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.     

Diversity and Abundance of Oil-Degrading Bacteria and Alkane Hydroxylase (alkB) Genes in the Subtropical Seawater of Xiamen Island

In this report, the diversity of oil-degrading bacteria and alkB gene was surveyed in the seawater around Xiamen Island. Forty-four isolates unique in 16S rRNA sequence were obtained after enrichment with crude oil. Most of the obtained isolates exhibited growth with diesel oil and crude oil. alkB genes were positively detected in 16 isolates by degenerate polymerase chain reaction (PCR). And for the first time, alkB genes were found in bacteria of Gallaecimonas, Castellaniella, Paracoccus, and Leucobacter. Additional 29 alkB sequences were retrieved from genomic DNA of the oil-degrading communities. Phylogenetic analysis showed that the obtained alkB genes formed five groups, most of which exhibited 60–80% similarity at the amino acid level with sequences retrieved from the GenBank database. Furthermore, the abundance of alkB genes in seawater was examined by real-time PCR. The results showed that alkB genes of each group in situ ranged from about 3 × 10³ to 3 × 10⁵ copies L⁻¹, with the homologs of Alcanivorax and Pseudomonas being the most predominant. Bacteria of Alcanivorax, Acinetobacter, and Pseudomonas are important oil degraders in this area; while those frequently reported in other area, like Oleiphilus spp., Oleispira spp., and Thalassolituus spp. were not found in our report. These results indicate that bacteria and genes involved in oil degradation are quite diverse, and may have restriction in geographic distribution in some species.     

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C₁ to C₄), while medium-chain (C₅ to C₁₆) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C₁ to C₄ alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with engineering MMO-like enzymes as well as investigating the fundamental question of whether enzymes unrelated to MMO can support growth on small alkanes.The most intensively studied medium-chain alkane hydroxylases are the AlkB enzymes (2, 20, 29), especially AlkB from Pseudomonas putida GPo1 (13, 28, 32, 35). While most members of the AlkB family act on C₁₀ or longer alkanes, some accept alkanes as small as C₅ (30). A recent study (12) indicated that AlkB from P. putida GPo1 may also be involved in propane and butane assimilation. AlkB selectively oxidizes at the terminal carbon to produce the 1-alkanols. No systematic protein engineering studies have been conducted on this di-iron integral membrane enzyme, although selection and site-directed mutagenesis efforts identified one amino acid residue that sterically determines long-chain alkane degradation (35).The most recent addition to the known biological alkane-hydroxylating repertoire is the CYP153 family of heme-containing cytochrome P450 monooxygenases. Although their activity was detected as early as 1981 (1), the first CYP153 was characterized only in 2001 (16). Additional CYP153 enzymes were identified and studied more recently (9, 10, 31). These soluble class II-type three-component P450 enzymes and the AlkB enzymes are the main actors in medium-chain-length alkane hydroxylation by the cultivated bacteria analyzed to date (31). CYP153 monooxygenases have been the subject of biochemical studies (9, 16, 19), and their substrate range has been explored (10, 14). Known substrates include C₅ to C₁₁ alkanes. The best-characterized member, CYP153A6, hydroxylates its preferred substrate octane predominantly (>95%) at the terminal position (9).Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101; camphor hydroxylase) and P450 BM3 (CYP102A; a fatty acid hydroxylase) (8, 36). The resulting enzymes, however, hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions; highly selective terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase (22). We wished to determine whether a small-alkane terminal hydroxylase could be obtained instead by directed evolution of a longer-chain alkane hydroxylase that exhibits this desirable regioselectivity. For this study, we chose to engineer AlkB from P. putida GPo1 and CYP153A6 from Mycobacterium sp. strain HXN-1500 (9, 33) to enhance activity on butane. Because terminal alkane hydroxylation is the first step of alkane catabolism in P. putida GPo1, we reasoned that it should be possible to establish an in vivo evolution system that uses growth on small alkanes to select for enzyme variants exhibiting the desired activities.The recombinant host Pseudomonas putida GPo12(pGEc47ΔB) was engineered specifically for complementation studies with terminal alkane hydroxylases and was used previously to characterize members of the AlkB and CYP153 families (26, 31). This strain is a derivative of the natural isolate P. putida GPo1 lacking its endogenous OCT plasmid (octane assimilation) (5) but containing cosmid pGEc47ΔB, which carries all genes comprising the alk machinery necessary for alkane utilization, with the exception of a deleted alkB gene (34). We show that this host can be complemented by a plasmid-encoded library of alkane hydroxylases and that growth of the mixed culture on butane leads to enrichment of novel butane-oxidizing terminal hydroxylases.     

Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.     

施用胶质芽胞杆菌菌剂对黑麦草根际土壤脲酶、磷酸酶及过氧化氢酶活性的影响

土壤酶活性是反映土壤肥力最为重要的生物学指标之一。采用稀释涂布平板法研究了从玉米（K02）、草地早熟禾（K05）、披碱草（K09）、多年生黑麦草（K11）、匍匐翦股颖（K12）根际土壤中分离到的5株胶质芽胞杆菌菌株对黑麦草根际土壤脲酶、磷酸酶及过氧化氢酶活性的影响。结果表明：在苗期、中期、收获期各处理黑麦草根际土壤脲酶、磷酸酶和过氧化氢酶活性均高于对照（P〈0.05）。总体来看,各处理黑麦草根际土壤脲酶和过氧化氢酶活性呈先增加后降低的变化趋势;土壤磷酸酶活性与对照（CK）相比除苗期处理K12外,其他处理均呈上升趋势,以处理K05磷酸酶活性最大。研究表明,施入胶质芽胞杆菌菌剂对黑麦草根际土壤脲酶、磷酸酶及过氧化氢酶活性有一定的积极作用,其结果可为生物钾肥的研制提供必要的数据。     

Novel Alkane Hydroxylase Gene (alkB) Diversity in Sediments Associated with Hydrocarbon Seeps in the Timor Sea,Australia

Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem''s response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation.Alkanes are saturated hydrocarbons that are widespread in marine environments due to a variety of anthropogenic and natural sources. They constitute the major fraction of hydrocarbon components found in crude oils and refined petroleum and are also produced by various marine organisms (e.g., zooplankton) as cellular components (2, 44). Alkanes are considered as pollutants, with short-chained alkanes acting as solvents toward cellular membranes and other lipid components (34) while longer-chained alkanes may contribute to the formation of oil films and slicks that may limit nutrient and oxygen exchange (21). Importantly, alkanes also serve as important carbon and energy sources for some microorganisms. In marine environments, alkanes succumb to various removal and dispersal processes such as dissolution, photochemical oxidation, evaporation, adsorption, and sedimentation. However, the greatest removal pathway for alkanes in marine sediments is via biodegradation by bacteria (13). This mechanism also mediates the transfer of oil-derived carbon to higher trophic levels (28, 37), and therefore these bacteria have an important role in carbon cycling in environments subject to long-term inputs of hydrocarbons such as marine seep-associated ecosystems. Alkane biodegradation is mediated by a diverse range of marine bacteria using various electron acceptors although degradation generally proceeds at greater rates under aerobic conditions than under anaerobic conditions, where the process is relatively slow (8, 26).In the presence of oxygen, well-characterized alkane oxidation pathways are initiated by an activation step whereby oxygen is introduced to the alkane substrate before further catabolic steps can proceed. A number of oxygen-dependent alkane hydroxylase enzyme systems have been discovered that catalyze this initial step including the soluble di-iron methane monooxygenases and the membrane-bound copper-containing methane monooxygenases, both of which act upon short-chain alkanes (i.e., C₁ up to C₈). Integral membrane non-heme iron alkane hydroxylases (the alk system) that are related to the well-characterized AlkB of Pseudomonas putida GPo1 (also known as Pseudomonas oleovorans TF4-1 I) act upon longer-chain alkanes (i.e., C₅ to C₁₆) (40). Other systems exist that include alkane-hydroxylating cytochrome P450 enzymes in addition to other enzyme systems that are known to exist based purely on chemical analyses of metabolites formed during alkane degradation experiments (22, 25, 29); however, knowledge pertaining to the enzymes and genes involved as well as their importance in the environment is limited. Only recently have genes involved in the degradation of long-chain alkanes (e.g., C₃₂ and C₃₆) been identified in Acinetobacter sp. strain DSM 17874 (39) though there is no information about the presence or importance of such enzymes in the environment.Although various chemical and microbiological aspects of petroleum oil and alkane biodegradation in marine systems have been relatively well studied, there is a general lack of knowledge concerning the diversity or abundance of the functional genes involved. The biochemical and molecular aspects of alkB genes and the enzymes they encode have been relatively well studied, and this has enabled the development of molecular tools for the study of alkB genes in the environment (19). Elevated levels of hydrocarbons or the introduction of hydrocarbons to environments has been shown to increase gene copy numbers, indicating the potential use of alkB genes as bioindicators of oil pollution and/or biodegradation (16, 33, 36, 43). However, to date only one study has used culture-independent molecular methods to examine the diversity of alkB genes in a marine environment (20), and no studies have examined hydrocarbon-degrading genes where natural hydrocarbon seepage occurs.In this study, the diversity and relative abundance of alkB genes were examined in sediments of the Timor Sea, a region where natural seeps are sources of widespread petroleum hydrocarbons. It was hypothesized that (i) novel alkB genes may exist in this unique tropical marine environment, (ii) that variations in gene diversity would be found in sediments with different hydrocarbon levels, and (iii) that the abundance of certain alkB gene types may reflect the levels of measured hydrocarbons in sediments, and therefore this assay could be used as a complementary tool for monitoring petroleum inputs into sediments of the Timor Sea.     

大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.     

Studies of the Efficacy of Alfalfa and Reed in the Phytoremediation of Hydrocarbon-Polluted Soil

The efficacy of plants as means of decontaminating hydrocarbon-polluted soil has been studied. Ditch reed (Phragmites australis) and alfalfa (Medicago sativa) markedly intensified processes of pollutant destruction, the effect being particularly pronounced in the case of polycyclic aromatic hydrocarbons. Comparative analysis of microflora in soils (including those devoid of plants and rhizosphere) demonstrated that, in addition to preventing a pollutant-induced decrease in the amount of heterotrophic microorganisms, the plants stimulated their development, significantly increasing the population of degraders. Effects of plants on major physiological groups of soil microorganisms under conditions of pollution were ambiguous. The rhizosphere microbial consortium of alfalfa was less susceptible to effects of pollutants than that of reed.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

骨髓基质细胞的分离、鉴定以及TH基因的转染与表达

目的是探索骨髓基质细胞的分离培养、鉴定及其接受并表达TH基因的能力。实验中通过密度梯度离心法成功地从成年SD大鼠骨髓中分离获得了骨髓基质干细胞 ,并用流式细胞仪对其进行鉴定 ,纯度可达 75 %。进一步采用复制缺陷型腺相关病毒载体介导的基因转染方法 ,将之改造成为携带lacZ与TH基因的工程细胞 ,经X gal染色和TH免疫组化检测 ,转染效率为 (74 .6± 19.4 ) %。实验结果表明骨髓基质细胞易于接受并表达外源基因 ,有望作为运载细胞应用于帕金森病的基因治疗。     

CYP21和CYP21P基因启动子序列对绿色荧光蛋白基因表达的影响

特异性扩增CYP21基因和CYP21P基因启动子区域－770bp--1bp片段,去除pEGFP-N1载体中的CMV启动子,构建含CYP21基因启动子的pEGFP-N1载体（pCYP21)和CYP21P基因启动子的pEGFP-N1载体（pCYP21P),分别将上述两种构建载体、野生型pEGFP-N1(阳性对照）质粒及阴性对照转染入肾上腺皮质来源的Y1细胞系中,用倒置荧光显微镜,以及激光共聚焦显微镜等方法观测绿色荧光蛋白的表达。转染后,在荧光倒置显微镜下首次发现Y1细胞中出现绿色荧光蛋白的时间阳性对照为3小时,pCYP21为7小时,pCYP21P与阳性对照（未转染任何载体的Y1细胞）始终未观测到绿色荧光蛋白。阳性对照和pCYP21的绿色荧光蛋白表达强于pCYP21,pCYP21P与阴性对照始终未观测到绿色荧光蛋白。阳性对照和pCYP21的绿色荧光蛋白在胞核中的荧光强度高于胞浆。上述结果进一步表明,含有CYP21和CYP21P两种基因启动子的GFP质粒在Y1细胞中表达存在显著差异。     

Oxidation of Methyl tert-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n-Octane-Grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K_s value (20 to 40 mM) for MTBE.     

Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.     

人雄激素芳香化酶基因（CYP19）结构及其表达调控研究进展

人雄激素芳香化酶与乳腺癌的发生和发展密切相关.综述了编码该酶的CYP19基因的结构、独特的组织特异性表达和调控CYP19基因表达的各种因素及其有关的调控机制.