Optimisation of hypocrellin production in Shiraia-like fungi via genetic modification involving a transcription factor gene and a putative monooxygenase gene.

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation.

Enzymatic synthesis of a CCK-4 tripeptide fragment.

OBJECTIVE: To synthesize a tripeptide derivative Phac-Met-Asp(OMe)-Phe -NH2, which is a fragment of the gastrin C-terminal tetrapeptide CCK-4, by enzymatic reaction. METHODS: Three free enzymes, alpha-chymotrypsin, papain and thermolysin from acyl donor Phac-Met-OCam was involved in three steps. The choice of appropriate enzymes and solvents was selected. RESULTS: All enzymatic reactions were obtained in reasonable yields(63%-92%). FAB-MS and FD-MS verified the correct molecular mass of the peptides. CONCLUSION: Studies on the alpha-chymotrypsin catalyzed coupling reaction between Phac-Met-OCam and H-Asp(OMe)2 have focused on the low water content media. By papain catalyzed saponification of Phac-Met-Asp(OMe)2, alpha-methyl ester of aspartic acid is selectively hydrolyzed to retain beta-methyl ester, and Phac-Met-Asp(OMe)-OH and H-Phe-NH2 can be coupled efficiently by thermolysin.