Two Novel Alkane Hydroxylase-Rubredoxin Fusion Genes Isolated from a Dietzia Bacterium and the Functions of Fused Rubredoxin Domains in Long-Chain n -Alkane Degradation

Abstract

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 KOB2⌬1 suggested the alkW1 could restore the growth of KOB2⌬1 on C 14 and C 16 n-alkanes and induce faster growth on C 18 to C 32 n-alkanes than alkW1⌬Rd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2⌬1 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.Alkane hydroxylation is the key step in alkane degradation in microorganisms, and alkane hydroxylases play an important role in the microbial degradation of alkanes (34). There are three classes of alkane hydroxylases in microorganisms, depending on the chain length of the alkane substrate. The soluble nonheme di-iron monooxygenases (sMMO) and membrane-bound particulate copper-containing enzymes (pMMO) are the main enzymes that catalyze the oxygenation of alkanes C 1 to C 5 in length (21). The integral-membrane alkane monooxygenase (AlkB)-related alkane hydroxylases (37) and cytochrome P450 enzymes (35) found in fungi and bacteria can oxidize long-chain alkanes with up to 16 carbon atoms. Among the members of the third class of enzymes, which can catalyze the oxidation of alkanes longer than C 18 , only one C 15 to C 36 alkane monooxygenase (LadA) found in Geobacillus thermodenitrificans NG80-2, which is distinct from other known AlkBtype alkane hydroxylases, has been cloned and the activities of purified LadA on alkanes with different chain lengths have been previously identified (8). In the AlkB system, three individual...

n -Alkane Chain Length Alters Dietzia sp. Strain DQ12-45-1b Biosurfactant Production and Cell Surface Activity

“…All experiments were performed in triplicate with various controls. Detailed experimental procedures and methods are described in the supplemental material.Strain DQ12-45-1b could degrade C 16 , C 24 , and C 36 n-alkanes for growth (see Table S1 in the supplemental material), as was previously reported (19,20). Along with the growth and degradation of alkanes, the surface tension of the culture broth decreased from approximately 62 mN m Ϫ1 to 27.78 Ϯ 1.97 and 45.97 Ϯ 2.19 mN m Ϫ1 at day 30 for C 16 and C 24 cultures, respectively, and to 48.66 Ϯ 0.51 mN m Ϫ1 at day 45 for the C 36 culture (see Fig.…”

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

“…However, alkanes with longer chain lengths have poor water solubility and most likely accumulate in the cell membrane (55). The AlkB-type AH is a membrane-integrated alkane hydroxylase, while CYP153 is a soluble AH present in the cytoplasm (9,24). It is convenient and energy saving for the membrane-integrated AlkB to oxidize long-chain n-alkanes in the membrane, as the resulting products may serve as inducers for the corresponding repressor in the cytoplasm.…”

“…For example, in the broad-spectrum n-alkane-degrading bacterium Dietzia sp. DQ12- 45-1b (19), the AlkB-type AlkW1 oxidized C 16 to C 40 n-alkanes, while CYP153 oxidized n-alkanes with shorter chain lengths (fewer than 14 carbon atoms) (15,24). Similar results were also observed in the Gram-negative bacterium Alcanivorax dieselolei B-5, expressing one P450 and two alkB genes, which could be induced by C 8 to C 16 and C 12 to C 26 n-alkanes, respectively (23).…”

Quantitative PCR analysis of diesel degrading genes of Acinetobacter calcoaceticus isolates

“…Interestingly, novel genes encoding AlkB-Rubredoxin fusion proteins were used in the hydroxylation of long-chain alkanes by Gram positive bacteria (Nie et al, 2011). In addition, rubredoxin-rubredoxin reductase systems are present in many other organisms that are unable to degrade alkanes, where they serve other functions such as rapid transport of reducing equivalents to the final receptor (Hagelueken et al, 2007).…”