Altered substrate specificity of the gluco‐oligosaccharide oxidase from Acremonium strictum

A gluco‐oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX‐VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX‐VN oxidized xylose, galactose, and N‐acetylglucosamine. Besides monosaccharides, GOOX‐VN oxidized xylo‐oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello‐oligosaccharides. Of three mutant enzymes that were created in GOOX‐VN to improve substrate specificity, Y300A and Y300N doubled k_cat values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX‐VN and its variants are particularly valuable for oxidative modification of cello‐ and xylo‐oligosaccharides. Biotechnol. Bioeng. 2011;108: 2261–2269. © 2011 Wiley Periodicals, Inc.     

两种枝顶孢霉胞内细菌的分离鉴定及种群演替

分离、鉴定了枝顶孢霉(Acremonium strictum Gams.)2种胞内细菌,探讨了细菌在其宿主真菌菌丝细胞内的种群演替规律。结果表明,2种胞内细菌分别为不动杆菌Epbas6菌株(Acinetobacter sp.Epbas6)和地衣芽孢杆菌(Bacillus licheniformis)。镜检分析和原始分离物菌落统计表明,不动杆菌和地衣芽孢杆菌的比例为77.5∶1;然而,4℃保藏6个月的真菌分离物中不动杆菌的数量大大减少,地衣芽孢杆菌占据优势,二者的比例变为1∶50.45;而保藏12个月的真菌分离物中只有地衣芽孢杆菌。根据2种细菌的16SrDNA保守序列设计引物,分别扩增了保藏6个月和12个月的枝顶孢霉分离物,发现保藏6个月的分离物能够扩增出2种细菌的保守序列,而保藏12个月的分离物只能扩增出地衣芽孢杆菌保守序列。研究结果说明在枝顶孢霉人工培养和菌种保藏过程中,2种细菌在其宿主真菌菌丝细胞内发生着复杂的生态互作,存在着动态种群演替过程。     

Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum

The strain of Acremonium strictum PTCC 5282 was applied to investigate the biotransformation of androst-1,4-dien-3,17-dione (I; ADD). Microbial products obtained were purified by preparative TLC and the pure metabolites were characterized on the basis of their spectroscopic features (¹³C NMR, ¹H NMR, FTIR, MS) and physical constants (melting points and optical rotations). The 15α-Hydroxyandrost-1,4-dien-3,17-dione (II), 17β-hydroxyandrost-1,4-dien-3-one (III), androst-4-en-3,17-dione (IV; AD), 15α-hydroxyandrost-4-en-3,17-dione (V), 15α,17β-dihydroxyandrost-1,4-dien-3-one (VI) and testosterone (VII) were produced during this fermentation. Formation of the 15α,17β-dihydroxy derivative of ADD is reported for the first time during steroid biotransformation. The bioconversion reactions observed were 1,2-hydrogenation, 15α-hydroxylation and 17-ketone reduction. From the time course profile of this biotransformation, ketone reduction and 1,2-hydrogenation were observed from the first day of fermentation while 15α-hydroxylation occurred from the third day. Optimum concentration of the substrate, which gave the maximum bioconversion efficiency, was 0.5 mg ml⁻¹ in one batch. The highest yield of the microbial products recorded in this work was achieved within the pH range 6.5–7.3 and at the temperature of 27 °C.     

Microbial hydroxylation of unsaturated, cyclic carboxylic acids protected as benzoxazoles

Microbial hydroxylation of 2-(cyclopent-1-enyl)benzoxazole (1) and 2-(cyclohex-1-enyl)benzoxazole (2) by Cunninghamella blakesleeana DSM 1906 and Bacillus megaterium DSM 32, respectively, gave chiral allylic alcohols 3-(benz-1,3-oxazol-2-yl)cyclopent-2-en-1-ol (3) and 3-(benz-1,3-oxazol-2-yl)cyclohex-2-en-1-ol (4) along with achiral ketones 3-(benz-1,3-oxazol-2-yl)cyclopent-2-en-1-one (5) and 3-(benz-1,3-oxazol-2-yl)cyclohex-2-en-1-one (6). Both allylic alcohols were produced in enantiomeric excesses higher than 99%. The determination of their absolute configurations (S in both cases) is described.     

Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282

The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.     

Microbial hydroxylation of o-bromophenylacetic acid: synthesis of 4-substituted-2,3-dihydrobenzofurans

Microbial hydroxylation of o-bromophenylacetic acid provided 2-bromo-5-hydroxyphenylacetic acid. This enabled a route to the key intermediate 4-bromo-2,3-dihydrobenzofuran for synthesizing a melatonin receptor agonist and sodium hydrogen exchange compounds. Pd-mediated coupling reactions of 4-bromo-2,3-dihydrobenzofuran provided easy access to the 4-substituted-2,3-dihydrobenzofurans.     

Microbial production of two new dihydroxylated androstenedione derivatives by Acremonium strictum

We found that Acremonium strictum NN106 converted 4-androstene-3, 17-dione (androstenedione) to 12 compounds. Among them, five products were isolated and found to be hydroxylated at the 11α-, 14α-, 7α, 11α-, 6β, 11α- or 6β, 14α-positions of androstenedione. 6β, 11α-Dihydroxy and 6β, 14α-dihydroxy derivatives of androstenedione have been obtained for the first time. From the time course profile of this transformation, sequential hydroxylation at the 6β-position followed by 11α- or 14α-monohydroxylation was observed. The oxidative product of the 6β, 14α-dihydroxy derivative was found to be the most potent inhibitor of human placentral aromatase.     

Interactions between Psilocybe fasciata and its companion fungus Acremonium strictum

Interactions between Psilocybe fasciata and its companion fungus Acremonium strictum were analysed. The conidia of A. strictum were observed on stipe, flesh and gill of P. fasciata, which suggested that A. strictum is the fungicolous fungi or mycophilic fungi of P. fasciata. The microscopic observations of the interacting hyphae of P. fasciata and A. strictum in dual culture and the negligible activities of chitinase and β-1,3-glucanase in inducing or non-inducing media of the pure and mixed cultures of the two fungi indicated that A. strictum is not the mycoparasite of P. fasciata. In addition, the co-existence, no pigmentation and dew formation in dual culture of both fungi were observed, which implied that the interference competition between the two fungi is weak. The activities of cellobiase, filter paper enzyme, endoglucanase and xylanase of pure and mixed cultures of P. fasciata and A. strictum were the same or similar, which may allow the co-existence of the two fungi. As a consequence of coevolution, the relationship between P. fasciata and A. strictum is commensalism: A. strictum showed no clear benefit to P. fasciata; however, P. fasciata may shelter A. strictum by its psychoactive tryptamines and may be helpful to conidium dispersal of A. strictum. The relationship between P. fasciata and A. strictum is different from that of A. strictum and other fungi.     

Microbial hydroxylation of epiandrosterone by Aspergillus candidus

In this work, Aspergillus candidus MRC 22634 converted epiandosterone 1 into 10 hydroxylated metabolites. A. candidus has been shown to hydroxylate 1 predominantly at C-11α, C-1α, and C-15β with minor hydroxylations occurring at C-14α and C-7α. Oxidation at C-3, reduction at C-17, and C-3 epimerization of some of the remaining substrate have also been shown. 15β-Hydroxylation and C-3 epimerization of 1 by a fungus were reported for the first time. Two of the metabolites, 1α,3α-dihydroxy-5α-androstan-17-one 4 and 15β,17β-dihydroxy-5α-androstan-3-one 7, were identified as new compounds.     

Microbial oxidation of cumene

Summary A total of 1229 cultures, including 230 actinomycetes, 508 other bacteria, 12 fungi and 479 yeasts were screened for their ability to oxidize the isopropyl side chain of 2-phenyl propane (cumene). Four strains of actinomycetes and six strains of bacteria but no yeasts were found positive in converting 2-phenyl propane to its oxygenated products. Eight strains oxidized cumene through the alkyl side chain producing 2-phenyl-1-propanol. TwoBacillus strains oxidized cumene to an oxygenated product.Pseudomonas oleovorans NRRL B-3429 exhibited the highest alkyl side chain oxidation activity. The optimum reaction conditions for strain B-3429 are: 25 °C, pH 6.5 and 48 h of reaction. Octane-grown cells of strain B-3429 produced higher product yields (about 7.2-fold) than the glucose-grown cells. Prolonged incubation resulted in an increase in 2-phenyl-1-propionic acid production at the expense of 2-phenyl-1-propanol. The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.1%. Reaction in the presence of methanol favored the accumulation of 2-phenyl-1-propionic acid and also increased the total yield. (The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 14.9%.) Structures of the reaction products were confirmed by GC/MS and GC/IR analyses. Products contained 92% R(–) isomer.     

Rapid identification of Acremonium lolii and Acremonium coenophialum endophytes through arbitrarily primed PCR

Abstract Using a random decamer 5'-CCGAGGTGAC-3' in an arbitrarily primed PCR, similar band patterns were observed between Acremonium lolii and A. coenophialum DNA, which were somewhat different from those formed by other fungal DNA. Despite sharing bands of around 0.7, 0.9 and 2.1 kb, A. lolii can be distinguished from A. coenophialum by the presence of an additional band at around 0.5 kb in the arbitrarily primed PCR.     

Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans

Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC₅₀ values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40 nM (³H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC₅₀ of progesterone 2.3 ± 0.1 × 10⁻⁷ M, EC₅₀ of androsten-3,17-dione 24 ± 2 × 10⁻⁷ M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons -naphthoflavone and ketoconazole exhibited EC₅₀ values of 0.3 ± 0.01 × 10⁻⁷ M and 27 ± 5 × 10⁻⁷ M, respectively, whereas β-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of -naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.     

The microbiological hydroxylation of patchoulol in Absidia coerulea and Mucor hiemalis

Patchoulol was subjected to transformation by Absidia coerulea AM93 and Mucor hiemalis AM450 strains. Both micro-organisms, which displayed differing vulnerability to the fungistatic action of the patchoulol, were capable of selective hydroxylations of the substrate. The major constituents of the mixtures after transformation were (8S)-8-hydroxypatchoulol and (9R)-9-hydroxypatchoulol; the transformation carried out by M. hiemalis resulted in the additional formation of (3R)-3-hydroxypatchoulol. The mixture of (8S)-8-hydroxypatchoulol and (9R)-9-hydroxypatchoulol can be used in the synthesis of patchoulenol, a compound with an odour very similar to that of a valuable fragrance, norpatchoulenol.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

甾体C7-羟基化研究进展

生物羟基化被用作研究甾体代谢机制和制备羟基甾体的工具,许多真菌微生物菌具有甾体C7-羟基化能力,而C7-羟基甾体具有许多重要的生物活性。在分子水平上,人们已经发现了7α-羟基化酶及其基因。就以上几个方面做一简单综述,并对此领域的发展趋势进行展望。     

Flexibility of the endogenous progesterone lactonisation pathway in Aspergillus tamarii KITA: transformation of a series of cortical steroid analogues

A range of cortical steroids have been transformed by the fungus Aspergillus tamarii, which has the ability to convert progesterone to testololactone in high yield through a four step enzymatic pathway. 16alpha,17alpha-Epoxyprogesterone underwent a rare epoxide opening resulting in a unique inversion of stereochemistry to give 16beta-hydroxy-17alpha-oxa-D-homo-androst-4-en-3,17-dione. The metabolism of deoxycorticosterone resulted in relatively efficient transformation to testololactone with no other products isolated. Transformation of 17alpha-hydroxyprogesterone yielded 17alpha-oxa-D-homo-androst-1,4-dien-3,17-dione, a lactone not previously isolated from A. tamarii. Cortexolone was transformed to the 20(R)-alcohol with no further transformation observed. Evidence is also presented for the presence of a highly flexible but stereospecific keto-reductase. All metabolites were isolated by column chromatography and were identified by 1H, 13C NMR, DEPT analysis and other spectroscopic data.     

Microbial degradation of chlorinated benzenes

Chlorinated benzenes are important industrial intermediates and solvents. Their widespread use has resulted in broad distribution of these compounds in the environment. Chlorobenzenes (CBs) are subject to both aerobic and anaerobic metabolism. Under aerobic conditions, CBs with four or less chlorine groups are susceptible to oxidation by aerobic bacteria, including bacteria (Burkholderia, Pseudomonas, etc.) that grow on such compounds as the sole source of carbon and energy. Sound evidence for the mineralization of CBs has been provided based on stoichiometric release of chloride or mineralization of (14)C-labeled CBs to (14)CO(2). The degradative attack of CBs by these strains is initiated with dioxygenases eventually yielding chlorocatechols as intermediates in a pathway leading to CO(2) and chloride. Higher CBs are readily reductively dehalogenated to lower chlorinated benzenes in anaerobic environments. Halorespiring bacteria from the genus Dehalococcoides are implicated in this conversion. Lower chlorinated benzenes are less readily converted, and mono-chlorinated benzene is recalcitrant to biotransformation under anaerobic conditions.     

DNA slows dissociation of progesterone receptor-steroid ligand complexes

Steroid ligands are known to affect the interactions of their respective receptors with DNA. In the present study, the possibility of DNA interference in progesterone receptor-ligand interactions was investigated. An oligonucleotide containing a hormone response element (HRE) was shown to decrease the dissociation rate of complexes of [3H]progesterone or [3H]16alpha,17alpha-cycloalkanoprogesterones with PRs from rabbit and rat uterine cytosol. The extent to which the oligonucleotide affected the dissociation constant varied from about 4- to 1.5-fold depending on the ligand structure and was ranked in the following order: progesterone>16alpha,17alpha-cyclopropanoprogesterone approximately 16alpha,17alpha-cyclopentanoprogesterone>/=16alpha,17alpha-cyclohex-2'-enoprogesterone approximately 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone>/=16alpha,17alpha-cyclohexanoprogesterone. The control oligonucleotide lacking HRE had a weak effect, if any, on the dissociation kinetics. No influence of the HRE-containing oligonucleotide on the equilibrium binding of ligands to PR was observed. The results suggest that the DNA partner affects binding of PR to its ligand.     

Microbial breakdown of halogenated aromatic pesticides and related compounds

Abstract Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.