Microbial transformation has proven to be an important resource in the production of various chemicals used in the pharmaceutical and agrochemical industries.1 Microorganisms, such as fungi have been widely used in biotransformation experimentsto yield bioactive metabolites. Many of these reactions are difficult to achieve by chemical synthetic protocols. As a result, fungi have played an integral role in the development of green chemistry and by extension the pharmaceutical industry.

The fungus Beauveria bassiana ATCC 7159 (B. sulfurescens or Sporotrichum sulfurescens) has gained popularity as a microbial oxidant, and therefore, was chosen by the research group as a suitable host to carry out biotransformation studies on several diterpenes analogues (1-16). Biotransformation of organic compounds are catalyzed by cytochrome P450 (CYP)enzyme inside the cells of the fungus.

General equation: RH + O2+ NADPH + H+→ ROH + H2O + NADP+

METHODOLOGY

1. Extraction and purification of diterpene; stemodin (1) from the plant Stemodia maritima:

a. The green and dried plant material were extracted with petrol and acetone respectively.

b. The extracts were purified using Flash Chromatography to afford stemodin (1) as the major compound.

2. Synthesis of diterpene analogues:

a. Stemodin(1)was used as a suitable starting material to prepare fifteen analogues (2-16)

3. Biotransformation of terpene analogues:

a.The medium was inoculated withagrowing culture of B.bassianafrom a slant (1 slant per 4 flasks)

b.The shaking culture was pulse fed after 24, 36, 48 and 60 h (10, 20, 30, 40% of the substrate), then shakenfor a period of 14 days

c.The contents of the flasks were filtered to separate the broth from the growing fungal biomass. Both the broth and biomass were extracted with ethyl acetate.

d.The extracts were purified by Flash Column Chromatography and the structures of the compounds were elucidated using Nuclear Magnetic Resonance (NMR)Spectroscopy.

RESULTS

Stemodin (1) and analogues (2-16)

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SUB-HEADING: Products of Biotransformation.

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CONCLUSIONS

• Beauveria bassiana ATCC 7159 has been proven to be an excellent fungus in biotransformation of terpenes, thereby yielding novel analogues.
• The Oxygen functionality at C-2 had more influence on the biotransformation reactions than that of the C-13 position.
• Thirteen products of transformation and one polyketide natural product were isolated and characterized from the fermentations.

RELEVANCE AND POTENTIAL APPLICATION

• Access to novel compounds (Ten novel compounds were isolated).
• Generationof a library of compounds for biological screening experiments.
• Leads to potential pharmaceutical drug(s).
• Structure activity relationship studies.
• The results can be used to further probe and assist in designing a model the active site of the hydroxylase Cyp enzyme in B. bassiana