Ursodeoxycholic acid production by Gibberella zeae mutants

Abstract

Ursodeoxycholic acid (UDCA) is a highly demanded pharmaceutical steroid widely used in medicine. An ascomycete Gibberella zeae VKM F-2600 is capable of producing UDCA by 7β-hydroxylation of lithocholic acid (LCA). The present study is aimed at the improvement of the fungus productivity. The original procedures for the protoplast obtaining followed by UV mutagenesis and screening of ketoconazole-resistant mutant clones have been applied. The highest yield of G. zeae protoplasts was obtained when using the mycelium in the active growth phase, ammonium chloride as an osmotic stabilizer and treatment of the fungal cells by the lytic enzymes cocktail from Trichoderma hurzanium. The conditions for effective protoplast regeneration and the UV-mutagenesis were found to provide 6-12% survival rate of the protoplasts with superior number of possible mutations. Three of 27 ketoconazole-resistant mutant clones obtained have been selected due to their increased biocatalytic activity towards LCA. The mutant G. zeae M23 produced 26% more UDCA even at relatively high LCA concentration (4 g/L) as compared with parent fungal strain, and the conversion reached 88% (w/w). The yield of UDCA reached in this study prefers those ever reported. The results contribute to the knowledge on ascomycete mutagenesis, and are of importance for biotechnological production of value added cholic acids.

Keywords: 7β-hydroxylation; Gibberella zeae; Lithocholic acid; Mutagenesis; Protoplasts; Ursodeoxycholic acid.

Fig. 1

Conversion of LCA to UDCA

Fig. 2

Influence of mycelium age (a), osmotic stabilizers (b), concentration of the lytic enzymes complex from T. harzianum (c) and digestion time (d) on G. zeae protoplast yield

Fig. 3

Phase-contrast micrographs of Gibberella zeae VKM F-2600 mycelium of second generation stage (18 h) grown in medium no. 3 (see Table 1) (a–c) and protoplasts released from 18 h fungal cells treated with T. harzianum lytic enzymes for 5 h (d–h): 100x (a), 400 (b) x, 1000x (c-h) (optical microscopy)

Fig. 4

Influence of different osmotic stabilizers on regeneration frequency of G. zeae protoplasts

Fig. 5

The regenerative morphology of the colonies of G. zeae protoplasts. 10 µL of protoplast suspension (0.5 × 10⁵/mL) was spread out on agar medium A supplemented with sucrose (1 M) and different concentrations of ketoconazole: 0 µM (a), 15 µM (b), 20 µM (c), 25 µM (d), 30 µM (e), 40 µM (f), 50 µM (g); 60 µM (h), 70 µM (i) and incubated at 28 °C for 12 days

Fig. 6

Effect of ketoconazole concentration (a) and UV exposure (b) on survival of G. zeae protoplasts

Fig. 7

Regeneration of G. zeae protoplasts exposed to UV irradiation. 10 µL of protoplast suspension (0.5 × 10⁵/mL) was spread out on agar medium A with sucrose (1 M): without UV exposure (a) or exposed to UV for 2 min (b), 3 min (c), 5 min (d), 6 min (e) and incubated at 28 °C for 12 days

Fig. 8

Comparative statistical analysis of LCA to UDCA conversion (144 h) by G. zeae parent (P) and mutant (M) strains (GC data of three independent experiments) (a). TLC profiles of 120 h (b) and 144 h (c) samples (~ 10 µg of steroids in the spot): S1, standard samples of LCA (0.5 mkg) and UDCA (7 mkg); S2, standard samples of LCA (1.0 mkg) and UDCA (8 mkg) (top to bottom); X–undefined metabolite (the structure of the product was not determined due to its low amount and concomitant formation of several metabolites). LCA initial concentration was 4 g/L