Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

A novel cytochrome P450, CYP53A15, was identified in the pathogenic filamentous ascomycete Cochliobolus lunatus. The protein, classified into the CYP53 family, was capable of para hydroxylation of benzoate. Benzoate is a key intermediate in the metabolism of aromatic compounds in fungi and yet basically toxic to the organism. To guide functional analyses, protein structure was predicted by homology modeling. Since many naturally occurring antifungal phenolic compounds are structurally similar to CYP53A15 substrates, we tested their putative binding into the active site of CYP53A15. Some of these compounds inhibited CYP53A15. Increased antifungal activity was observed when tested in the presence of benzoate. Some results suggest that CYP53A15 O-demethylation activity is important in detoxification of other antifungal substances. With the design of potent inhibitors, CYP53 enzymes could serve as alternative antifungal drug targets.

SummaryCytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Aims: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. Methods and Results: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. Conclusion: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. Significance and Impact of the Study: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.