Flavomyces fulophazii

2.2. Amounts of compounds in the in vitro cultures of F. fulophazii

Amounts of all identified compounds were determined by HPLC-MS in the lyophilized cultures of ten Hungarian and seven Mongolian F. fulophazii isolates grown in three replicates (Supplementary Table S4, Fig. S8). Among tetramic acids and azaphilones, vermelhotin and flavochlorine A were found to be the main compounds, respectively, in all samples. The highest amounts of vermelhotin were determined in cultures of the Hungarian and Mongolian isolates HF-3 (5.3 mg /g) and MF-7 (5.5 mg /g), respectively (data are averages, calculated from contents of three replicate cultures).

In order to isolate vermelhotin by preparative HPLC, three-three lyophilized cultures of the isolate HF-3 and those of MF-7 were pooled and extracted (six cultures, in total). Since the total weight of these pooled cultures was 1.09 g, 5.9 mg vermelhotin could be isolated as the calculated maximum yield (CMY). The preparative HPLC isolation could be regarded to be effective, according to a comparison of the CMY of vermelhotin (5.9 mg) with the amount of vermelhotin isolated from the lyophilized cultures (4.8 mg), thus suggesting the practical utility of F. fulophazii in high-yield vermelhotin production. Accumulation of the vermelhotin derivatives (dihydroxyvermelhotin, hydroxyvermelhotin, methoxyvermelhotin and oxovermelhotin) showed a close correlation with that of vermelhotin, resulting in their highest amounts also in the cultures of isolates HF-3 and MF-7 (Supplementary Table S4). Among these vermelhotin derivatives, hydroxyvermelhotin was determined to be the most abundant compound, reaching its maximum levels of 1.1 mg /g and 1.0 mg/g in the cultures of isolates HF-3 and MF-7, respectively (average values, calculated from contents of three parallel cultures). Accordingly, in addition to vermelhotin, hydroxyvermelhotin could also be isolated from the pooled cultures of isolates HF-3 and MF-7 by preparative HPLC.

A relative high-level accumulation of the azaphilone flavochlorine A was detected in cultures of isolates MF-3 (MF-3B, 4.2 mg /g), HF-1 (HF-1A, 3.1 mg /g) and HF-9 (HF–9B, 2.2 mg /g). However, flavochlorine A levels were at least one order of magnitude smaller in the corresponding parallel cultures of these isolates. Consequently, in order to isolate flavochlorine A, cultures of different isolates (detailed above), containing the highest levels of this compound, were pooled and extracted. Accumulation of azaphilones B–G, occurring as minor compounds relative to flavochlorine A, showed a close correlation with the accumulation of flavochlorine A.

We found the metabolite production of F. fulophazii highly variable, resulting in differences in the amounts of compounds among the isolates and also among the parallel cultures of one isolate.