.1.2. Nomenclature and structure of PUFAs … 14
• 1.1.3. Health related aspects of PUFAs … 17
• 1.2. PUFAs containing Single Cell Oils … 23
• 1.2.1. Overview … 23
• 1.2.2. PUFAs rich oil-producing microorganisms … 24
• 1.2.3. Characteristics of protist thrasutochytride, T. aureum … 27
• 1.3. Biosynthesis of PUFAs … 33
• 1.3.1. Overview … 33
• 1.3.2. Desaturation and elongation process … 36
• 1.3.3. Polyketide-like synthesis of PUFAs … 40
• 1.4. Objectives of Research … 42
• CHAPTER II. Molecular cloning of front-end (Δ4 and Δ5) desaturases from Thraustochytrium aureum and their functional expression in Pichia pastoris … 43
• 2.1. Introduction … 43
• 2.2. Materials and Methods … 46
• 2.2.1. Strain and medium … 46
• 2.2.2. Extraction of genomic DNA … 46
• 2.2.3. Amplification and cloning of Δ5- and Δ4-desatrase genes … 48
• 2.2.4. Expression of TauDES5 and TauDES4 in P. pastoris … 48
• 2.2.5. Fatty acid analysis … 49
• 2.3. Results and Discussion … 50
• 2.3.1. Total nucleic acid extraction … 50
• 2.3.2. Amplification and cloning of Δ5- and Δ4-desatrase genes … 53
• 2.3.3. Expression of plasmids containing Δ5- and Δ4-desaturases in P. pastoris … 62
• 2.3.4. Desaturases with Δ5- and Δ4-regioselectivities from T. aureum … 69
• CHAPTER III. Identification and characterization of multi-functional elongation activities from Thraustochytrium aureum … 71
• 3.1. Introduction … 71
• 3.2. Material and Methods … 75
• 3.2.1. Strain and growth conditions … 75
• 3.2.2. PCR-based cloning of Δ5-elongase gene … 75
• 3.2.3. Expression of TauELO in P. pastoris … 76
• 3.2.4. Fatty acid analysis … 77
• 3.3. Results and Discussion … 78
• 3.3.1. Cloning of TauELO gene … 78
• 3.3.2. Expression of TauELO5 in P. pastoris … 82
• 3.3.3. Characterization of Δ6-elongation of TauELO … 87
• 3.3.4. Characterization of Δ9-elongation of TauELO … 90
• 3.3.5. Characterization of monosaturated elongation of TauELO … 93
• 3.3.6. Phylogenetic analysis of PUFA elongase … 95
• CHAPTER IV. Co-expression of △5-desaturase, multi-functional elongase and △4-desaturase from the protist Thraustochytrium aureum and production of ω6-DPA and DHA in the methylotrophic yeast Pichia pastoris … 98
• 4.1. Introduction … 98
• 4.2. Material and Methods … 102
• 4.2.1. Strain and growth conditions … 102
• 4.2.2. PCR-based cloning of TauELO, TauDES4 and TauDES5 genes … 102
• 4.2.3. Vector construction and expression in P. pastoris … 106
• 4.2.4. Fatty acid analysis … 110
• 4.3. Results and Discussion … 111
• 4.3.1. Heterologous expression of TauELO … 111
• 4.3.2. Co-expression of TauELO and TauDES5 … 112
• 4.3.3. Co-expression of TauELO, TauDES5, and TauDES4 … 115
• CHAPTER V. REFERENCE … 117