Bikaverin

Fungal naphthoquinones, like red bikaverin, are of interest due to their growing applications in designing pharmaceutical products. Though considerable work has been done on the elucidation of bikaverin biosynthesis pathway in Fusarium fujikuroi, very few reports are available regarding its bioproduction in F. oxysporum. We are hereby proposing a putative metabolic pathway for bikaverin bioproduction in a wild F. oxysporum strain by cross-linking the pigment profiles we obtained under two different fermentation conditions with literature. Naphthoquinone pigments were extracted with a pressurized liquid extraction method, and characterized by HPLC-DAD and UHPLC-HRMS. The results led to the conclusions that the F. oxysporum LCP531 strain was able to produce bikaverin and its various intermediates, e.g., pre-bikaverin, oxo-pre-bikaverin, dinor-bikaverin, me-oxo-pre-bikaverin, and nor-bikaverin, in submerged cultures in various proportions. To our knowledge, this is the first report of the isolation of these five bikaverin intermediates from F. oxysporum cultures, providing us with steady clues for confirming a bikaverin metabolic pathway as well as some of its regulatory patterns in the F. oxysporum LCP531 strain, based on the previously reported model in F. fujikuroi. Interestingly, norbikaverin accumulated along with bikaverin in mycelial cells when the strain grew on simple carbon and nitrogen sources and additional cofactors. Along bikaverin production, we were able to describe the excretion of the toxin beauvericin as main extrolite exclusively in liquid medium containing complex nitrogen and carbon sources, as well as the isolation of ergosterol derivate in mycelial extracts, which have potential for pharmaceutical uses. Therefore, culture conditions were also concluded to trigger some specific biosynthetic route favoring various metabolites of interest. Such observation is of great significance for selective production of pigments and/or prevention of occurrence of others (aka mycotoxins).

Microbial pigments have a distinguished potential for applications in food and pharmaceutical industries, stimulating the research in this field. The present study evaluated the ideal conditions for extracting bikaverin (red pigment) from the biomass ofFusarium oxysporumCCT7620. Among the solvents tested, ethyl acetate extraction resulted in the highest bikaverin concentration and the kinetic study revealed a saturation in bikaverin concentration from 256 min on. Based on a preliminary economic study, three sequential extractions with ethyl acetate was considered the ideal protocol to recover bikaverin. After extraction, chromatographic methods were tested to purify bikaverin. The use of silica gel or Sephadex (open column) could not successfully purify bikaverin, but the semi-preparative HPLC resulted in a bikaverin-enriched fraction with a purity degree equivalent to the commercial analytical standard. This work provides relevant information regarding the extraction and purification of bikaverin, which may be useful for other downstraming processes.

Bikaverin is a reddish pigment produced by different fungal species, most of them from the genus Fusarium, with antibiotic properties against certain protozoa and fungi. Chemically, bikaverin is a polyketide with a tetracyclic benzoxanthone structure, resulting from the activity of a specific class I multifunctional polyketide synthase and subsequent group modifications introduced by a monooxygenase and an O-methyltransferase. In some fungi, bikaverin is found with smaller amounts of a precursor molecule, called norbikaverin. Production of these metabolites by different fungal species depends on culture conditions, but it is mainly affected by nitrogen availability and pH. Regulation of the pathway has been investigated in special detail in the gibberellin-producing fungus Fusarium fujikuroi, whose genes and enzymes responsible for bikaverin production have been recently characterized. In this fungus, the synthesis is induced by nitrogen starvation and acidic pH, and it is favored by other factors, such as aeration, sulfate and phosphate starvation, or sucrose availability. Some of these inducing agents increase mRNA levels of the enzymatic genes, organized in a coregulated cluster. The biological properties of bikaverin include antitumoral activity against different cancer cell lines. The diverse biological activities and the increasing information on the biochemical and genetic basis of its production make bikaverin a metabolite of increasing biotechnological interest.