Artificial reconstruction of two cryptic angucycline antibiotic biosynthetic pathways

Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.     

Protein Engineering of the Progesterone Hydroxylating P450‐Monooxygenase CYP17A1 Alters Its Regioselectivity

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17?C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active‐site residues were identified and systematically mutated to alanine. In whole‐cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α‐hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.     

The Oxygen Dilemma: A Severe Challenge for the Application of Monooxygenases?

Monooxygenases are promising catalysts because they in principle enable the organic chemist to perform highly selective oxyfunctionalisation reactions that are otherwise difficult to achieve. For this, monooxygenases require reducing equivalents, to allow reductive activation of molecular oxygen at the enzymes' active sites. However, these reducing equivalents are often delivered to O₂ either directly or via a reduced intermediate (uncoupling), yielding hazardous reactive oxygen species and wasting valuable reducing equivalents. The oxygen dilemma arises from monooxygenases' dependency on O₂ and the undesired uncoupling reaction. With this contribution we hope to generate a general awareness of the oxygen dilemma and to discuss its nature and some promising solutions.     

Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol

Hyroxytyrosol (HTyr), an important phenol present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anticarcinogenic activities. This work describes the synthesis of HTyr via double hydroxylation of 2-phenylethanol (PEA) employing toluene monooxygenases (TMOs) as biocatalysts. Wild-type TMOs were initially evaluated for their ability to oxidise PEA and structurally-related substrates, providing better understanding of the factors responsible for controlling the regiospecificity. Both the length of the alkyl side chain and the presence of the hydroxyl group were found to influence the activity, possibly by interfering with the substrates’ entrance into the active site. Directed evolution of toluene-4-monooxygenase of Pseudomonas mendocina KR1 led to the discovery of variant TmoA S395C with a 15-fold increase in PEA hydroxylation rate. Saturation-mutagenesis at position TmoA I100 resulted in the finding of novel HTyr-producing variants I100A, I100S and I100G.