克雷伯氏肺炎菌中影響1,3-丙二醇生產相關基因之研究

The study of 1,3-propanediol production by engineering glycerol metabolic related genes in Klebsiella pneumoniae

周大中(Ta-Chung Chou)

指導教授 ： 蘇伯琦

元智大學/工程學院/生物科技與工程研究所/碩士(2009年)

摘要

1,3- 丙二醇是一種重要的有機化工原料， 最重要的用途是作為聚酯
（polyester）、聚醚（polyether）和聚亞氨酯（polyurethanes）的單體。由它合成的聚酯有獨特的性質和優異的性能，而且可以使聚酯塑膠具有易於自然循環的可生物降解特性。目前已知有許多微生物諸如克雷伯氏菌屬之Klebsiella pneumoniae、 Klebsiella oxytoca，梭狀芽孢桿菌屬之Clostridium butyricum，檸檬酸桿菌屬之Citrobacter freundii，Enterobacter agglomerans，嗜酸乳酸桿菌屬之Lactobacillus brevis 等等皆可生合成1,3-丙二醇。本研究利用同源性染色體基因重組技術將克雷伯氏肺炎菌（Klebsiella pneumoniae）中，甘油氧化代謝途徑之重要基因dhaD、dhaK 剔除，剔除之菌株將無法產生甘油代謝後之副產物且試圖將代謝途徑轉向有效利用甘油合成1,3-丙二醇。根據實驗結果顯示dhaD、dhaK剔除菌株TC100，在氧化代謝甘油途徑中，乳酸、2,3-丁二醇與酒精等副產物被順利剔除。另外在產量上，野生型菌株與突變株TC100 之1,3-丙二醇產量分別為5.59（g/L）與1.91 （g/L）；TC100 並沒有如預期般因缺少生成部分副產物而產生更多的1,3-丙二醇。
因此本研究持續以提升菌株生產1,3-丙二醇為目的，將其代謝甘油路徑中直接與1,3-丙二醇生產有關的dhaB 與dhaT 基因進行過量表現，利用基因剪接技術將兩基因接至表現質體上並將其分別轉入野生型菌株與TC100。進一步利用酵素活性分析證實過量表現的dhaB 與dhaT 活性皆較無過量表現此二酵素之對照組菌株為高。在利用液相層析法中比較各菌株間1,3-丙二醇的產量關係，研究結果顯示過量表現dhaB 之菌株TCKP01，其每單位菌量之1,3-丙二醇產量大約高出野生型菌株1.01% （5.62g/L）、過量表現dhaT 之菌株TCKP05中，每單位菌量之1,3-丙二醇產量大約高出野生型菌株1.01%；在TC100 方面，
過量表現dhaB 之菌株TC101，其每單位菌量之1,3-丙二醇產量高出TC100 約20.4%、過量表現dhaT 之菌株TC105，每單位菌量之1,3-丙二醇產量高出TC100約56.6%。因此本研究證實將dhaD、dhaK 剔除有助於減少副產物的產生以利純化1,3-丙二醇的過程與將dhaB 或dhaT 分別加以過量表現，將有益於增加單位菌量之1,3-丙二醇產量。這些重組菌株對於工業生產1,3-丙二醇有很大的助益。

關鍵字

克雷伯氏肺炎菌 ； 1,3-丙二醇 ； 細菌血紅素

並列摘要

1,3-propanediol (1,3-PD) is an important organic compound in chemical industry,and this compound could potentially be used for many synthesis reactions, such as a monomer to produce polyesters, polyethers and polyurethanes. The polyesters which were synthesized by 1,3-PD has unique and outstanding performance; moreover, 1,3-PD may enable the polyesters easy to have the characteristic of natural circulation and biodegradation . In recent study, there are many different microorganisms that produce 1,3-propanediol, such as Klebsiella pneumoniae, Klebsiella oxytoca, Clostridium butyricum, Citrobacter freundii, Enterobacter agglomerans and Lactobacillus brevis. In this study, in order to facilitate the conversion of glycerol to
1,3-PD in K. pneumoniae, we employed the homologous recombination to knock out two important genes, dhaD and dhaK, in glycerol oxidation pathway to eliminate side
products during glycerol metabolism. The result indicated that although the side products in glycerol oxidation pathway such as lactate, 2,3-butanediol and ethanol
were successfully eliminated in mutant strain TC100, the productivity of 1,3-propanediol of wild type (WT) and TC100 were 5.59(g/L) and 1.91(g/L), respectively.
To further promote the 1,3-PD productivity, we directly overexpressed the genes dhaB and dhaT, involved in 1,3-PD formation, by transforming expression vector into
TC100. We verified that the enzyme activities of DhaB or DhaT were higher in the recombinant strains TC101 (TC100/pBAD-dhaB) and TC105 (TC100/pBAD-dhaT)
compared with TC100. Using high performance liquid chromatography, the 1,3-PD productivity of TCKP01(WT/pBAD-dhaB) and TCKP05(WT/pBAD-dhaT) were both 1.01% higher than wild type strain respectively. Onther the other hand, the 1,3-PD productivity of TC101 and TC105 were 20.4% and 56.6% higher than TC100. In this study, we corroborated that deletion of dhaD and dhaK led to reduce side products
and overexpression of dhaB and dhaT facilitated the 1,3-PD productivity in K. pneumoniae. The recombinant strain(s) would be a great industrial application in the production of 1,3-PD.

並列關鍵字

Klebsiella pneumoniae ； 1,3-Propanediol ； Vitreoscilla hemoglobin

參考文獻

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