Smits T H, Röthlisberger M, Witholt B, van Beilen J B
Institute of Biotechnology, ETH Hönggerberg, Zürich, Switzerland.
Environ Microbiol. 1999 Aug;1(4):307-17. doi: 10.1046/j.1462-2920.1999.00037.x.
We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C6-C11) or long-chain n-alkanes (C12-C16), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n-alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases.
我们基于多个高度保守的序列基序，开发了用于聚合酶链反应（PCR）扩增与食油假单胞菌GPo1和不动杆菌属ADP1烷烃羟化酶相关基因的高度简并寡核苷酸。在所有能够在中链（C6 - C11）或长链正构烷烃（C12 - C16）上生长的革兰氏阴性菌以及三分之二的革兰氏阳性菌中，均获得了预期大小的PCR产物。对PCR片段进行克隆和测序后发现，其编码的肽段与食油假单胞菌GPo1烷烃羟化酶的相应片段具有43.2 - 93.8%的序列同一性。无法在正构烷烃上生长的菌株未产生与烷烃羟化酶基因具有同源性的PCR产物。以PCR产物为探针，克隆了乙酸钙不动杆菌EB104和恶臭假单胞菌P1的烷烃羟化酶基因。这两个基因分别使不动杆菌属ADP1的一个烷烃羟化酶阴性突变体和一个含有除alkB外所有食油假单胞菌alk基因的大肠杆菌重组体能够在正构烷烃上生长，表明克隆的基因确实编码烷烃羟化酶。
