Ouyang Shao-Ping, Sun Shang-Yu, Liu Qian, Chen Jinchun, Chen Guo-Qiang
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China.
Appl Microbiol Biotechnol. 2007 Feb;74(1):43-9. doi: 10.1007/s00253-006-0637-6. Epub 2006 Oct 5.
Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.
甲苯双加氧酶（TDO）催化芳香族化合物的不对称顺式二羟基化反应。为了实现苯向苯顺式二醇的高效生物转化，以恶臭假单胞菌KT2442、施氏假单胞菌1317和嗜水气单胞菌4AK4作为宿主来表达TDO基因tod。构建了质粒pSPM01，它是携带来自质粒pKST11的tod的广宿主质粒pBBR1MCS - 2的衍生物，并将其导入上述三株菌株中。检测了它们催化苯转化为苯顺式二醇（即简称为DHCD的顺式 - 3,5 - 环己二烯 - 1,2 - 二醇）的能力。在优化的培养基和生长条件下进行摇瓶培养时，重组恶臭假单胞菌KT2442（pSPM01）、施氏假单胞菌1317（pSPM01）和嗜水气单胞菌4AK4（pSPM01）产生的苯顺式二醇分别为2.68、2.13和1.17 g/L。相比之下，大肠杆菌JM109（pSPM01）和大肠杆菌JM109（pKST11）分别产生0.45和0.53 g/L的DHCD。当在6升发酵罐中进行生物转化时，恶臭假单胞菌KT2442（pSPM01）中DHCD的产量约为60 g/L；这是迄今为止报道的最高DHCD产量。
