Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp D7
Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp D7
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DOI：
10.1099/00221287-145-9-2393
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发表时间：
1999-09-01
期刊：
影响因子：
2.8
通讯作者：
Davies, J
中科院分区：
文献类型：
--
作者：
Chow, KT;Pope, MK;Davies, J
芳香酸的非氧化脱羧的遗传学在原核生物和真核生物中都知之甚少。尽管在许多微生物中观察到了作用于各种底物的此类反应，但据我们所知，编码负责这些过程的酶的基因尚未在文献中报道。在此，描述了从土壤中分离的链霉菌（Streptomycessp.D7），其有效地将4-羟基-3-甲氧基苯甲酸（香草酸）转化为2-甲氧基苯酚（愈创木酚）。蛋白质二维凝胶分析表明，几个蛋白质的合成响应香草酸。其中之一的特征在于部分氨基末端测序，导致从基因组DNA λ噬菌体文库克隆的基因簇，由三个ORF，vdcB（602 bp），vdcC（1424 bp）和vdcD（239 bp）。蛋白质序列的比较表明，该产品的vdcB（201 aa）是类似的苯丙烯酸脱羧酶的酵母，推定的产品vdcC（475 aa）和vdcD（80 aa）是类似的假设蛋白质的未知功能从各种微生物，并发现在一个类似的集群在枯草芽孢杆菌。北方印迹分析显示，香草酸诱导的细胞中的2.5 kb的mRNA转录本的合成，这表明该簇是在一个单一的诱导型启动子的控制下。整个vdc基因簇在变铅青链霉菌1326中作为异源宿主的表达导致该菌株获得非氧化性地将香草酸脱羧为愈创木酚的能力。链霉菌D 7菌株和重组链霉菌S.然而，表达VDC基因簇的Lividans 1326不使结构相似的芳香酸脱羧，这表明该系统对香草酸是特异性的。这种分解代谢系统可能是有用的，作为一个组件的途径工程研究集中在生产有价值的化学品从林业和农业副产品。
The genetics of non-oxidative decarboxylation of aromatic acids are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous micro-organisms acting on a variety of substrates, the genes encoding enzymes responsible for these processes have not, to our knowledge, been reported in the literature. Here, the isolation of a streptomycete from soil (Streptomyces sp. D7) which efficiently converts 4-hydroxy-3-methoxybenzoic acid (vanillic acid) to 2-methoxyphenol (guaiacol) is described. Protein two-dimensional gel analysis revealed that several proteins were synthesized in response to vanillic acid. One of these was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic DNA lambda phage library, consisting of three ORFs, vdcB (602 bp), vdcC(1424 bp) and vdcD (239 bp). Protein sequence comparisons suggest that the product of vdcB (201 aa) is similar to phenylacrylate decarboxylase of yeast; the putative products of vdcC (475 aa) and vdcD (80 aa) are similar to hypothetical proteins of unknown function from various micro-organisms, and are found in a similar cluster in Bacilius subtilis. Northern blot analysis revealed the synthesis of a 2.5 kb mRNA transcript in vanillic-acid-induced cells, suggesting that the cluster is under the control of a single inducible promoter. Expression of the entire vdc gene cluster in Streptomyces lividans 1326 as a heterologous host resulted in that strain acquiring the ability to decarboxylate vanillic acid to guaiacol non-oxidatively. Both Streptomyces sp, strain D7 and recombinant S. lividans 1326 expressing the vdc gene cluster do not, however, decarboxylate structurally similar aromatic acids, suggesting that the system is specific for vanillic acid. This catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural by-products.