Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST
Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST
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DOI：
10.1128/aem.63.6.2232-2239.1997
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发表时间：
1997-06-01
影响因子：
4.4
通讯作者：
Zennaro, E
中科院分区：
文献类型：
--
作者：
Beltrametti, F;Marconi, AM;Zennaro, E
测定了编码苯乙烯氧化成苯乙酸的荧光假单胞菌ST的4，377-bp染色体区域的核苷酸序列。在该区域中鉴定出四个开放阅读框架，命名为styA、styB、styC和styD。序列分析和生物转化测定，进行分批和连续培养，使我们能够确定测序基因的功能。styA和styB编码负责苯乙烯转化为环氧苯乙烯的苯乙烯单加氧酶; styC编码该途径的第二种酶，将环氧苯乙烯转化为苯乙醛的环氧苯乙烯异构酶; styD基因产生将苯乙醛氧化为苯乙酸的苯乙醛脱氢酶。StyA基因全长415个氨基酸，与荧光假单胞菌和铜绿假单胞菌的对羟基苯甲酸羟化酶以及恶臭假单胞菌的水杨酸羟化酶具有较弱的同源性，提示StyA可能是一种黄素腺嘌呤二核苷酸结合单加氧酶。StyB与质粒pJP 4编码的2，4-二氯苯酚-6-单加氧酶的羧基端部分同源，而styC与任何已知的蛋白质没有明显的同源性。第四个开放阅读框styD编码502个氨基酸，与几种真核和原核乙醛脱氢酶高度同源。基因的顺序对应于分解代谢步骤的顺序。先前提出的环氧苯乙烯还原酶基因的存在，直接将环氧苯乙烯转化为2-苯基乙醇（A。M. Marconi，F. Beltrametti，G. Bestetti，F. Solinas，M. Ruzzi，E. Galli和E. Zennaro，Appl. Environ. Microbiol. 61：121-127，1996），尚未通过测序和在连续培养物中进行的生物转化测定法证实。插入序列IS 1162的拷贝，属于IS 21样家族的元素，被确定为紧接苯乙烯分解代谢基因的下游。
The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas flourescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P.flourescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to he partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A. M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformatlon assays performed in continuous cultures. A copy of the insertion sequence IS1162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.