Alkane Monooxygenase Research Articles

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ± 7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ± 1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.

Bacterial seeding potential of digestate in bioremediation of diesel contaminated soil

Petroleum contaminated soils constitute an environmental problem which may be solved with the help of bioremediation. Soil bioaugmentation with petroleum degrading bacteria is an efficient clean-up strategy. Currently scientific interest focuses on finding sources of microbial agents able to degrade hydrocarbons which may serve as species pools for enrichments during inoculum preparation. Seven microbial enrichments were obtained from various digestates, from a petroleum contaminated soil and from mix of both: soil and digestate. Diesel degrading capacities of microorganisms from digestates, which may serve as inoculum and nutrient source for bioremediation, were assessed. Additionally, the presence and abundance of alkane monooxygenase encoding genes (alkB) was estimated in enrichments. The highest proportion of alkB genes was found for enrichments originating from composted digestate of organic fraction of municipal solid waste and petroleum contaminated soil having the most efficient diesel removal rates (78 and 77% diesel removal, respectively). Enrichments obtained from digestate mixed with soil have a lower performance than single source enrichments. Strains belonging to Rhodococcus and Achromobacter genus were found in all enrichments, and Rhodococcus dominated in enrichment with the higher diesel degradation potential. All those results indicate a great potential of digestate as a source of diesel degraders for soil bioremediation.

Engineering of an oleaginous bacterium for the production of fatty acids and fuels.

Production of free fatty acids (FFAs) and derivatives from renewable non-food biomass by microbial fermentation is of great interest. Here, we report the development of engineered Rhodococcus opacus strains producing FFAs, fatty acid ethyl esters (FAEEs) and long-chain hydrocarbons (LCHCs). Culture conditions were optimized to produce 82.9 g l-1 of triacylglycerols from glucose, and an engineered strain with acyl-coenzyme A (CoA) synthetases deleted, overexpressing three lipases with lipase-specific foldase produced 50.2 g l-1 of FFAs. Another engineered strain with acyl-CoA dehydrogenases deleted, overexpressing lipases, foldase, acyl-CoA synthetase and heterologous aldehyde/alcohol dehydrogenase and wax ester synthase produced 21.3 g l-1 of FAEEs. A third engineered strain with acyl-CoA dehydrogenases and alkane-1 monooxygenase deleted, overexpressing lipases, foldase, acyl-CoA synthetase and heterologous acyl-CoA reductase, acyl-ACP reductase and aldehyde deformylating oxygenase produced 5.2 g l-1 of LCHCs. Metabolic engineering strategies and engineered strains developed here may help establish oleaginous biorefinery platforms for the sustainable production of chemicals and fuels.

Stabilization and scale-up of photosynthesis-driven ω-hydroxylation of nonanoic acid methyl ester by two-liquid phase whole-cell biocatalysis.

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O2 via photosynthetic water oxidation. Thus far, research on photosynthesis-driven bioprocesses mainly focuses on strain development and the proof of principle in small-scale biocatalytic reaction setups. This study investigates the long-term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω-hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity-independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two-liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7-fold increased initial specific activities (5.6 ± 0.1 U/gCDW ) and 7.6-fold increased specific yields on biomass (3.8 ± 0.1 mmolH-NAME /gCDW ) as compared with single aqueous phase biotransformations. Finally, the whole-cell biotransformation system was successfully scaled from glass tubes to a stirred-tank photobioreactor. This is the first study reporting the application of the two-liquid phase concept for efficient phototrophic whole-cell biocatalysis.

Development of plant-microbe phytoremediation system for petroleum hydrocarbon degradation: An insight from alkb gene expression and phytotoxicity analysis

Aim of present work was to assess in-planta association potential of isolated endophytic bacterial strain Pseudomonas sp. (J10) (KY608252) with two cultivars of Lolium perenne L. (small & jumbo) and Arabidopsis thaliana L. for total petroleum hydrocarbon (TPH) degradation, alkane monooxygenase (alkb) gene expression and phytotoxicity analysis. A plant-microbe phytoremediation system was established to investigate the bacteria's ability to colonize the plant body and quantification of alkb gene to help withstand TPH stress in soil as well as in hydroponics. A real-time PCR method was developed to analyze bacterial colonization and survival within the plant body. Analysis revealed that J10 efficiently colonized all the tested plant species and expressed alkb gene under hydrocarbon stress ranging between 3.7 × 102–3.9 × 106 in A. thaliana and L. perenne (small), respectively. The colonization was more pronounced in soil as compared to hydroponic system. J10 inoculation reduced phytotoxicity and suggested that inoculation had a positive effect on plant growth under stress conditions as compared to control. L. perenne (small) showed significant TPH removal efficiency (45.6%) followed by L. perenne jumbo (24.5%) and A. thaliana (6.2%). In hydroponics, L. perenne (small) degraded about 28.2% TPH followed by L. perenne (jumbo) as 24.4%. Potential of the indigenously isolated plant endophytes may be exploited further for phytoremediation efficiency and industrial applications.

Biosurfactant-Producing Capability and Prediction of Functional Genes Potentially Beneficial to Microbial Enhanced Oil Recovery in Indigenous Bacterial Communities of an Onshore Oil Reservoir.

Microbial enhanced oil recovery (MEOR) is a bio-based technology with economic and environmental benefits. The success of MEOR depends greatly on the types and characteristics of indigenous microbes. The aim of this study was to evaluate the feasibility of applying MEOR at Mae Soon Reservoir, an onshore oil reservoir experiencing a decline in its production rate. We investigated the capability of the reservoir's bacteria to produce biosurfactants, and evaluated the potentials of uncultured indigenous bacteria to support MEOR by means of prediction of MEOR-related functional genes, based on a set of metagenomic 16s rRNA gene data. The biosurfactant-producing bacteria isolated from the oil-bearing sandstones from the reservoir belonged to one species: Bacillus licheniformis, with one having the ability to decrease surface tension from 72 to 32mN/m. Gene sequences responsible for biosurfactant (licA3), lipase (lipP1) and catechol 2,3-dioxygenase (C23O) were detected in these isolates. The latter two, and other genes encoding MEOR-related functional proteins such as enoyl-CoA hydratase and alkane 1-monooxygenase, were predicted in the bacterial communities residing the reservoir's sandstones. Exposure of these sandstones to nutrients, consisting of KNO3 and NaH2PO4, resulted in an increase in the proportions of some predicted functional genes. These results indicated the potentials of MEOR application at Mae Soon site. Using the approaches demonstrated in this study would also assist evaluation of the feasibility of applying MEOR in oil reservoirs, which may be enhanced by an appropriate nutrient treatment.

Establishment of BmoR-based biosensor to screen isobutanol overproducer

BackgroundIsobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4–C6 linear alcohols and C3–C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library.ResultsFirstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment.ConclusionsThis work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.

Biodegradation of naphthalene, BTEX, and aliphatic hydrocarbons by Paraburkholderia aromaticivorans BN5 isolated from petroleum-contaminated soil

To isolate bacteria responsible for the biodegradation of naphthalene, BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylene), and aliphatic hydrocarbons in petroleum-contaminated soil, three enrichment cultures were established using soil extract as the medium supplemented with naphthalene, BTEX, or n-hexadecane. Community analyses showed that Paraburkholderia species were predominant in naphthalene and BTEX, but relatively minor in n-hexadecane. Paraburkholderia aromaticivorans BN5 was able to degrade naphthalene and all BTEX compounds, but not n-hexadecane. The genome of strain BN5 harbors genes encoding 29 monooxygenases including two alkane 1-monooxygenases and 54 dioxygenases, indicating that strain BN5 has versatile metabolic capabilities, for diverse organic compounds: the ability of strain BN5 to degrade short chain aliphatic hydrocarbons was verified experimentally. The biodegradation pathways of naphthalene and BTEX compounds were bioinformatically predicted and verified experimentally through the analysis of their metabolic intermediates. Some genomic features including the encoding of the biodegradation genes on a plasmid and the low sequence homologies of biodegradation-related genes suggest that biodegradation potentials of strain BN5 may have been acquired via horizontal gene transfers and/or gene duplication, resulting in enhanced ecological fitness by enabling strain BN5 to degrade all compounds including naphthalene, BTEX, and short aliphatic hydrocarbons in contaminated soil.

Genetic and Physiological Characteristics of a Novel Marine Propylene-Assimilating Halieaceae Bacterium Isolated from Seawater and the Diversity of Its Alkene and Epoxide Metabolism Genes.

The Gram-negative marine propylene-assimilating bacterium, strain PE-TB08W, was isolated from surface seawater. A structural gene analysis using the 16S rRNA gene showed 96, 94, and 95% similarities to Halioglobus species, Haliea sp. ETY-M, and Haliea sp. ETY-NAG, respectively. A phylogenetic tree analysis showed that strain PE-TB08W belonged to the EG19 (Chromatocurvus)-Congregibacter-Haliea cluster within the Halieaceae (formerly Alteromonadaceae) family. Thus, strain PE-TB08W was characterized as a newly isolated Halieaceae bacterium; we suggest that this strain belongs to a new genus. Other bacterial characteristics were investigated and revealed that strain PE-TB08W assimilated propylene, n-butane, 1-butene, propanol, and 1-butanol (C3 and C4 gaseous hydrocarbons and primary alcohols), but not various other alcohols, including methane, ethane, ethylene, propane, and i-butane. The putative alkene monooxygenase (amo) gene in this strain was a soluble methane monooxygenase-type (sMMO) gene that is ubiquitous in alkene-assimilating bacteria for the initial oxidation of alkenes. In addition, two epoxide carboxylase systems containing epoxyalkane, the co-enzyme M transferase (EaCoMT) gene, and the co-enzyme M biosynthesis gene, were found in the upstream region of the sMMO gene cluster. Both of these genes were similar to those in Xanthobacter autotrophicus Py2 and were inductively expressed by propylene. These results have a significant impact on the genetic relationship between terrestrial and marine alkene-assimilating bacteria.

Differential Protein Expression During Growth on Medium Versus Long-Chain Alkanes in the Obligate Marine Hydrocarbon-Degrading Bacterium Thalassolituus oleivorans MIL-1.

The marine obligate hydrocarbonoclastic bacterium Thalassolituus oleivorans MIL-1 metabolizes a broad range of aliphatic hydrocarbons almost exclusively as carbon and energy sources. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on medium- (n-C14) or long-chain (n-C28) alkanes. During growth on n-C14, T. oleivorans expresses an alkane monooxygenase system involved in terminal oxidation including two alkane 1-monooxygenases, a ferredoxin, a ferredoxin reductase and an aldehyde dehydrogenase. In contrast, during growth on long-chain alkanes (n-C28), T. oleivorans may switch to a subterminal alkane oxidation pathway evidenced by significant upregulation of Baeyer-Villiger monooxygenase and an esterase, proteins catalyzing ketone and ester metabolism, respectively. The metabolite (primary alcohol) generated from terminal oxidation of an alkane was detected during growth on n-C14 but not on n-C28 also suggesting alternative metabolic pathways. Expression of both active and passive transport systems involved in uptake of long-chain alkanes was higher when compared to the non-hydrocarbon control, including a TonB-dependent receptor, a FadL homolog and a specialized porin. Also, an inner membrane transport protein involved in the export of an outer membrane protein was expressed. This study has demonstrated the substrate range of T. oleivorans is larger than previously reported with growth from n-C10 up to n-C32. It has also greatly enhanced our understanding of the fundamental physiology of T. oleivorans, a key bacterium that plays a significant role in natural attenuation of marine oil pollution, by identifying key enzymes expressed during the catabolism of n-alkanes.

Characterization of a biosurfactant-producing Leclercia sp. B45 with new transcriptional patterns of alkB gene

To investigate the hydrocarbon-degrading ability, biosurfactant-producing capacity, and alkane monooxygenase system of Leclercia spp. A bacterial strain classified as Leclercia sp. B45 was isolated, and its biosurfactant-producing capacity, hydrocarbon-degrading ability, and alkane hydroxylase (alkB) gene transcriptional patterns were evaluated by TLC, FTIR, GC-MS, and RT-qPCR, respectively. Strain B45 showed active biosurfactant-producing ability, which was preferentially induced by C16. The extracted biosurfactant tolerated a wide range of salinity, pH, and temperature. The degradation rate of n-decane (C10), n-hexadecane (C16), and octacosane (C28) by strain B45 could reach 92.6%, 94.1%, and 67.8%, respectively. Furthermore, the alkB transcription levels in the strain B45 with C10, C16, or C28 as a carbon source were distinctly higher than those of the control group during the late exponential and stationary phases. The relative alkB transcript copy number decreased with the increase in alkane chain length, which is consistent with B45 strain biodegradation kinetics. Leclercia sp. B45 showed excellent n-alkane degradation performance and biosurfactant-producing capacity. Meanwhile, the alkB gene in Leclercia sp. B45 is likely to represent a novel gene, whose transcription level was significantly upregulated when induced by n-alkane. These results provide new insights into alkane metabolism mechanism in Leclercia sp. B45.

Evaluation of aromatic hydrocarbon decomposition catalyzed by the dioxygenase system and substitution of ferredoxin and ferredoxin reductase.

In this study, the catalytic activity and kinetic characteristics of the aromatic hydrocarbon dioxygenase system and the possibility of substituting its ferredoxin and ferredoxin reductase components were evaluated. The genes encoding toluene dioxygenase and toluene dihydrodiol dehydrogenase were cloned from Pseudomonas putida F1, and the corresponding enzymes were overexpressed and purified to homogeneity. Oxidative hydroxylation of toluene to cis-toluene dihydrodiol was catalyzed by toluene dioxygenase, and its subsequent dehydrogenation to 3-methylcatechol was catalyzed by toluene dihydrodiol dehydrogenase. The specific activity of the dioxygenase was 2.82U/mg-protein, which is highly remarkable compared with the values obtained in previous researches conducted with crude extracts or insoluble forms of enzymes. Kinetic parameters, as characterized by the Hill equation, were vmax = 497.2μM/min, KM = 542.4μM, and nH = 2.2, suggesting that toluene dioxygenase has at least three cooperative binding sites for toluene. In addition, the use of alternative ferredoxins and reductases was examined. Ferredoxin cloned from CYP153 could transfer electrons to the iron sulfur protein component of toluene dioxygenase. The ferredoxin could be reduced by ferredoxin, rubredoxin, and putidaredoxin reductases of CYP153, alkane-1 monooxygenase, and camphor 5-monooxygenase, respectively. The results provide useful information regarding the effective enzymatic biotreatment of hazardous aromatic hydrocarbon contaminants.

Medium Chain and Long Chain Alkanes Hydroxylase Producing Whole Cell Biocatalyst From Marine Bacteria

Alkanes are major component of crude oil that could be hydrolyzed by the enzyme of alkane hydroxylase. The are three types of alkane hydroxylase based on the chain length of alkane such as short-chain length/SCL (C2-C4), medium-chain length/MCL (C5-C17), and long-chain length/LCL (C>18). The aims of this study were to characterize and identify alkanes-degrading bacteria from these bacteria. The 30 strains from marine were grown on MCL (Pentane-C5H12, Decane-C10H22, and Pentadecane-C15H32) and LCL (n-Paraffin-C12H19C17 and branch of Pristane-C19H40). The study showed twenty-nine isolates have the ability to degrade alkanes compounds, whereas 14 isolates have grown ability on MCL and LCL medium, 11 isolates have the ability to grow on MCL and n-LCL, 3 isolates have the ability only to grow on MCL medium and 1 isolate has the ability only grow on n-LCL medium. The growth test result indicated that 29 isolates have medium-chain alkane monooxygenase and long-chain alkane hydroxylase. Based on 16S rDNA gene analysis, we obtained twenty nine of oil- degrading bacteria, namely a-proteobacteria (57 %), g-proteobacteria (30 %), Flavobacteria (7 %), Bacilli (3%) and Propionibacteriales (3 %). g-Proteobacteria and a-proteobacteria which seems to play an important role in the alkane biodegradation.

Impacts of n-alkane concentration on soil bacterial community structure and alkane monooxygenase genes abundance during bioremediation processes

Petroleum hydrocarbons, mainly consisting of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), are considered as priority pollutants and biohazards in the environment, eventually affecting the ecosystem and human health. Though many previous studies have investigated the change of bacterial community and alkane degraders during the degradation of petroleum hydrocarbons, there is still lack of understanding on the impacts of soil alkane contamination level. In the present study, microcosms with different n-alkane contamination (1%, 3% and 5%) were set up and our results indicated a complete alkane degradation after 30 and 50 days in 1%- and 3%-alkane treatments, respectively. In all the treatments, alkanes with medium-chain length (C11-C14) were preferentially degraded by soil microbes, followed by C27-alkane in 3% and 5% treatments. Alkane contamination level slightly altered soil bacterial community, and the main change was the presence and abundance of dominant alkane degraders. Thermogemmatisporaceae, Gemmataceae and Thermodesulfovibrionaceae were highly related to the degradation of C14- and C27-alkanes in 5% treatment, but linked to alkanes with medium-chain (C11-C18) in 1% treatment and C21-alkane in 3% treatment, respectively. Additionally, we compared the abundance of three alkane-monooxygenase genes, e.g., alk_A, alk_P and alk_R. The abundance of alk_R gene was highest in soils, and alk_P gene was more correlated with alkane degradation efficiency, especially in 5% treatment. Our results suggested that alkane contamination level showed non-negligible effects on soil bacterial communities to some extents, and particularly shaped alkane degraders and degrading genes significantly. This study provides a better understanding on the response of alkane degraders and bacterial communities to soil alkane concentrations, which affects their biodegradation process.

Medium Chain and Long Chain Alkanes Hydroxylase Producing Whole Cell Biocatalyst From Marine Bacteria

Alkanes are major component of crude oil that could be hydrolyzed by the enzyme of alkane hydroxylase. The are three types of alkane hydroxylase based on the chain length of alkane such as short-chain length/SCL (C2-C4), medium-chain length/MCL (C5-C17), and long-chain length/LCL (C>18). The aims of this study were to characterize and identify alkanes-degrading bacteria from these bacteria. The 30 strains from marine were grown on MCL (Pentane-C5H12, Decane-C10H22, and Pentadecane-C15H32) and LCL (n-Paraffin-C12H19C17 and branch of Pristane-C19H40). The study showed twenty-nine isolates have the ability to degrade alkanes compounds, whereas 14 isolates have grown ability on MCL and LCL medium, 11 isolates have the ability to grow on MCL and n-LCL, 3 isolates have the ability only to grow on MCL medium and 1 isolate has the ability only grow on n-LCL medium. The growth test result indicated that 29 isolates have medium-chain alkane monooxygenase and long-chain alkane hydroxylase. Based on 16S rDNA gene analysis, we obtained twenty nine of oil- degrading bacteria, namely a-proteobacteria (57 %), g-proteobacteria (30 %), Flavobacteria (7 %), Bacilli (3%) and Propionibacteriales (3 %). g-Proteobacteria and a-proteobacteria which seems to play an important role in the alkane biodegradation.

Integrated methodological approach reveals microbial diversity and functions in aerobic groundwater microcosms adapted to vinyl chloride.

Vinyl chloride (VC), a known human carcinogen, is often formed in groundwater (GW) by incomplete reductive dechlorination of chlorinated ethenes. An integrated microbial ecology approach involving bacterial enrichments and isolations, carbon stable-isotope probing (SIP) and metagenome and genome sequencing was applied to ethene-fed GW microcosms that rapidly transitioned to aerobic growth on VC. Actinobacteria, Proteobacteria and Bacteroidetes dominated the microbial communities in ethene- and VC-grown cultures. SIP with 13C2-VC demonstrated that Nocardioides spp. significantly participated in carbon uptake from VC (52.1%-75.7% enriched in heavy fractions). Sediminibacterium, Pedobacter and Pseudomonas spp. also incorporated 13C from VC into genomic DNA. Ethene- and VC-assimilating Nocardioides sp. strain XL1 was isolated. Sequencing revealed a large (∼300 kbp) plasmid harboring genes encoding alkene monooxygenase and epoxyalkane: coenzyme M transferase, enzymes known to participate in aerobic VC and ethene biodegradation. The plasmid was 100% identical to pNOCA01 found in VC-assimilating Nocardioides sp. strain JS614. Metagenomic analysis of enrichment cultures indicated other bacteria implicated in carbon uptake from VC possessed the genetic potential to detoxify epoxides via epoxide hydrolase or glutathione S-transferase (Pseudomonas) and/or metabolize VC epoxide breakdown products and downstream VC metabolites. This study provides new functional insights into aerobic VC metabolism within a GW microbial community.

Functional Genetic Diversity and Culturability of Petroleum-Degrading Bacteria Isolated From Oil-Contaminated Soils.

In this study, we compared the culturability of aerobic bacteria isolated from long-term oil-contaminated soils via enrichment and direct-plating methods; bacteria were cultured at 30°C or ambient temperatures. Two soil samples were collected from two sites in the Shengli oilfield located in Dongying, China. One sample (S0) was close to the outlet of an oil-production water treatment plant, and the other sample (S1) was located 500 m downstream of the outlet. In total, 595 bacterial isolates belonging to 56 genera were isolated, distributed in Actinobacteria, Firmicutes, Bacterioidetes, and Proteobacteria. It was interesting that Actinobacteria and Firmicutes were not detected from the 16S rRNA gene clone library. The results suggested the activation of rare species during culture. Using the enrichment method, 239 isolates (31 genera) and 96 (22 genera) isolates were obtained at ambient temperatures and 30°C, respectively, from S0 soil. Using the direct-plating method, 97 isolates (15 genera) and 163 isolates (20 genera) were obtained at ambient temperatures and 30°C, respectively, from two soils. Of the 595 isolates, 244 isolates (41.7% of total isolates) could degrade n-hexadecane. A greater number of alkane-degraders was isolated at ambient temperatures using the enrichment method, suggesting that this method could significantly improve bacterial culturability. Interestingly, the proportion of alkane degrading isolates was lower in the isolates obtained using enrichment method than that obtained using direct-plating methods. Considering the greater species diversity of isolates obtained via the enrichment method, this technique could be used to increase the diversity of the microbial consortia. Furthermore, phenol hydroxylase genes (pheN), medium-chain alkane monooxygenases genes (alkB and CYP153A), and long-chain alkane monooxygenase gene (almA) were detected in 60 isolates (11 genotypes), 91 isolates (27 genotypes) and 93 isolates (24 genotypes), and 34 isolates (14 genotypes), respectively. This study could provide new insights into microbial resources from oil fields or other environments, and this information will be beneficial for bioremediation of petroleum contamination and for other industrial applications.

Draft Genome Sequence of n-Alkane-Utilizing Acinetobacter sp. Strain BS1, Isolated from Ethane Oxidation Culture.

ABSTRACTHere, we report the draft whole-genome sequence of a bacterial strain, Acinetobacter sp. strain BS1, isolated from black soil during ethane oxidation culture. Medium- or long-chain alkane oxidation-related genes were identified; however, the short-chain alkane monooxygenase was not detected.

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli.

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M.aq ; the strain is also known as Marinobacter hydrocarbonoclasticus VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A M.aq is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR M.aq is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A M.aq operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from Pseudomonas putida GPo1. Using a recombinant Escherichia coli strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14 alkanols and C12 1,12-alkanediol by whole-cell biotransformation.IMPORTANCE To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Complete genome sequence of Tsukamurella sp. MH1: A wide-chain length alkane-degrading actinomycete

Tsukamurella sp. strain MH1, capable to use a wide range of n-alkanes as the only carbon source, was isolated from petroleum-contaminated soil (Pitești, Romania) and its complete genome was sequenced. The 4,922,396 bp genome contains only one circular chromosome with a G + C content of 71.12%, much higher than the type strains of this genus (68.4%). Based on the 16S rRNA genes sequence similarity, strain MH1 was taxonomically identified as Tsukamurella carboxydivorans. Genome analyses revealed that strain MH1 is harboring only one gene encoding for the alkB-like hydroxylase, arranged in a complete alkane monooxygenase operon. This is the first complete genome of the specie T. carboxydivorans, which will provide insights into the potential of Tsukamurella sp. MH1 and related strains for bioremediation of petroleum hydrocarbons-contaminated sites and into the environmental role of these bacteria.