Overhage Jörg, Steinbüchel Alexander, Priefert Horst
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, D-48149 Münster, Germany.
Appl Environ Microbiol. 2002 Sep;68(9):4315-21. doi: 10.1128/AEM.68.9.4315-4321.2002.
The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.
基因座ehyAB、calA和calB分别编码丁香酚羟化酶、松柏醇脱氢酶和松柏醛脱氢酶，它们参与假单胞菌属菌株HR199中丁香酚分解代谢的第一步，通过聚合酶链反应（PCR）扩增这些基因座，并将其组合构建一个分解代谢基因盒。该基因盒被克隆到新设计的广宿主载体pBBR1-JO2（pBBR1-JO2ehyABcalAcalB）中，并转移到真养产碱菌H16。携带该质粒的真养产碱菌H16重组菌株表达了具有功能活性的丁香酚羟化酶、松柏醇脱氢酶和松柏醛脱氢酶。来自指数生长后期的真养产碱菌H16（pBBR1-JO2ehyABcalAcalB）细胞被用作生物催化剂，用于将丁香酚生物转化为阿魏酸。在未经进一步优化的情况下，每升培养物每小时丁香酚的最大转化率达到2.9 mmol，20小时内阿魏酸的产率为93.8 mol%（基于丁香酚）。
