by

 Debra K Hobson and David S Wales

 (This article originally appeared in JSDC, 1998, 114, 42-44.)

Anthraquinone compounds, historically of crucial importance in the dyestuffs industry, have also been identified as secondary metabolites produced by fungi. Secondary metabolites are low molecular weight natural products that have a restricted taxonomic distribution, possess no obvious function in cell growth and are synthesised for a finite period by cells that are no longer undergoing balanced growth. Primary metabolism includes all of the anabolic and catabolic processes that are finely balanced to keep the organism alive. It is essentially an identical process for all living organisms.

Many compounds now referred to as secondary metabolites were known and widely exploited prior to modern biotechnology and were mainly obtained from plants (Figure 1). Examples of use are as dyes, perfumes, spices, poisons, pharmacological agents and cosmetics.

The occurrence of large scale secondary metabolism is not common to all living organisms, but restricted to certain taxonomic groups. Among animals, arthropods, coral reef coelenterates and other marine invertebrates (e.g. sea urchins and starfish) are the most prolific producers of secondary metabolites. Within the plant kingdom a great abundance of secondary metabolites have been isolated. Few secondary metabolites have been reported in blue-green algae, whereas a rich and extensive secondary metabolism has been observed in bacteria and fungi.

Secondary metabolites are thought to have specialised survival functions in nature, being particularly numerous in organisms occupying densely inhabited environments and are believed to have a prominent role in the coexistence and coevolution of species allowing interaction within a community (Table 1).

Despite their diversity, secondary metabolites are all produced from a few key intermediates of primary metabolism. Primary and secondary metabolism are intimately related, with secondary metabolites depending on precursors and energy generated through primary metabolism (Figure 2).

The anthraquinone secondary metabolite alizarin is derived from the madder plant. It was exploited as a colorant by man long before the structure was elucidated by Graebe and Liebermann in the 1860s. The anthraquinonoid colorants form the basis of many of the modern synthetic dyes and, after the azo class, they form the second most important group of organic colorants listed in the Colour Index today. Anthraquinone dyes tend to predominate in the violet, blue and green hue sectors in the disperse, vat, and acid application classes of dyes, although they have also made important contributions to mordant, solvent and reactive dyes and also to pigments.

Anthraquinone disperse dyes still predominate in the dyeing of cellulose acetate and polyamide substrates, and find use with polyester where other dyes have limitations in application, for example, meeting the extremely high light fastness requirements for very pale colours or for the dyeing of polyester upholstery trim in the automotive sector. However, anthraquinone dyes are at present in decline. This essentially is not a result of any shortcoming of these compounds as dyes, but chiefly because their chemical production is expensive and environmentally unfriendly. Several methods of anthraquinone production have been employed depending on the resources available, but all processes require strong acids, alkalis, high temperatures and heavy metal catalysts.

The dyestuff industry is suffering from increases in costs of feedstock and energy for dye synthesis. It is also under increasing pressure to minimise the damage to the environment caused by production process and its effluents. Apart from the basic need to control the acid, alkali and colour in effluent, producers also have to meet increasingly stringent requirements for the control of heavy metals (such as mercury, cadmium, copper, nickel, lead, zinc and chromium), all of which adds to the cost of the dye.

The industry is continuously looking for cheaper, more environmentally friendly routes to existing dyes; biotechnology may provide an answer. Anthraquinones have been isolated from a number of fungi including Drechslera, Trichoderma, Aspergillus and Curvularia strains. Most of these fungi produce a mixture of anthraquinones. This is typical of the enzymes of secondary metabolism which are believed to be less specific than those of primary metabolism. Aspergillus cristatus, for example, has been reported to produce as many as 15 different anthraquinones. A strain of Curvularia lunata has been identified that produces only three anthraquinones: chrysophanol, helminthosporin and cynodontin, with cynodontin, the 1,4,5,8-tetrahydroxy-3-methylanthraquinone making up over 70% of the mix (Figure 3).

An anthraquinone of 70% purity can be chemically converted to a modern dyestuff, and therefore fermentation of C. lunata is the proposed method for producing cheap, environmentally friendly anthraquinone dyestuff intermediates. Exploiting the fungal synthesis of anthraquinones has several advantages over chemical methods. The medium in which the fungal culture grows contains no expensive chemicals. The fermentation is carried out at low temperature (30 °C) and neutral pH so that the expensive, fuel-consuming high temperatures and environmentally unfriendly strong acids and alkalis of the chemical synthesis are not required. The purity of the cynodontin is the key factor making the exploitation of this organism for dye production possible.

Cynodontin extracted from the biomass of C. lunata has been successfully converted to two anthraquinone biodyes (CI Disperse Blue 7 and CI Acid Green 28), each with a methyl group in the 3-position (Figure 4). The properties of these biologically derived dyes applied to knitted polyamide were compared with those of conventional dyes, and found to be identical to all important respects.

Extraction of cynodontin from the mycelia of C. lunata was later found not to be necessary for conversion to the biodye. The ability to produce biodyes from cynodontin retained within the mycelia is a particularly important factor when considering the additional cost of an extraction process to dye production.

To date impressive yields of cynodontin have been achieved, although these cannot be considered high enough to warrant full-scale commercial production for which at least a ten-fold increase is required. It is possible that further manipulation of the nutrients supplied to the organism during its growth and production will result in improved yields. Further work is proposed to identify and manipulate the genes required for anthraquinone synthesis so that the anthraquinone produced by the fungi could be specifically designed rather than being left to chance. Yields could be increased by genetically promoting production of the anthraquinone synthase enzyme system.

Cynodontin is a useful anthraquinone since it is tinctorially the strongest and most bathochromic of the hydroxyanthraquinones. Anthraquinones containing three or more substituents are used extensively as commercial dyes. However, other anthraquinones may act as intermediates to a wider range of dyes. Anthraquinone compounds with chloro or amino substitutions would be useful for dye production but are not common among fungal anthraquinones. It is possible that these compounds may be engineered by genetic modification, or by adding to the pathway that is naturally present.

The authors thank the Editor of the Journal of the Society of Dyers and Colourists (JSDC) for granting permission to republish this article on-line.

• Competitive weapons against other fungi, bacteria, plants, insects etc.

• Metal chelating agents

• Agents that influence spore germination

• Structural and extracellular protective agents

• Agents of host fungal symbiosis

• Reserve pool of new pathways

Figure 1  Secondary metabolites exploited by man

Figure 2  Roles of low molecular weight metabolites in primary and secondary metabolites

Figure 3  Anthraquinones produced by Curvularia lunata

Figure 4  Some anthraquinone dyes

Copyright © 1998:  Hobson & Wales / JSDC

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