Pseudomonas mosselii TO7生產聚羥基烷酸酯及其合成途徑之研究

Abstract

由微生物產生的中鏈聚羥基烷酸酯 (medium-chain-length Polyhydroxyalkanoates, MCL-PHAs) 柔韌有彈性，適合開發應用於軟組織工程或藥物傳輸等生醫材料。本研究針對一株中鏈PHA高產量生產菌Pseudomonas mosselii TO7之PHA合成代謝途徑進行探討。以0.5%辛酸為碳源，1 g/L之 (NH4)2SO4為氮源，培養基初始pH值為9，經一階段培養方式培養40小時後，所收獲P. mosselii TO7菌體具有最佳PHA產量，為菌體乾重45%以上，主要組成是聚羥基辛酸酯，於PHA比例高達 95％。利用廉價碳源棕櫚核仁油或大豆油生成中鏈聚羥基烷酸酯，由棕櫚核仁油產生的PHA結晶度較高。利用丙烯酸 (acrylic acid) 及2-溴正辛酸 (2-bromooctanoic acid)兩種抑制劑証實P. mosselii TO7主要由脂肪酸經β氧化途徑合成PHA，由醣類經脂肪酸生合成途徑僅能產生少量PHA。P. mosselii TO7 的PhaC1Pm為第二型PHA聚合酶，將P. mosselii TO7擁有的兩個PHA聚合酶基因，以PHA基因缺失株P. putida GPp104 PHA-分別進行PHA累積表現，顯示P. mosselii TO7利用八至十二碳脂肪酸為碳源時，主要作用酵素為PhaC1 Pm。(R)-立體特異性烯酰輔酶A水合酶於菌體內利用脂肪酸合成PHA為重要酵素之一，因此選殖出P. mosselii TO7 PHA (R)-立體特異性烯酰輔酶A水合酶 (phaJ4Pm)，以phaJ4Pm分別於Escherichia coli LS5218利用十二烷酸為碳源，P. mosselii TO7利用辛酸為碳源，進行異源表現與過度表現，結果皆顯示PhaJ4Pm可提升PHA產量及PHA的八碳單體組成。以trans-2-octenoyl-CoA分析PhaJ4Pm的酵素活性，証實PhaJ4Pm 具有水合酶功能。令PhaJ4Pm與P. putida GPo1 PHA聚合酶1 (poly(3-hydroxyalkanoate) polymerase 1, PhaC1Pp) 進行偶合反應，証實經PhaJ4Pm催化之產物為R-specific。先前本研究室已知第二型PHA聚合酶PhaC1Pp之L 484位置可能為影響基質專一性的重要胺基酸。為闡明第二型PHA聚合酶影響基質專一性的重要胺基酸，本研究進行PhaC1Pp之L484位置飽和突變實驗，証實野生株leucine置換為valine時，對酵素基質專一性的改變最為明顯。以人類環氧化物水解酶 (epoxide hydrolase from Human) 為模板建立酵素3D模型，顯示出484位置上之胺基酸可能由於接近酵素催化殘基histidine 479而影響了PHA的合成。本研究探討P. mosselii TO7所生產中鏈聚羥基烷酸酯之特性及其合成代謝途徑中，PhaC1Pm，PhaC2Pm及PhaJ4Pm三酵素對PHA合成影響；並提供第二型PHA聚合酶PhaC1 Pp影響基質專一性重要胺基酸位置之分析。

The properity of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) possessing flexibility and elasticity are potentially used in cardiovascular, soft tissue engineering and drug delivery. This study investigated the PHA biosynthestic pathways of high-yield medium-chain-length PHAs producing strain Pseudomonas mosselii TO7. P. mosselii TO7 can produce PHA at a yield of more than 45% of its dry weight in mineral salt medium containing 0.5% octanoic acid and 1g/L (NH4)2SO4 after 40 hours incubation. The 3-hydroxyoctanoate monomer produced by shake flask cultivation in one stage made up 95% of the polymer composition. Initial culture medium pH level of 9 lead to increased PHA biosynthesis. P. mosselii TO7 can utilize palm kernel oil or soybean oil to produce PHA, however, using palm kernel oil having relatively higher crystallinity. The inhibitors studies including acrylic acid and 2-bromooctanoic acid indicated that β-oxidation of fatty acids is mainly used to synthesize PHA in P. mosselii TO7. Two class II PHA polymerase genes phaC1Pm and phaC2Pm of P. mosselii TO7 were heteroexpression in PHA-negative mutant P. putida GPp104 PHA-. The result demonstrated that when P. mosselii TO7 used 8-12 carbon fatty acids as a carbon source, the main enzyme was PhaC1Pm. The (R)-specific enoyl-CoA hydratase from P. mosselii TO7 (PhaJ4Pm) was identified, owing to (R)-specific enoyl-CoA hydratase play an important role for PHA biosynthesis from fatty acids in microorganism. Hetereoexpression of PhaJ4Pm in Escherichia coli LS5218 when feeding 12 carbon fatty acids, the result showed increased PHAs production and 3-hydroxyoctanoate monomer composition of PHA. Similar results also have appeared in P. mosselii TO7 when overexpression of PhaJ4Pm used 8 carbon fatty acids as a sole carbon source. Analysis of PhaJ4Pm enzyme activity using trans-2-octenoyl-CoA confirmed that PhaJ4Pm functions as a hydrase. A coupling reaction between PhaJ4Pm and PHA polymerase 1 from P. putida GPo1 (PhaC1Pp) displayed the PhaJ4-catalyzed products with R-specific stereospecificity. Our previous study indicated that class II PHA polymerase PhaC1Pp at mutation at L484V may affect the substrate specificity of enzyme. To explain substrate-specific related amino acid residue in class II PHA polymerase, PhaC1Pp was used. Saturation mutagenesis at residue leucine 484 in PhaC1Pp confirmed when the wild type leucine was switched with valine, a change in substrate-specificity was most significant. Constructing 3D enzyme models showed the amino acid residues at 481, 482, and 484 may influence PHA biosynthesis due to close to the enzyme catalytic residue His479. This study explores the characteristics of PHA produced from P. mosselii TO7, the effects of PhaC1Pm, PhaC2Pm and PhaJ4Pm on PHA biosynthesis, and providing analysis of substrate-specific related amino acid residue in a class II PHA polymerase PhaC1 Pp.