Heterologous expression of cytochrome P450 monooxygenases from Aspergillus terreusand Cryptococcus neoformans

[摘要] English: Cytochrome P450 monooxygenases (CYP450) are heme-thiolate proteins which are present in all biological kingdoms. They catalyse the addition of a single oxygen atom to a non-activated carbon atom. The metabolic system of filamentous fungi is maintained by numerous enzymes catalysing the production of bioactive compounds. Genome sequencing projects have revealed the presence of a large collection of CYP450 families within the fungal kingdom. Although some fungal CYP450s are involved in essential reactions such as xenobiotic degradation and membrane ergosterol synthesis, there is still a large collection of uncharacterized fungal CYP450s. The aim of this study was to accomplish and optimize expression and then investigate the substrate specificity of the self-sufficient Aspergillus terreus CYP505E3 as well as five CYP450s from Cryptococcus neoformans. In the case of the Cryptococcus CYP450s the ultimate aim was to establish their possible role in Cryptococcal prostaglandin production.Self-sufficient A. terreus CYP450s were reported by Vatsyayan and co-workers (2008) to show activity towards alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents and steroids. The genome of A. terreus comprises 125 CYP450s with two probable self-sufficient CYP450s namely CYP505A19 and CYP505E3. Omissions within the CYP505A19 sequence makes it unlikely to be active leaving the CYP505E3 as probably the only functional self-sufficient CYP450 in A. terreus. The nucleotides on the N-terminus of A. terreus CYP505E3 were modified to enhance the expression level in Escherichia coli. The recovery of CYP505E3 in the soluble fraction of cell free extract was significantly improved by using expression conditions established through a number of Plackett-Burman experiments. A low temperature, low FeCl3, high culture volume and prolonged incubation time contributed to the improved recovery.The CYP505E3 within the whole cells catalysed sub-terminal hydroxylation of hexylbenzoic acid (HBA) to produce ω-1 OH-HBA, ω-2 OH-HBA and ω-4 OH-HBA. However, no hydroxylated products were produced from the other substrates tested. The activity of self-sufficient A. terreus CYP505E3, towards HBA was similar to that CYP505E3, towards HBA was similar to that of Bacillus megaterium CYP102A1 and Fusarium oxysporum CYP505A1 indicating that CYP505E3 is probably also a fatty acid hydroxylase. The opportunistic yeast pathogen Cryptococcus neoformans is known to produce authentic immunomodulatory prostaglandin E2 from arachidonic acid (AA). However, because of the absence of cyclooxygenase (COX) and lipoxygenase (LOX) homologues in its genome, the mechanism used by the organism to produce prostaglandins remains unclear. Five CYP450s and a CYP450 reductase (CPR) are present within the genome of the organism. The probable roles of these CYP450s in Cryptococcal prostaglandin production were investigated.The C. neoformans CYP450 genes were cloned into pET28 and into a pETDuet plasmid into which the CPR had already been cloned. These plasmids were used to investigate the expression of the CYP450s as well as the CPR using E. coli BL21 (DE3) as well as strains carrying plasmids for the co-expression of tRNAs that are rare in E. coli or for the molecular chaperones groES and groEL. The CO difference spectra of all the cloned CYP450s did not produce the expected absorbance peak at 450 nm. However, SDS-PAGE analysis indicated possible expression with protein bands which corresponded with the expected sizes. Three of the CYP450s gave acceptable expression when they were expressed using just pET28 without co-expression of the additional tRNA or molecular chaperones. The co-expression of C. neoformans CYP450 with groES-groEL molecular chaperones eliminated low molecular weights protein bands produced in the case of the CYP450s and the CPR.The C. neoformans CYP450s co-expressed with the CPR and groES-groEL molecular chaperones had barely detectable or no activity when tested with arachidonic acid and hexylbenzoic acid. The challenges encountered with heterologous expressions of C. neoformans CYP450s in an E. coli host, could be resolved using an alternative host such as Saccharomyces cerevisiae.

[发布机构] University of the Free State