Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase

AIMS: To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance. METHODS AND RESULTS: Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.     

Biotransformation of indole derivatives by mycelial cultures

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.     

Production of indole diterpenes by Aspergillus alliaceus

Production of two related indole diterpenes (differing by a dimethyl leucine side chain) by Aspergillus alliaceus was improved through several pilot scale fermentations. Media were optimized through focus primarily on initial increases, as well as mid-cycle additions, of carbon and nitrogen sources. Fermentation conditions were improved by varying ventilation conditions using various combinations of air flowrate and back-pressure set points. Production improvements were quantified based on total indole diterpene concentration as well as the ratio of the major-to-minor by-product components. Those changes with a positive substantial impact primarily on total indole diterpene concentration included early cycle glycerol shots and enhanced ventilation conditions (high air flowrate, low back-pressure). Those changes with a significant impact primarily on ratio included higher initial cerelose, soybean oil, monosodium glutamate, tryptophan, or ammonium sulfate concentrations, higher broth pH, and enhanced ventilation conditions. A few changes (higher initial glycerol and monosodium glutamate concentrations) resulted in less notable and desirable titer or ratio changes when implemented individually, but they were adopted to more fully realize the impact of other improvements or to simplify processing. Overall, total indole diterpene titers were improved at the 600 L pilot scale from 125-175 mg/L with a ratio of about 2.1 to 200-260 mg/L with a ratio of about 3.3-4.5. Thus, the ability to optimize total indole diterpene titer and/or ratio readily exists for secondary metabolite production using Aspergillus cultures.     

Oxidation of indole and its derivatives by heme-independent chloroperoxidases]

Indole, indolylacetic acid, and tryptophan were oxidized by cloroperoxidases isolated from strains of Streptomyces lividans and Pseudomonas pyrrocinia. Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were extracted from the reaction medium.     

Combinatorial synthesis of libraries of indole derivatives

The synthesis of two indole derivative libraries is described. 2-Acyl-3-amino-indoles 4 can easily be accessed by treatment of the intermediates 3 with bases in a one-pot reaction sequence whereas the reaction of the isolated intermediates 5 (R(3)=aromatic-, heteroaromatic, or cycloalkyl) with acid chlorides yielded the novel indole derivatives 6. The products were purified by reversed phase column chromatography and obtained in multi-milligram quantities in acceptable yields.     

Determination of substituted indole derivatives by ion suppression-reverse-phase high-performance liquid chromatography

A method for the separation of substituted indole derivatives has been developed by the use of ion suppression-reversed-phase high-performance liquid chromatography (IS-HPLC). Signal response, selectivity (alpha), retention times (tR), and capacity factors (k') were monitored by varying the mobile phase with respect to methanol composition and pH. Chromatographic parameters including tR, k', K (distribution coefficients), alpha, number of theoretical plates, height equivalent to theoretical plates, and column resolution were calculated and assessed in regard to the suitability of four stationary phases in the analysis of 20 substituted indole derivatives. This work has established the chromatographic foundation needed to analyze over 10 specific enzyme reactions involved in the microbial and plant metabolism of auxins. Quantitative studies on the stability of auxins were possible by employing IS chromatography (ISC). The application of the developed IS chromatographic technique was employed to detect indole-3-acetic acid derived from L-tryptophan in a fluorescent pseudomonad culture.     

Production of indole alkaloids by surface immobilized C. roseus cells

A technique was developed to surface immobilize plant cells and was scaled up in laboratory size bioreactors. This technique was shown not to hinder the biosynthetic potential of Catharanthus roseus immobilized cells and to induce a partial release (300 mug/L) of serpentine into the culture medium contrary to suspension cultured cells. The release pattern seemed to follow the biosynthesis trends of the product. This release mechanism could be stimulated by a factor of 10 within 2 h by increasing the pH of the culture from 5.0 to 5.5.     

Photobiotransformation of indole to its value-added derivatives by Rhodobacter sphaeroides OU5

A purple non-sulfur anoxygenic phototrophic bacterium, Rhodobacter sphaeroides OU5 was able to photobiotransform indole in the presence of various organic substrates to its value-added derivatives tryptophan, tryptamine, indole lactic acid and indigo, which are of high commercial value. The product formed varied with the precursors provided in the medium. Received 21 August 1997/ Accepted in revised form 10 January 1998     

The oxidation of indole derivatives catalyzed by horseradish peroxidase is highly chemiluminescent

The indole moeity is present in many substances of biological occurrence. Its metabolism, in most cases, involves an oxidative pathway. This study reports the oxidation of a series of indole derivatives, including several of biological origin, catalyzed by horseradish peroxidase in the presence of H2O2. Chemiluminescence emission was observed in most cases and its intensity and spectral characteristics were correlated with structural features of the substrates. The structures of the main products were determined. The participation of molecular oxygen and superoxide ion in the reaction was demonstrated and a general mechanism for product formation proposed. Since the oxidation of 2-methylindole proved to be highly chemiluminescent, its potentiality as a developing system for peroxidase-based assays was tested and showed to be very effective.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

Synthesis and biological activity of 8beta-substituted hydrocodone indole and hydromorphone indole derivatives.

The 8beta-unsubstituted and substituted analogues of hydrocodone indole and hydromorphone indole were synthesized and their binding affinities to opioid receptors were determined. Introduction of an 8beta-methyl group into the indolomorphinan nucleus increased affinity at all opioid receptors. 6,7-Dehydro-4,5alpha-epoxy-8beta-methyl-6,7,2',3'-indolomorphinan (9) was found to be a delta antagonist with subnanomolar affinity (0.7 nM) for the delta-opioid receptor, and to have good delta-selectivity (mu/delta=322).