Plaggenborg R, Overhage J, Steinbüchel A, Priefert H
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):528-35. doi: 10.1007/s00253-003-1260-4. Epub 2003 Mar 27.
Pseudomonas putida KT2440 is a physiologically extremely versatile non-pathogenic bacterium that is applied as a "biosafety strain" in biotechnological processes, as authorized by the USA National Institute of Health. Analysis of the P. putida KT2440 whole-genome sequence revealed the genetic organization of the genes fcs, ech, and vdh, which are essential for ferulic acid conversion to vanillic acid via vanillin. To confirm the physiological function of these structural genes as feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh), respectively, they were cloned and expressed in Escherichia coli. Recombinant strains harboring fcs and ech were able to transform ferulic acid to vanillin. The enzyme activities of Fcs and Vdh were determined in protein extracts of these cells. The essential involvement of fcs, ech and vdh in the catabolism of ferulic acid in P. putida KT2440 was proven by separately inactivating each gene by insertion of Omega-elements. The corresponding mutant strains KT2440 fcsOmegaKm, KT2440 echOmegaKm, and KT2440 vdhOmegaKm were not able to grow on ferulic acid. The potential application of P. putida KT2440 and the mutant strains in biotechnological vanillin production process is discussed.
恶臭假单胞菌KT2440是一种生理功能极其多样的非致病细菌，经美国国立卫生研究院授权，它被用作生物技术过程中的“生物安全菌株”。对恶臭假单胞菌KT2440全基因组序列的分析揭示了fcs、ech和vdh基因的遗传组织，这些基因对于阿魏酸通过香草醛转化为香草酸至关重要。为了分别证实这些结构基因作为阿魏酰辅酶A合成酶（Fcs）、烯酰辅酶A水合酶/醛缩酶（Ech）和香草醛脱氢酶（Vdh）的生理功能，将它们克隆并在大肠杆菌中表达。携带fcs和ech的重组菌株能够将阿魏酸转化为香草醛。在这些细胞的蛋白质提取物中测定了Fcs和Vdh的酶活性。通过插入Omega元件分别使每个基因失活，证明了fcs、ech和vdh在恶臭假单胞菌KT2440阿魏酸分解代谢中的重要作用。相应的突变菌株KT2440 fcsOmegaKm、KT2440 echOmegaKm和KT2440 vdhOmegaKm不能在阿魏酸上生长。讨论了恶臭假单胞菌KT2440和突变菌株在生物技术香草醛生产过程中的潜在应用。
