Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites

Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2–6, while M. phaseolina transformed compound 1 into polar metabolites 7–11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12–14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1–14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC₅₀ values between 49.0 and 84.9 μM.     

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase¹ and isopenicillin N (IPN) synthase¹ respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P₇ and P₈) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules. Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999     

��-Glucan content and hydration properties of filamentous fungi

The aim of this work was to isolate and identify filamentous fungi from several sources to study the dietary fiber and ??-glucan content. The fungal hydration properties such as water absorption and water holding capacities were also evaluated. Total dietary fiber of isolates exhibited a noticeable variability from 16 to 53% and the highest values were obtained for the genera Paecilomyces and Penicillium, a fact consistent with a higher content of ??-glucans (24 and 17%, respectively), higher than previously reported for Basidiomycetes and yeast. We observed a large decrease (75%) in the water holding capacity when the mycelia were dried. Isolates of filamentous fungi with greater water holding capacity also exhibited greater absorption capacity. Paecilomyces variotii and Penicillium nalgiovense had the best hydration properties. Our results contribute to the search for new unconventional ingredients providing a high protein and ??-glucans content. The addition of these dried mycelia could change the hydration properties in the food system.     

Differential RNA accumulation of two β-tubulin genes in arbuscular mycorrhizal fungi

RNA was isolated from spores of different arbuscular mycorrhizal (AM) fungi and used for RT-PCR with degenerate primers for beta-tubulin genes. PCR products were cloned and the sequence of several clones was analysed for each fragment. Comparison of sequences identified two loci for beta-tubulin genes with different GC content and codon usage. Btub1 sequences were most similar to beta-tubulin genes from the Oomycota, while Btub2 sequences showed highest similarity to sequences from the Zygomycota. RT-PCR experiments were carried out to monitor RNA accumulation patterns of Btub1 and Btub2 in asymbiotic germinating spores and in symbiotic extraradical hyphae of three different AM fungi. This indicated that Btub1 is constitutively expressed in Gigaspora rosea, but down-regulated during symbiosis in Glomus mosseae and Glomus intraradices. In contrast, Btub2 showed constitutive expression in the two Glomus species, but down-regulation in G. rosea. Further analysis of different fungi indicated that Btub2 primers could be used to specifically monitor RNA accumulation of AM fungi in environmental samples.     

Biosynthesis of β-glucans in fungi

Glucans are the most abundant polysaccharides present in fungi. The present review provides updated information on the structure and synthesis of -glucans in fungal cells. Synthesis of these polymers made up of B1,3 chains with a variable degree of B1,6 branching involves several reactions: initiation, chain elongation and branching, of which the most studied one is the elongation step. This reaction, catalyzed by the so-called glucan synthetases, utilizes UDPG as sugar donor. Properties of glucan synthetases are extremely variable depending on the fungal species, and their developmental stage. Because of the importance of these polysaccharides it is anticipated that comprehension of their mechanism of synthesis, is important for the understanding of cell wall assembly and cell growth and morphogenesis, as well as for the design of specific antifungal drugs.Abreviations UDPG uridine-diphospho-glucose - GDPG guanosine-diphospho-glucose - ADPG adenosine-diphospho-glucose - MW molecular weight - mic minimal inhibitory concentration - d.p. degree of polymerization - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.     

Isolation and Characterization of Mutations in the β-Tubulin Gene of SACCHAROMYCES CEREVISIAE

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.     

Quantification and characterization of β-lactam resistance genes in 15 sewage treatment plants from East Asia and North America

The emerging antibiotic resistance genes in the aquatic environment have aroused public concern. As β-lactam is the most widely used group of antibiotics, β-lactam resistance genes were selected to investigate their distribution and diversity in the activated sludge from 15 geographically different sewage treatment plants (STPs) of China, Singapore, USA, and Canada. Specific PCR and quantitative real-time PCR (q-PCR) were used to investigate the occurrence and abundance of nine β-lactam resistance genes. Five genes (OXA-1, OXA-2, OXA-10, ampC, and TEM-1) were detected in most of the sludge collected, while three genes (mecA, CTX-M-1, and SME) were not found in any sludge sample. The total abundances of the six detected β-lactam resistance genes in the 15 STPs varied from 5.34?×?10(1) copies/ng DNA (ampC) to 5.49?×?10(4) copies/ng DNA (OXA-1). Overall, OXA-1 had the highest total concentration, followed by IMP and OXA-10. Noticeably, the abundances of TEM-1 in Chinese STPs were generally higher than those in the STPs of other countries, while the abundances of OXA-2 and IMP in the STPs of North America were much greater than those of East Asia. A total of 78 clones carrying β-lactam resistance genes were randomly selected from six clone libraries for phylogenetic diversity analysis; the similarity of these cloned genes to known β-lactam resistance genes with sequence identities ranged from 96% to 100%. Furthermore, OXA-1, ampC, and IMP were found to be more diverse than the other β-lactam resistance genes.     

Biosynthesis of β-lactam nuclei in yeast

We have engineered brewer's yeast as a general platform for de novo synthesis of diverse β-lactam nuclei starting from simple sugars, thereby enabling ready access to a number of structurally different antibiotics of significant pharmaceutical importance. The biosynthesis of β-lactam nuclei has received much attention in recent years, while rational engineering of non-native antibiotics-producing microbes to produce β-lactam nuclei remains challenging. Benefited by the integration of heterologous biosynthetic pathways and rationally designed enzymes that catalyze hydrolysis and ring expansion reactions, we succeeded in constructing synthetic yeast cell factories which produce antibiotic cephalosporin C (CPC, 170.1 ± 4.9 μg/g DCW) and the downstream β-lactam nuclei, including 6-amino penicillanic acid (6-APA, 5.3 ± 0.2 mg/g DCW), 7-amino cephalosporanic acid (7-ACA, 6.2 ± 1.1 μg/g DCW) as well as 7-amino desacetoxy cephalosporanic acid (7-ADCA, 1.7 ± 0.1 mg/g DCW). This work established a Saccharomyces cerevisiae platform capable of synthesizing multiple β-lactam nuclei by combining natural and artificial enzymes, which serves as a metabolic tool to produce valuable β-lactam intermediates and new antibiotics.     

cDNA cloning of β-tubulin gene and organization of tubulin genes in Vigna radiata (mung bean) genome

We isolated an almost full-length cDNA clone containing -tubulin gene from a partial cDNA library of mung bean using chicken cDNA as probe. Cross-hybridization with chicken -tubulin cDNA and positive hybridization-selection and translation of mung bean mRNA established that this clone contains -tubulin sequences. We studied the organization of tubulin genes in mung bean. In this plant tubulin genes are organized in tandem repeats of alternating - and -tubulin genes. The 5.6 kb basis repeat unit which contains both - and -tubulin genes is repeated twenty times per haploid genome.Abbreviations SDS sodium dodecyl sulphate - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA, pH 7.4     

Computational and functional analysis of β-lactam resistance in Zymomonas mobilis

Zymomonas mobilis, a Gram-negative ethanologenic non-pathogenic bacterium, is reported to exhibit resistance to high concentrations of β-lactam antibiotics. In the present study, Z. mobilis was found to be resistant to I-IV generations of cephalosporins and carbapenems, i.e. narrow, broad and extended spectrum β-lactam antibiotics. We have analysed the genome of Z. mobilis (GenBank accession No.: NC 006526) harbouring multiple genes coding for β-lactamases (BLA), β-lactamase domain containing proteins (BDP) and penicillin binding proteins (PBP). The conserved domain database analysis of BDPs predicted them to be members of metallo β-lactamase superfamily. Further, class C specific multidomain AmpC (β-lactamase C) was found in the three β-lactamases. The β-lactam resistance determinants motifs, HXHXD, KXG, SXXK, SXN, and YXN are present in the BLAs, BDPs and PBPs of Z. mobilis. The predicted theoretical pI and aliphatic index values suggested their stability. One of the PBPs, PBP2, was predicted to share functional association with rod shape determining proteins (GenBank accession Nos. YP_162095 and YP_162091). Homology modelling of three dimensional structures of the β-lactam resistance determinants and further docking studies with penicillin and other β-lactam antibiotics indicated their substrate-specificity. Semi-quantitative PCR analysis indicated that the expression of all BLAs and one BDP are induced by penicillin. Disk diffusion assay, SDS-PAGE and zymogram analysis confirms the substrate specificity of the β-lactam resistance determinants. This study gives a broader picture of the β-lactam resistance determinants of a non-pathogenic ethanologenic Z. mobilis bacterium that could have implications in laboratories since it is routinely used in many research laboratories in the world for ethanol, fructooligosaccharides, levan production and has also been reported to be present in wine and beer as a spoilage organism.     

Positive and Negative Selection in the β-Esterase Gene Cluster of the Drosophila melanogaster Subgroup

We examine the pattern of molecular evolution of the β-esterase gene cluster, including the Est-6 and ψEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For ψEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of ψEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of ψEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the β-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function. [Reviewing Editor: Dr. Martin Kreitman]