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살리실산의 슈도모나스 종들에서의 medium-chain-length 폴리히드록시알칸산(MCL-PHA) 생합성 저해 및 MCL-PHA 구성 모노머의 조절 현상에 관한 연구

Two Roles of Salicylic Acid in the Accumulation of Polyhydroxyalkanoic Acid in Pseudomonas spp.: Inhibition of PhaG-Mediated Monomer-Unit Incorporation and Modulation of Monomer-Unit Distribution,

심지훈 (Shim, Ji Hoon, 경상대학교 대학원)

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초록/요약moremore
Salicylic acid(SA), without being metabolized, was found to inhibit the synthesis of polyhydroxyalkanoic acid(PHA) in the two different types of pseudomonads Pseudomonas fluorescens BM07 and Pseudomonas aeruginosa BM114. BM07 strain accumulates only medium-chain-length(MCL)-PHA whereas BM114 strain ...
Salicylic acid(SA), without being metabolized, was found to inhibit the synthesis of polyhydroxyalkanoic acid(PHA) in the two different types of pseudomonads Pseudomonas fluorescens BM07 and Pseudomonas aeruginosa BM114. BM07 strain accumulates only medium-chain-length(MCL)-PHA whereas BM114 strain accumulates a blend of MCL- and short-chain-length(SCL)-PHA. The two strains showed totally different reaction against SA addition; SA inhibited the growth of BM07 but not BM114. SA strongly and sensitively inhibited PHA synthesis in BM07 strain only when it was growth-associated in the presence of additional nitrogen but weakly inhibited under inactive growth condition such as nitrogen limitation. For the cells grown with 70 mM fructose, an increasing addition of SA(up to 4 mM) induced a selective complementary change in the mol% of two monomer-units, 3-hydroxydodecanoate(C12) and 3-hydroxy-cis-5-dodecenoate(C12:1), in the PHA while the mole ratios of the other MCL-monomer-units remained unchanged. For BM07 cells grown with 50 mM fructose plus 5 mM 11-phenoxyundecanoic acid(11-POU), the addition of 1.5 mM SA significantly shifted the major constituting monomer-units to the longer ones; 3-hydroxy-5-phenoxyvalerate(5POHV) from 58 to 25 mol%; 3-hydroxy-7-phenoxyheptanoate(7POHH) from 26 to 43 mol%; 3-hydroxy-9-phenoxynonanoate(9POHN) from 5 to 18 mol%. When grown on octanoic and decanoic acid, P. aeruginosa BM114 accumulated PHA composed of 3-hydroxybutyrate(3HB), 3-hydroxycaproic acid(3HC), 3-hydroxyoctanoic acid (3HO) and 3-hydroxydecanoic acid(3HD). Solvent fractionation and characterization of the resulting PHA revealed that the PHA was a blend of two polymers, P(3HB) and P(3HC-co-3HO-co-3HD). The addition of 3 to 5 mM SA selectively inhibited the synthesis of P(3HC-co-3HO-co-3HD) by ~80% of the control content in a one-step cultivation, where total PHA synthesis was inhibited by ~25%. However, P. aeruginosa BM114 grown with nonanoic or undecanoic acid also produced PHA containing 3HB-unit in addition to 3-hydroxyacids with odd number of carbons, 3-hydroxyvalerate(3HV) and higher acids. Organic solvent fractionation revealed that the PHA was a mixture of 45% P(3HB-co-3HV) and 55% P(3-hydroxyheptanoate (3HH)-co-3-hydroxynonanoate(3HN)-co-3-hydroxyundecanoate(3HU)). Similarly, SA inhibited the accumulation of only MCL-PHA, not SCL-PHA. In addition, 2-bromooctanoic acid(2-BrOA), known as a PHA-synthesis inhibitor showed similar inhibition characteristics in carbon-source dependency but more sensitively than SA. Thus, to some extents, it seems possible to change the monomer-unit ratio of the PHA produced in Pseudomonas spp. using the inhibitory effect of SA
목차moremore
I. 서론 1
1.1 Polyhydroxyalkanoic acid(PHA) 1
1.2 PHA 합성 경로 2
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I. 서론 1
1.1 Polyhydroxyalkanoic acid(PHA) 1
1.2 PHA 합성 경로 2
1.3 PHA 합성 저해제의 연구 3
1.4 연구목적 및 연구내용 6
II. 재료 및 실험 방법 8
2.1 시약 8
2.2 사용 균주와 배양 배지 8
2.3 세균으로부터 폴리에스터의 생합성 8
2.4 폴리에스터의 순수분리 9
2.5 기체 크로마토그라피(Gas Chromatography, GC) 분석 10
2.6 폴리에스터의 fractionation 분석 10
2.7 시차주사 열분석(Differential Scanning Calorimetry, DSC) 11
2.8 핵자기공명(Nuclear Magnetic Resonance, NMR) 분광학 실험 11
III. 결과 및 고찰 12
3.1 P. fluorescens BM07에서 salicylic acid에 의한 PHA 합성의 저해 및 PHA 모노머 조성의 조절 12
3.1.1 Fructose에서 배양한 P. fluorescens BM07에서 salicylic acid가 세포성장과 PHA 합성에 미치는영향 12
3.1.2 5 mM 11-POU와 50 mM fructose의 공동기질에서 배양한 P. fluorescens BM07에서 salicylic acid가 세포성장과 PHA 합성에 미치는 영향 14
3.1.3 3 mM 11-POU와 50 mM fructose의 공동기질에서 배양한 P. fluorescens BM07에서 salicylic acid가 세포성장과 PHA 합성에 미치는 영향 18
3.1.4 Octanoic acid와 fructose의 공동기질에서 배양한 P. fluorescens BM07에서 salicylic acid가 세포성장과 PHA 합성에 미치는 영향 23
3.2 SCL-PHA와 MCL-PHA를 동시에 합성하는 균주 P. aeruginosa BM114에서 salicylic acid에 의한 MCL-PHA의 특이적 저해 24
3.2.1 분리한 균주의 특징 24
3.2.2 P. aeruginosa BM114에서 SCL-PHA와 MCL-PHA의 blend-type 폴리머 합성 24
3.2.3 P. aeruginosa BM114에서 salicylic acid가 세포성장과 PHA 합성에 미치는 영향 32
3.2.4 P. aeruginosa BM114에서 2-bromooctanoic acid가 세포성장과 PHA 합성에 미치는 영향 36
IV. 결론 40