[논문]지질생산 및 유전자 조작의 잠재적 자원으로서의 토착 미세조류 Chlamydomonas reinhardtii K01의 분리 및 특성

김은경; 조대현; 서상익; 이창준; 김희식; 서현효

doi:10.5352/jls.2022.32.3.202

초록
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반수체 단세포 진핵생물인 녹조류 Chlamydomonas reinhardtii는 미세조류와 유전자 변형을 이용하여 고부가가치 물질을 생산하는 세포 공장으로 연구자와 산업계에서 오랫동안 사용되어 왔다. 현재 대부분의 기초 및 산업 연구에 사용되는 외래종인 C. reinhardtii를 대체하기 위하여 충청과 전라지역 12개의 담수에서 채취한 시료에서 Chlamydomonas로 추정되는 미세조류 K01을 분리하였다. 분리균주 K01의 형태학적 분석과 18S rDNA 유전자 서열(NCBI 수탁번호 KC166137)의 계통발생학적 분석을 통하여 분리균주 K01는 Chlamydomonas reinhardtii로 동정 되었다. 분리균주 C. reinhardtii K01의 성장 및 지질 함량은 TAP 배지에서 3개의 야생 균주 및 4개의 돌연변이 균주와 비교하였다. 이들 균주 중 C. reinhardtii K01 균주가 배양 3일째 1.74×10⁷ cells/ml개의 세포수가 관찰되었다. 이는 비교 균주 중 가장 높은 세포 수(1.20×10⁷ cells/ml)를 나타내어 UTEX2244와 비교했을 때 1.5배 빠른 성장속도를 보였다. 분리균주 C. reinhardtii K01의 지질함량(20.67%)은 야생형 균주의 지질함량과 유사하였다. 분리균주 Chlamydomonas reinhardtii K01의 지방산 성분은 7종의 분양 균주에서 확인된 지방산 중 oleate (C18:1)가 검출되지 않았다. 분리균주 C. reinhardtii K01은 nitrate (NaNO₃)를 질소원으로 포함하는 BG11배지에서 배양 6일 동안 0.87 g/l까지 세포밀도가 증가하였으나, 분양균주인 7종의 Chlamydomonas의 경우 모든 균주에서 세포증식이 거의 일어나지 않았다.

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The green alga Chlamydomonas reinhardtii, a unicellular haploid eukaryote, has long been used by researchers and industries as a cell factory to produce high value-added microalgae substances using genetic modification. Microalga K01, presumed to be Chlamydomonas, was isolated from 12 freshwater sam...

The green alga Chlamydomonas reinhardtii, a unicellular haploid eukaryote, has long been used by researchers and industries as a cell factory to produce high value-added microalgae substances using genetic modification. Microalga K01, presumed to be Chlamydomonas, was isolated from 12 freshwater samples from the Chungcheong and Jeolla regions to replace C. reinhardtii, an introduced species currently used in most basic and industrial research. The isolated K01 strain was identified as C. reinhardtii through morphological and phylogenetic studies of the 18S rDNA gene sequence (NCBI accession number KC166137). The growth and lipid content of the isolated C. reinhardtii K01 were compared with three wild and four mutant strains in TAP medium, and it was found that the K01 strain could produce 1.74×107 cells/ml by the third day of culture. The growth rate of C. reinhardtii K01 was 1.5 times faster than UTEX2244, which showed the highest number of cells (1.20×107 cells/ml) among the compared strains. The lipid content of the isolated C. reinhardtii K01 (20.67%) was similar to those of the wild strains, although the fatty acid oleate C18:1 was not detected in the isolated strain but was identified in the seven others. The cell density of the isolated strain increased to 0.87 g/l during a six-day culture in BG11 medium, where nitrate (NaNO3) was introduced as a nitrogen source, while the seven acquired strains showed almost no cell proliferation.

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표/그림 (8)

표 Table 1. Chlamydomonas reinhardtii strains used for this study
그림 Fig. 1. Light microscopy image of isolated strain K01.
그림 Fig. 2. The phylogenetic tree of isolated strain K01 18SrDNA gene sequence. the scale bar denotes nucleotide substitutions per site.
그림 Fig. 3. Comparison of cell growth of the isolated strain Chlamydomonas. reinhardtii K01 and the 7 types of acquired strains Chlamydomonas in TAP medium. The solid line is the wild-type strains and the dotted line is the mutant strains. Symbols : (-●-) K01; isolated strain, (-▲-) 2243; UTEX 2243, (-◆-)2244; UTEX 2244, (-■-) W14; UTEX W14, (-○-) sta6-p, (-△-) sta-6; (-◇-) BAFJ5C2; (-□-) I7.
그림 Fig. 4. Comparison of dry cell weight and lipid content of the isolated strain Chlamydomonas.reinhardtii K01 and the 7 types of acquired strains Chlamydomonas in TAP medium.
그림 Fig. 5. Comparison of biomass and lipid productivity of the isolated strain Chlamydomonas.reinhardtii K01 and the 7 types of acquired strains Chlamydomonas in TAP medium.
그림 Fig. 6. Comparison of (A) microscopic comparison of intracellular neutral lipid accumulation with nile red staining. Neutral lipids are observed as yellow droplets in the cells and red color is due to the self- autofluorescence of chlorophyll. The size bar shown 10 micrometers. (B) FAME composition of the isolated strain Chlamydomonas reinhardtii K01 and the 7 types of acquired strains Chlamydomonas.
그림 Fig. 7. Comparison of cell growth of the isolated strain C. reinhardtii K01 and the 7 acquired strains of Chlamydomonas in TAP medium containing ammonium (NH₄Cl) and BG11 medium containing nitrate (NaNO₃) as nitrogen sources. Symbols : (▩) TAP medium, (■) BG11 medium.

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제안 방법

The isolated microalgae were identified by examining their morphological and genetic characteristics. A morphological study was conducted using an optical microscope (Nikon Eclipse 80i, Nikon, Japan), and genetic identification was carried out by amplifying the gene sequence of 18S rDNA through polymerase chain reaction (PCR). For PCR, GO Taq DNA polymerase (GoTaq Green Master Mix 2×, Promega, USA) was introduced, and genomic DNA extracted with a DNA extraction kit (Wizard® Genomic DNA Purification Kit, Promega, USA) was used as a template.
In order to compare the culture characteristics and lipid productivity of the isolated strain C. reinhardtii K01 with previously isolated Chlamydomonas, three wild strains (UTEX 2243, UTEX2244, and W14) were acquired from UTEX (Culture Collection of Algae at the University of Texas in Austin, Texas), a microalgae distribution institution, and four mutant strains (Sta6-parent, Sta6, BAFJ5C2, and I7) were acquired from the Chlamycollection (Chlamydomonas Resource Center, University of Minnesota), and a comparative experiment was performed (Table 1).
1). For the molecular genetic identification of the isolated strain K01, the genome was extracted and the 18S rDNA sequence was amplified to obtain a sequence of 1,480 bp, and its homologous features were checked through the NCBI BLAST system. Phylogenetic analysis results showed that the 18S rDNA sequence of the isolated K01 was 99% similar to that of C.

이론/모형

Total lipid contents were determined for C. reinhardtii K01 and the acquired strains of Chlamydomonas by modifying the Bligh and Dryer method [2]. A culture solution volume of 10 ml was centrifuged at 5,000x g for 30 min.

성능/효과

One of the isolated algae was the microalga K01, which was presumed to be Chlamydomonas due to its mobility. The observation results of the isolated microalga K01 under optical microscope revealed that it was a green alga, and its morphology was similar to that of Chlamydomonas (Fig. 1).
0, and it was quite similar to Chlamydomonas reinhardtii. Therefore, the isolated strain K01 was named Chlamydomonas reinhardtii K01 (Fig. 2).
Therefore, C. reinhardtii K01 showed the highest maximum cell number at the fastest rate. In the case of the mutant strains sta6 and sta6-P, slow growths of 6.
When each microalga was cultured in TAP medium for 6 days, the neutral lipids accumulation pattern of the isolated C. reinhardtii K01 was observed to be similar to the wild strains rather than the mutant strains, which accumulated neutral lipids (Fig. 6A).
The fatty acid compositions of the isolated strain C. reinhardtii K01 and the 7 acquired strains of Chlamydomonas were investigated after detecting the fatty acids through GC (Fig. 6B).
6B). It was found that the fatty acids in the FAME extracted from the 7 acquired strains commonly contained caprylate (C8:0), laurate (C12:0), palmitate (C16:0), hexadecadienoate (C16:2), hexadecadienoate (C16:3), stearate (C18:0), oleate (C18:1), linoleate (C18:2), and linoleate (C18:3). However, oleate (C18:1) was not detected in the isolated strain C.
The dry cell weights of the isolated C. reinhardtii K01 and UTEX2243 in the TAP medium containing ammonium (NH4Cl) were similar at 1.49 g/l and 1.53 g/l, respectively, and the dry cell weight of the mutant strain was the lowest at 0.61 g/l. Such results implied that both the isolated strain C.
The poor cell growth of the 7 acquired strains could confirm that no nitrate reductase genes (nit1, nit2) in the genotypes of the acquired wild UTEX type strains were found. It was assumed that these strains could not utilize nitrate as a nitrogen source or their utilization efficiency was very low.
The poor cell growth of the 7 acquired strains could confirm that no nitrate reductase genes (nit1, nit2) in the genotypes of the acquired wild UTEX type strains were found. It was assumed that these strains could not utilize nitrate as a nitrogen source or their utilization efficiency was very low. Nitrate reductase was preserved in the isolated strain C.
reinhardtii K01, confirmed by its growth characteristics made capable by utilizing nitrate unlike the 7 acquired strains. The results also confirmed that the indigenous isolated strain C. reinhardtii K01 could grow in more diverse media compared to the existing Chlamydomonas.

참고문헌 (18)

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