- 學位論文
以甘油為碳源的1, 3-丙二醇生產菌之基因選殖研究
Cloning of the recombinant strains to produce 1, 3-propanediol from glycerol
摘要
於1,3-丙二醇在各行業中廣泛的被使用，為高價值且需求量大之化學品。本研究探討如何利用低成本的甘油做為原料，經E. coli菌種醱酵，生產出高經濟價值的1,3-丙二醇。由於E. coli菌體中，缺乏直接將甘油代謝成1,3-丙二醇之路徑，因此須透過基因重組來建構生產菌。將甘油轉換成1,3-丙二醇的過程，需要兩種二醇氧化還原酶 (1,3-PDOR)與甘油脫水酶(GDHt)。甘油經甘油脫水酶反應後，生成3-羥基丙醛 (3-HPA)，再經1,3-丙二醇氧化還原酶催化，生成1,3-丙二醇。本研究首先將取自E. coli K12的yqhD基因與Klebsiella pneumonia rhinoscleromatis ATCC 13884之dhaB兩段基因同時建構於pGEX-2TK質體上，並用IRES串接，使兩段基因可同時表現。此質體命名為pGEX-2TK_yqhD_dhaB，並轉殖於E. coli BL21中表現。含pGEX-2TK_yqhD_dhaB之E. coli BL21重組菌，經IPTG誘導後，可大量表達1,3-丙二醇氧化還原酶；然而甘油脫水酶的表現量卻不明顯。針對重組菌之細胞粗萃液中，分析1,3-丙二醇氧化還原酶的活性。誘導前之樣品活性為4.6 U/g protein，誘導後3小時的樣品活性為13.4 U/g protein。此外，也利用含兩組T7啟動子之pETDuet質體，將yqhD及dhaB基因安插於其中，利用 T7啟動子同時表現。含pETDuet_dhaB_yqhD重組質體之E .coli BL21 (DE3)，經IPTG誘導，重組菌之粗萃液，1,3-丙二醇氧化還原酶活性，誘導前之樣品活性為5.1 U/g protein，誘導後3小時的樣品活性為29.4 U/g protein。再者建構pBAD33_dhaB重組質體，欲與pGEX-2TK_yqhD以雙質體方式於E. coli BL21中共表現。含pGEX-2TK_yqhD_dhaB之E.coli BL21重組菌，置於含甘油20 g/L醱酵培養基中培養，經24小時醱酵後，醱酵液中可測得0.4 g/L的1,3-丙二醇，產率約為0.05 g /g glycerol，甘油脫水酶的表現仍不明顯，造成1,3-丙二醇的產率偏低。
並列摘要
Despite of its high cost, the chemical 1,3-propanediol has been highly demanded and widely used in industries. This study investigated on how to take advantage of low-cost glycerol as raw material for the production of 1,3-propanediol with high economic value via E. coli fermentation. The conversion of glycerol to 1,3-propanediol requires the use of two enzymes：1,3-propanediol oxidoreductase (1,3-PDOR) and glycerol dehydratase (GDHt). The latter produces 3-hydroxypropionaldehyde (3-HPA) from glycerol, whereas the former converts 3-HPA to 1,3-propanediol. This study utilized the recombinat DNA technology to clone gene yqhD, from E. coli K12 and dhaB from the Klebsiella pneumonia rhinoscleromatis ATCC 13884 onto plasmid pGEX-2TK. These genes were connected with IRES to allow simultaneous over-expression. This recombinant plasmid, namely pGEX-2TK_yqhD_dhaB, was subsequently transformed into E. coli BL21. The recombinant strain, E. coli BL21_pGEX-2TK_yqhD_dhaB, was found capable of overexpressing 1,3-propanediol oxidoreductase after IPTG induction. However, the expression of GDHt was not obvious. Analysis of the crude extract of recombinant proteins for 1,3-PDORI activity before and after 3 hours of induction yielded 4.6 U/g protein and 13.4 U/g protein, respectively. In addition, genes yqhD and dhaB were also inserted into plasmid pETDuet containing two series of T7 promoters for overexpression. The 1,3-PDORI activity for recombinant strain E. coli BL21 (DE3) containing pETDuet_dhaB_yqhD before and after 3 hours of induction was found to be 5.1 U/g protein and 29.4 U/g protein, respectively. Then, we construct the plasmid pBAD33_dhaB, and E. coli BL21 containing plasmid of pGEX-2TK_yqhD and pBAD33_dhaB. After 24 hours of flask fermentation of E.coli BL21 harboring pGEX-2TK_yqhD_dhaB. In culture medium containing 20 g/L of glycerol, 0.4 g/L of 1,3-propanediol was obtained. This is equivalent to a yield of approximately 0.05 g/g glycerol. The expression of GDHt were insignificant, which lead to extremely low production rate of 1,3-propanediol.