Structure and biosynthesis of fungal cell walls: Methodological approaches

Fungal cell walls possess a characteristic chemical composition differentiating fungal cells from other cell types. For this reason, the mechanisms involved in cell-wall formation represent a potential target for selective antifungal drugs. Understanding the structure and biosynthesis of fungal cell walls opens the ways for design of effective drugs for treating fungal diseases. This article reviews the history methods employed in chemical and structural analysis of fungal cell walls and in studies concerning their formation.     

Carbon metabolism under conditions of regulated penicillin biosynthesis]

Carbon metabolism of P. chrysogenum under conditions of periodical addition of the nutrients was studied. It was found that a proper rate of the carbon source addition to the culture was of significant importance for intensive biosynthesis. The use of carbon for the energetic and constructive needs was not the same at different fermentation periods.     

Fresh approaches to antibiotic production

New antibiotics are needed, (a) to control diseases that are refractory to existing ones either because of intrinsic or acquired drug resistance of the pathogen or because inhibition of the disease is difficult, at present, without damaging the host (fungal and viral diseases, and tumours), (b) for the control of plant pathogens and of invertebrates such as helminths, insects, etc., and (c) for growth promotion in intensive farming. Numerous new antibiotics are still being obtained from wild microbes, especially actinomycetes. Chemical modification of existing compounds has also had notable success. Here we explore the uses, actual and potential, of genetics to generate new antibiotics and to satisfy the ever-present need to increase yield. Yield improvement has depended in the past on mutation and selection, combined with optimization of fermentation conditions. Progress would be greatly accelerated by screening random recombinants between divergent high-yielding strains. Strain improvement may also be possible by the introduction of extra copies of genes of which the products are rate-limiting, or of genes conferring beneficial growth characteristics. Although new antibiotics can be generated by mutation, either through disturbing known biosyntheses or by activating 'silent' genes, we see more promise in interspecific recombination between strains producing different secondary metabolities, generating producers of 'hybrid' antibiotics. As with proposals for yield improvement, there are two major strategies for obtaining interesting recombinants of this kind: random recombination between appropriate strains, or the deliberate movement of particular biosynthetic abilities between strains. The development of protoplast technology in actinomycetes, fungi and bacilli has been instrumental in bringing these idealized strategies to the horizon. Protoplasts of the same or different species can be induced to fuse by polyethylene glycol. At least in intraspecific fusion of streptomyces, random and high frequency recombination follows. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol, so that the deliberate introduction of particular genes into production strains can be realistically envisaged. Various kinds of DNA cloning vectors are being developed to this end. Gene cloning techniques also offer rich possibilities for the analysis of the genetic control of antibiotic biosynthesis, knowledge of which is, at present, minimal. The information that should soon accrue can be expected to have profound effects on the application of genetics to industrial microbiology.     

New approaches to antibiotic discovery

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use ‘helper’ strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic’ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.     

Optimization of the nutrient medium makeup for the biosynthesis of bleomycine antibiotic using a mathematical method of experimental design]

The fermentation medium for bleomycin biosynthesis was optimized with the help of a mathematical method for experiment modelling. With the use of the schemes of orthogonal latin squares the optimal concentrations of the sources of nitrogen, carbon and mineral salts were determined and the negative effect of cupric sulphate on the antibiotic biosynthesis was shown. The antibiotic production on the developed medium was 3.7 times higher than that on the initial medium.     

Novel approaches to the biosynthesis of vanillin

Microorganisms able to produce vanillin in excess of 6g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun.     

Methodological procedures for detecting antibiotic producers among cultures of the family Micromonosporaceae]

Data on intensification of the search for active cultures among Micromonospora are presented. It was shown that the frequency of detecting the antibiotic-producing cultures among Micromonospora under conditions of fermentation on the corn-glucose medium inoculated with agar blocks amounted to 35 per cent. The use of nutrient media of different composition for growing submerged inoculum of Micromonospora demonstrated that the rate of its growth reached maximum on the peastarch medium. The use of this medium for growing submerged seed material for fermentation in the corn-glucose medium increased the frequency of detecting active cultures from 35 to 43.1 per cent. The assay of Micromonospora antibiotic activity twice, i.e. in 96 and 240 hours of the fermentation process increased the frequency of detecting active cultures up to 57.1 per cent and revealing greater variety of antibiotics. Fermentation of Micromonospora cultures simultaneously on 6 different nutrient media inoculated with submerged seed mycelium and assay of the activity for 2 times, i. e. in 96 and 240 hours allowed a detection of up to 76.2 per cent of active strains out of the total number of the isolates.     

Effect of penicillin precursors on antibiotic biosynthesis in various strains]

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.     

Approaches to intensification and prolongation of antibiotic biosynthesis

World Journal of Microbiology and Biotechnology -     

Synthetic biological approaches to natural product biosynthesis

Small molecules produced in Nature possess exquisite chemical diversity and continue to be an inspiration for the development of new therapeutic agents. In their host organisms, natural products are assembled and modified using dedicated biosynthetic pathways. By rationally reprogramming and manipulating these pathways, unnatural metabolites containing enhanced structural features that were otherwise inaccessible can be obtained. Additionally, new chemical entities can be synthesized by developing the enzymes that carry out these complicated chemical reactions into biocatalysts. In this review, we will discuss a variety of combinatorial biosynthetic strategies, their technical challenges, and highlight some recent (since 2007) examples of rationally designed metabolites, as well as platforms that have been established for the production and modification of clinically important pharmaceutical compounds.     

Catabolite regulation of antibiotic biosynthesis

Conclusion In view of the possible mediation of carbon catabolite repression of antibiotic biosynthesis by phosphorylated substances, the concept of the role of phosphorus in the regulation of secondary metabolism should be re-evaluated. Many conclusions are based on an analogy with the effect of phosphorus in animal or plant cells (for review cf. Martin 1977). However, in contrast with plant and animal physiology, the production physiology of actinomycetes, typical soil microorganisms, was studied under nonphysiological conditions of nutrient over-supply in a submerged culture. This is analogous e.g. to space biology which studies the physiology of organisms in the state of weightlessness. Both disciplines have an extremely high significance but the elucidation of basic biological regularities is to be carried out back on Earth. In conclusion we may state that a critical evaluation of our contemporary knowledge seem to support the hypothesis that the antibiotic biosynthesis in actinomycetes, as well as the spore formation in bacilli — both regulated by the mechanism of catabolite repression — has its ecological significance, i.e. makes it possible for the organism to survive under conditions when vegetative growth is limited. For this reason this aotivity was preserved in the course of evolution as a protective mechanism of microbial populations and remained a part of the genome of many species.     

New approaches to the study of developing wood

Two recent papers illustrate contrasting approaches to studying gene expression during development of the xylem, the tissue that transports water and solutes around higher plants. The two methods used, studying single cells differentiating in vitro and collecting samples from across the region around the cambium of poplar trees, have both revealed genes that have altered expression during xylem development.     

Spontaneous variability of antibiotic biosynthesis in relation to morphogenetic processes in Actinomyces chromogenes var. trienicus]

Available data on possible relationship between the antibiotic activity of actinomycetes and the level of their differentiation, especially with their spore-formation ability, present certain interest with respect to possible relationship between the synthesis of antibiotics and the formation of secondary structures. The study of spontaneous stable variants of Actinomyces chromogenes var. trienicus demonstrates that all sporogenous variants produce the same complex of antibiotics as does the original population. The loss of the ability to synthesize antibiotics is observed only in the phenotypically different dwarf variant (VI). The impaired differentiation (the loss of spore-formation ability) is accompanied by disturbances in the antibiotic synthesis: asporogenous variants are either inactive or produce only 1 antibiotic from the complex synthesized by the original population. Changes in the structure of spore chains do not probably correlate with qualitative and quantitative measurable changes in the antibiotic synthesis. The statistic evidence is suggestive of the fact that the variant with a more complex profile and topography of aerial mycelium displays a higher activity.