Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, PR China.
Appl Environ Microbiol. 2011 Oct;77(20):7279-88. doi: 10.1128/AEM.00203-11. Epub 2011 Aug 26.
Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C(8) to C(32) n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2Δ1 suggested the alkW1 could restore the growth of KOB2Δ1 on C(14) and C(16) n-alkanes and induce faster growth on C(18) to C(32) n-alkanes than alkW1ΔRd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C(32) alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C(32) in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C(32)) in the Gram-negative bacterium.
从一株能够以原油和长度为 6 到 40 个碳原子的正构烷烃作为唯一碳源生长的 Dietzia 菌株 DQ12-45-1b 中克隆了两个烷烃羟化酶-还原酶融合基因同源物（alkW1 和 alkW2）。AlkW1 和 AlkW2 均具有完整的膜烷烃单加氧酶（AlkB）保守结构域和还原酶（Rd）保守结构域，它们融合在一起。系统发育分析表明，这两个 AlkB 融合的 Rd 结构域与革兰氏阳性细菌中的烷烃羟化酶-还原酶融合基因簇中的所有 Rd 一起形成了一个新的第三簇，并且该第三簇与已知的 AlkG1 和 AlkG2 型 Rd 相去甚远。当细胞以 C(8) 到 C(32) 正构烷烃作为唯一碳源生长时，DQ12-45-1b 中 alkW1 基因的表达被诱导，但未检测到 alkW2 基因的表达。在荧光假单胞菌 KOB2Δ1 的 alkB 缺失突变体中进行的功能异源表达表明，alkW1 可以恢复 KOB2Δ1 在 C(14) 和 C(16) 正构烷烃上的生长，并比 alkW1ΔRd（alkW1 的 Rd 结构域缺失突变基因）诱导更快的生长，alkW1ΔRd 也比 KOB2Δ1 本身诱导更快的生长。此外，革兰氏阴性荧光假单胞菌 CHA0 的 AlkB 与革兰氏阴性荧光假单胞菌 CHA0 和革兰氏阳性 Dietzia sp. DQ12-45-1b 的 Rd 的人工融合显著增加了 C(32) 烷烃的降解，与单独使用 AlkB 相比有所增加。总之，从 Dietzia 属克隆的 alkW1 基因编码了一种烷烃羟化酶，它可以增加对长度达 C(32)的正构烷烃的生长和降解，其融合的还原酶结构域是维持功能所必需的。此外，革兰氏阳性和阴性细菌的烷烃羟化酶和还原酶基因的融合可以增加革兰氏阴性细菌中长链正构烷烃（如 C(32)）的降解。
