CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.     

Identification of an l-dopa decarboxylase gene from Sorangium cellulosum So ce90

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.     

Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases.     

Investigation of cytochromes P450 in myxobacteria: excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

The exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed.     

Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce12 and So ce56: development of a mariner-based transposon mutagenesis system

Myxobacteria increasingly gain attention as a source of bioactive natural products. The genus Sorangium produces almost half of the secondary metabolites isolated from these microorganisms. Nevertheless, genetic systems for Sorangium strains are poorly developed, which makes the identification of the genes directing natural product biosynthesis difficult. Using biparental and triparental mating, we have developed methodologies for DNA transfer from Escherichia coli via conjugation for the genome sequencing model strain So ce56 and the secondary metabolite multiproducing strain So ce12. The conjugation protocol developed for strain So ce56 is not applicable to other Sorangium strains. Crucial points for the conjugation are the ratio of E. coli and Sorangium cellulosum cells, the choice of liquid or solid medium, the time used for the conjugation process and antibiotic selection in liquid medium prior to the plating of cells. A mariner-based transposon containing a hygromycin resistance gene was generated and used as the selectable marker for S. cellulosum. The transposon randomly integrates into the chromosome of both strains. As a proof of principle, S. cellulosum So ce12 transposon mutants were screened using an overlay assay to target the chivosazole biosynthetic gene cluster.     

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.     

Genome Mining in Sorangium cellulosum So ce56: IDENTIFICATION AND CHARACTERIZATION OF THE HOMOLOGOUS ELECTRON TRANSFER PROTEINS OF A MYXOBACTERIAL CYTOCHROME P450*

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.The cytochrome P450 (CYP)² enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1–4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7). Interestingly, members of both the [2Fe-2S] and the non-[2Fe-2S] ferredoxins have been reported to sustain cytochrome P450 catalyzed reactions. The latter group is further subdivided into mono- and dicluster ferredoxins (i.e. the [3Fe-4S] or [4Fe-4S] and the [3Fe-4S] + [4Fe-4S] or [4Fe-4S] + [4Fe-4S] ferredoxins). Remarkably, cytochrome P450 systems depending on non-[2Fe-2S] ferredoxins have been found exclusively in bacteria to date (8, 9).To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10).Sorangium cellulosum So ce56 is a genome-sequenced myxobacterial model strain. Because of their biotechnological potential as producers of secondary metabolites, the myxobacteria attract attention from both the academic community and the pharmaceutical industry. To date, more than 100 new basic structures and some 500 derivatives have been reported (11), with almost half of the newly discovered natural products being isolated from the genus Sorangium (11, 12). The potent anti-cancer agent epothilone, for example, was discovered from S. cellulosum So ce90 (13, 14). Epothilone is one of so far seven known myxobacterial compounds, the biosynthesis of which involves cytochromes P450 (15). Besides the epothilones, these are the antifungal leupyrrins (16) and the cytotoxic spirangienes (17) (also from S. cellulosum), the antibiotic myxovirescin from Myxococcus (18), the electron transport inhibitor stigmatellin (19) and the antibiotic aurafuron (20) from Stigmatella aurantiaca, and the antifungal ajudazols from Chondromyces crocatus (21).The recently genome-sequenced myxobacterium S. cellulosum So ce56 (12) shows great potential for biotechnological applications, as judged on the basis of its capacity for the production of secondary metabolites. Three biologically active compounds have been described so far, namely the fungicidal chivosazoles, the macrolide antibiotic etnangien, and the iron chelator myxochelin (12). Moreover, the bioinformatic analysis of the So ce56 genome has revealed numerous biosynthetic gene clusters of yet unknown function (11, 12). With a size of more than 13 Mbp, the genome of S. cellulosum So ce56 is to date the largest sequenced prokaryotic genome (12). It has been shown to harbor 21 cytochrome P450 genes. In light of the significance of S. cellulosum as a viable source of bioactive secondary metabolites (14) and the role of cytochromes P450 in the synthesis of natural products (2), it is of great interest to elucidate the function of these enzymes.Therefore, the investigation of the S. cellulosum So ce56 cytochrome P450 systems opens a fascinating field not only with regard to basic research but also to exploit the biotechnological potential of this model strain. To achieve this goal it is important to provide a functional electron transport chain. Thus, the main objective of this work was to identify a myxobacterial ferredoxin/ferredoxin reductase couple able to support reactions catalyzed by S. cellulosum So ce56 cytochromes P450.     

Production of an exopolysaccharide bioflocculant by Sorangium cellulosum

AIMS: To isolate a new exopolysaccharide bioflocculant produced by the myxobacterium Sorangium cellulosum NUST06, and to characterize its chemical composition and expolysaccharide production relative to carbon source. METHODS AND RESULTS: Exopolysaccharide levels and biomass production by S. cellulosum NUST06 were analysed relative to carbon source. Glucose in the medium at a level of 3 g l(-1) completely inhibited cell growth and exopolysaccharide production, but low concentrations of glucose (1-2 g l(-1)) could stimulate cell utilization of starch. The chemical composition and flocculating activity of the NUST06 exopolysaccharide was investigated. The flocculant comprised 38.3% proteins and 58.5% carbohydrates, of which glucose, mannose and glucuronic acid were present at 51.3%, 39.2% and 10.5%, respectively. The flocculating activity of the NUST06 flocculant depended strongly on cations. CONCLUSIONS: It is feasible to produce an exopolysaccharide bioflocculant by the strain NUST06 in a mineral salts medium using starch as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain may be advantageous for commercial bioflocculant production and may enrich existing knowledge of myxobacteria.     

响应面法优化纤维堆囊菌So ce90原生质体的制备条件

采用响应面法优化纤维堆囊菌So ce90的原生质体制备条件。选择溶菌酶浓度,酶解时间和酶解温度作为考察因子,在单因素试验的基础上,采用Box—Benhnken中心组合设计方法进行三因素三水平试验设计。以原生质体生成率作为响应值,进行多元二次响应回归分析,得出纤维堆囊菌So ce90原生质体制备的最佳条件为：溶菌酶浓度1．40mg／mL,酶解时间为160min,酶解温度为30℃,响应模型预测的原生质体最高生成率为87．78％,在最优的条件下进行验证试验,得到原生质体生成率为86．23％,与预测值间的相对误差为1．76％,表明采用响应面法优化纤维堆囊茵So ce90原生质体的制备条件是可行的。     

Repeated batch production of epothilone B by immobilized Sorangium cellulosum

Production of extracellular epothilone B, one of the potent anticancer agents, by free and immobilized Sorangium cellulosum was studied using the repeated batch culture process. The concentration of alginate used in immobilization was directly related to the mass transfer rate of nutrients, mechanical stability, and the epothilone B production yield. With the optimized 3% (w/v) calcium alginate carrier, a prolonged repeated batch culture was investigated for the 5 repeated batches for 24 days. The maximum productivity of epothilone B obtained from the alginate-immobilized cells was 5.03 mg/l/day, which is 3 times higher than that of free cells (1.68 mg/l/day).     

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 μg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.     

产Epothilone B纤维堆囊菌的选育与发酵优化

目的:获得高产Epothilone B菌株和最佳营养条件及发酵条件。方法:采用紫外线照射的方法对实验室保存的1株纤纤堆囊菌AHB125进行诱变处理,并采用单因素和正交实验优化培养基中的碳源、氮源和发酵条件。结果:经诱变后获得1株遗传性能稳定变异菌株(SC4-56),Epothilone B产量为14.6mg/L,比初始菌株高195%;该菌株最佳碳源和氮源为6%玉米淀粉和3%蛋白胨,产量分别为20.5和21.8 mg/L;最佳发酵条件为:转速160r/min,温度32℃,接种量8%(V/V),装液量90 mL/500mL,优化后经验证实验,Epothilone B达28.24 mg/L。结论:获得了1株高产量变异菌株,并确定了最佳生产条件。     

Characterisation,genome size and genetic manipulation of the myxobacterium <Emphasis Type=Italic>Sorangium cellulosum</Emphasis> So ce56

In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.     

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

Background  

(+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli.     

Selective production of epothilone B by heterologous expression of propionyl-CoA synthetase in Sorangium cellulosum

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. cellulosum containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.     

C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.