Alkane hydroxylase homologues in Gram-positive strains

We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.     

Alkane activation by P450 oxygenases

The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.     

Alkane chemotaxonomy of Arbutus

Alkane analyses of Arbutus (Ericaceae) leaves of the three species (A. texana, A. menziesii and A. arizonica) native to the United States show that two morphologically overlapping but allopatric taxa (A. texana and A. menziesii) have substantially different alkane profiles at the unusually low C., level. These data supply additional evidence supporting continued recognition of them as separate species.     

Alkane activation by P450 oxygenases

The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.     

Alkane and wax ester production from lignin-related aromatic compounds

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin-related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off-line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/g_coumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /g_coumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value-added products.     

Alkane hydroxylases involved in microbial alkane degradation

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.     

Microbial Oxidation of the Isoprenoid Alkane Pristane

Microorganisms capable of utilizing pristane (2,6,10,14-tetramethylpentadecane) as a sole source of carbon and energy were isolated from soil specimens. A strain BPM 1613, tentatively classified in the genus Nocardia, accumulated several oxidation products of pristane in the culture fluid. Silica gel chromatography of the ethyl ether extract from the culture fluid yielded pristanol and pristanic acid as major products and pristyl pristanate and pristyl aldehyde as minor products. The confirmations on the estimated structures of these oxidation products are described in detail.     

Alkane distribution in epicuticular wax of epacridaceae

Alkane distribution pattern was determined in the epicuticular wax of leaves of 39 species and in the flower wax of three species of Epacridaceae. Uniform patterns were observed within some genera (Monotoca, Styphelia) whereas in others no common pattern was apparent. There was no difference between the alkane patterns of the two subfamilies Epacrideae and Styphelieae. The pattern was not affected by the presence of new growth, and there was no correlation with the season, habitat, climate, nature of soil, or the presence or absence of flowers.     

烃的微生物摄取机制研究进展

综述了近期在烃的微生物摄取机制方面的研究进展。对提高环境生物整治效果而言,微生物对非水溶性底物,尤其是烃的摄取机制是重要的课题。随着理论认识的深入和研究手段的丰富,在该领域已有了更多发现和结论。介绍了表面活性剂对烃摄取的影响,细胞表面性质的调整和烃的跨膜输送等方面的近期研究结果;同时,提供了细胞亚微结构分析,细胞趋化性等相关证据,指出了在该领域尚待解决的问题。     

Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.     

Alkane Oxidation by a Particulate Preparation from Candida

The oxidation of decane by a cell-free particulate preparation from Candida intermedia was studied. Decane is oxidized to decanoate via decanol and decanaldehyde. Oxidation of decane to decanol requires molecular oxygen. Decanol is oxidized to decanaldehyde by a nicotinamide adenine dinucleotide-linked dehydrogenase differing greatly in specificity from ordinary yeast alcohol dehydrogenase. Decanaldehyde is oxidized to decanoate by a nicotinamide adenine dinucleotide-linked dehydrogenase that oxidizes long-chain aldehydes but not short-chain aldehydes. The enzymes that oxidize decane, decanol, and decanaldehyde are all induced when decane is present in the medium. These enzymes are apparently located in the cell membrane.     

Genetic Analysis of Mating Type and Alkane Utilization in Saccharomycopsis lipolytica

The mating reaction in Saccharomycopsis lipolytica is shown to be bipolar through the analysis of whole meiotic tetrads. Genetic evidence is provided that alkane metabolism proceeds through a fatty acid or a fatty acid intermediate since several mutants unable to grow on n-tetradecane were also unable to utilize palmitic acid.     

铜绿假单胞菌对长链烷烃的摄取模式

研究了一株铜绿假单胞菌(CGMCC 1.1785)摄取长链烷烃的模式。铜绿假单胞菌1．1785能够以固态的长链烷烃为唯一碳源生长,在培养过程中产生表面活性代谢物。烃与水相的界面面积是细菌生长重要的影响因素,说明传质限制的存在。由于该菌不能够利用鼠李糖脂增溶的烃作为碳源,因此添加鼠李糖脂能够强化烃摄取的主要原因是烃界面的扩大。细胞表面疏水性从开始的急剧升高到后来的不断下降,说明在不同生长阶段细胞对烃的摄取模式是不同的。由此认为,铜绿假单胞菌1.1785既没有通过表面活性剂介导模式获取烃,也并非完全通过直接接触模式获取烃。据此提出该菌采用了一种运动接触的烃摄取模式,其趋化运动能力在这种摄取过程中起到重要作用。     

液态烷烃氧载体对粘红酵母发酵产番茄红素的影响

研究正己烷、正十二烷、正十六烷3种液态烷烃作为氧载体对粘红酵母生长和番茄红素合成的影响,发现氧载体不仅使菌体生物量提高,同时使单位细胞的番茄红素产率增大,从而提高粘红酵母合成番茄红素的能力。3种液态烷烃中正十二烷作为氧载体效果较好,试验结果表明,在发酵第0 h时添加4%的正十二烷,细胞生物量达到16.49 g/L,番茄红素合成量达到42.32 mg/L分别比对照组提高了26.2%和50.17%。     

Alkane inducible proteins in Geobacillus thermoleovorans B23

Background  

Initial step of β-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution.     

Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis

Background

Psychrophiles are presumed to play a large role in the catabolism of alkanes and other components of crude oil in natural low temperature environments. In this study we analyzed the functional diversity of genes for alkane hydroxylases, the enzymes responsible for converting alkanes to more labile alcohols, as found in the genomes of nineteen psychrophiles for which alkane degradation has not been reported. To identify possible mechanisms of low temperature optimization we compared putative alkane hydroxylases from these psychrophiles with homologues from nineteen taxonomically related mesophilic strains.

Results

Seven of the analyzed psychrophile genomes contained a total of 27 candidate alkane hydroxylase genes, only two of which are currently annotated as alkane hydroxylase. These candidates were mostly related to the AlkB and cytochrome p450 alkane hydroxylases, but several homologues of the LadA and AlmA enzymes, significant for their ability to degrade long-chain alkanes, were also detected. These putative alkane hydroxylases showed significant differences in primary structure from their mesophile homologues, with preferences for specific amino acids and increased flexibility on loops, bends, and α-helices.

Conclusion

A focused analysis on psychrophile genomes led to discovery of numerous candidate alkane hydroxylase genes not currently annotated as alkane hydroxylase. Gene products show signs of optimization to low temperature, including regions of increased flexibility and amino acid preferences typical of psychrophilic proteins. These findings are consistent with observations of microbial degradation of crude oil in cold environments and identify proteins that can be targeted in rate studies and in the design of molecular tools for low temperature bioremediation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1120) contains supplementary material, which is available to authorized users.     

烷烃降解基因alk研究进展

很多微生物都可以利用直链烷烃作为唯一碳源和能源 ,目前对该代谢机理的遗传学研究已相当深入。其典型菌株Pseudomonasputida可利用C6～C1 2的烷烃 ,编码这些参与烷烃降解的酶的基因位于两个基因簇alkBFGHJKL和alkST上 ,且此代谢途径受一个正反馈调节机制及两个不同的全局控制信号调控。其它可降解烷烃菌株的烷烃氧化基因与P .Putida中相应基因有较高的同源性。国内外的研究表明 ,alk基因可望应用于生物清污、微生物提高石油采收率及精细化工等诸多领域 。     

Alkane distribution in epicuticular wax of some heath plants in Norway

Alkane distribution patterns were determined in the epicuticular wax of eight species of Ericaceae and of Empetrum hermaphroditum. Some uniformity in patterns was noted in the subfamily Arbutoideae, whereas diversity prevailed in Rhododendroideae and Vaccinioideae. Environmental conditions exert a strong effect on the alkane pattern in Loiseleuria procumbens.