Brandt K, Thewes S, Overhage J, Priefert H, Steinbüchel A
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Germany.
Appl Microbiol Biotechnol. 2001 Sep;56(5-6):724-30. doi: 10.1007/s002530100698.
During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.
在筛选能够将丁香酚转化为香草醛的细菌过程中，分离出了菌株OPS1，通过16 s rDNA序列分析将其鉴定为一种新的假单胞菌属物种。当这种细菌在丁香酚上生长时，在培养上清液中鉴定出了中间产物松柏醇、阿魏酸、香草酸和原儿茶酸。通过对假单胞菌属菌株HR199的丁香酚阴性突变体SK6165进行互补，在假单胞菌属菌株OPS1的基因组文库中鉴定出了编码丁香酚羟化酶（ehyA、ehyB）的基因，该酶催化了这一生物转化的第一步。EhyA和EhyB与假单胞菌属菌株HR199的丁香酚羟化酶亚基的氨基酸同一性分别为57%和85%，与恶臭假单胞菌的对甲酚甲基羟化酶相应亚基的同一性高达34%和54%。此外，最近报道的荧光假单胞菌E118的丁香酚脱氢酶的α亚基和β亚基的氨基末端序列与EhyA和EhyB的相应区域吻合良好。在ehyB的下游，鉴定出一个开放阅读框，其推导的氨基酸序列与天青蛋白的同一性高达71%，很可能代表丁香酚羟化酶的生理电子受体的基因（azu）。通过PCR扩增丁香酚羟化酶基因，将其克隆并在大肠杆菌中进行功能表达。
