Overhage Jörg, Steinbüchel Alexander, Priefert Horst
Institut für Molekulare Mikrobiologie und Biotechnologie der Westfälischen Wilhelms-Universität Munster, Corrensstrasse 3, D-48149 Münster, Germany.
J Biotechnol. 2006 Sep 18;125(3):369-76. doi: 10.1016/j.jbiotec.2006.03.024. Epub 2006 May 4.
To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.
为了利用丁香酚作为廉价底物用于生物技术生产芳香族化合物，将来自简单青霉CBS 170.90的香草醇氧化酶基因（vaoA）克隆到适合革兰氏阳性菌的表达载体中，并在耐香草醛的革兰氏阳性菌株阿维链霉菌属HR167中表达。携带重组质粒pRLE6SKvaom的重组菌株表现出1.1U/g蛋白质的比香草醇氧化酶活性。此外，该菌株获得了以丁香酚作为唯一碳源生长的能力。在以丁香酚为碳源生长过程中，检测到中间产物松柏醇、松柏醛、阿魏酸、愈创木酚和香草酸作为分泌化合物排出，而香草醛仅能检测到痕量。阿维链霉菌属HR167（pRLE6SKvaom）的静息细胞从丁香酚产生松柏醇，最大转化率约为2.3 mmol/h/l培养物，在未经进一步优化的16小时生物转化后，获得的最大松柏醇浓度为4.7 g/1。除了松柏醇，还检测到痕量的松柏醛和阿魏酸。
