Interfakultäres Institut für Mikrobiologie und Infektionsmedizin

Research projects

Polyketide engineering

Polyketides (PK) are important secondary metabolites, widely used as antibiotics, antifungals, and drugs for other clinical applications. PK compounds are biosynthesized by complex multifunctional enzymes with modular architecture, the polyketide synthases (PKSs).

In typical modular type I PKSs, acyltransferase domains (ATs) select and load malonyl-CoA derived precursors onto acyl carrier proteins (ACPs). The AT domains are either embedded in the PKS (cis-ATs) or encoded by distinct genes and function in trans as discrete enzymes (trans-ATs). After the AT-dependent loading step, ketosynthases (KSs) catalyze the condensation of ACP-linked extender units. Optional domains, such as ketoreductases (KRs), dehydratases (DHs), enoylreductases (ERs), or methyltransferases (MTs) further process the polyketide chain. In most cases, the product is released from the assembly line by a thioesterase (TE) domain and modified by post-PKS tailoring enzymes.

Notably, the AT domain determines which extender unit is incorporated into the growing polyketide chain. Consequently, the extender unit specificity of the AT cumulatively dictates large portions of the final polyketide structure, and could be leveraged to improve the pharmacological properties of polyketide-based chemical entities through alteration of the precursors and/or the AT(s). These features make the ATs attractive for rational engineering of polyketide assembly lines.

For trans-AT type PKSs, only few examples of assembly lines utilizing nonmalonyl-CoA extender units have been reported. One of the best studied biosynthetic pathways, which involves a trans-AT type PKS and incorporates a branched unit into its product, is that of the antibiotic kirromycin from Streptomyces collinus Tü 365.

In our studies, we were particularly interested in the unusual and complex trans- and cis-PKS/NRPS assembly line of kirromycin and demonstrated that the pathway recruits two discrete ATs, KirCI and KirCII (Figure). We have shown that KirCI provides the biosynthetic machinery with malonate. The second AT, KirCII, was verified to transfer ethyl malonate onto the kirromycin ACP5 (Kirr-ACP5) and thus introduces the C-28 ethyl branch into the backbone of the antibiotic. More recently, in vitro studies revealed that this enzyme accepts other nonmalonyl-CoA substrates such as allylmalonyl-CoA, propargylmalonyl-CoA, and to a lesser extent azidoethyl- and phenylmalonyl-CoA.

Alkyne-containing molecules, such as propargyl-derived compounds provide functional groups for further derivatization by “click chemistry”. The term “click chemistry” describes rapid, selective, high-yield reactions of chemical moieties under mild, aqueous conditions.

Encouraged by the promising spectrum of substrate specificity of KirCII, we decided to study the flexibility of the discrete AT in vivo to exploit its features for polyketide engineering.

In our kirromycin bioderivatization approach, the producer strain S. collinus Tü 365 was engineered to facilitate the utilization of non-natural polyketide precursors. The activated precursors (allyl- and propargyl-malonyl-CoA) were introduced into the polyketide backbone, which resulted in the production of novel derivatives, allyl- and propargyl-kirromycin (Figure).

Our results demonstrated that after the extender unit substitution, which is accomplished relatively early in the assembly process, the downstream NRPS and PKS modules accept the un-natural side chains and continue the product biosynthesis. These findings uncover the flexibility of a mixed PKS/NRPS assembly line toward the un-natural building blocks and encourage the further exploration of trans-AT-based polyketide engineering. Therefore, the KirCII/ACP5 system may have the potential to become a valuable tool, enabling the regiospecific incorporation of chemical handles into polyketides, which can serve as anchors for “click” chemistry. The combination of this “bio-derivatization” approach and “click” chemistry methods enables the generation of novel molecular probes and analogues of compounds, which would be otherwise difficult to access by using exclusively conventional organic chemistry. Thus, our current projects focuses on the implementation of the KirCII/ACP5 tool in heterologous assembly lines to achieve structural diversity and generate novel products with improved pharmacokinetic properties or new bioactivities.

Aminoglycoside engineering

Aminoglycosides (AGs) are an old class of antibiotic compounds, mostly applied in cases of infections that are caused by Gram-negative pathogenic bacteria. In contrast to the known and established antibiotic therapy, the potential use of AGs for treatment of human genetic diseases, human immunodeficiency and viral infections was first recently discovered and reported.

The promising AG-features became a great motivation to study this class of natural products in order to understand the intracellular production process. The obtained information and its application in directed genetic engineering approaches will enable the production of AG derivatives with possibly new therapeutic properties. Hence, our research group is mainly interested in the biosynthetic- and biosynthesis-related steps of selected AGs. The gained knowledge will be used to design strategies for strain engineering as well as for directed pathway modification and AG derivatization.