Biosynthetic pathways for natural products have explored a vast chemical ‘space’ during evolution but synthetic biologists seek to expand this space even further. Modular architectures of biosynthetic pathways seem to favor evolutionary diversification – they also invite synthetic biologists to reshuffle and engineer their parts into non-natural arrangements. Structurally tailored natural products resulting from such efforts could potentially benefit medicinal chemistry since natural products often show biological activities. While the pioneering works of natural product engineers have afforded many promising examples of ‘unnatural natural products’, they have also highlighted a need for more straightforward and reliable engineering tools.
We investigate the modular pathways biosynthesizing nonribosomal peptides, such as the antibiotics gramicidin S and penicillin or the immunosuppressant cyclosporin. We aim to develop more robust methods for repurposing nonribosomal peptide synthetases (NRPSs). Our efforts build on mechanistic studies of engineered synthetases, novel high throughput screening methods and their application in laboratory evolution experiments. Directed evolution recapitulates the evolutionary process described by Charles Darwin in a targeted and accelerated fashion and may allow creation of artificial peptide synthetases rivaling the catalytic prowess of natural enzymes.
Ongoing Projects
On the molecular level, nonribosomal peptide synthetases (NRPSs) are chains of enzymatic domains acting concertedly to assemble a peptide from amino acid building blocks. When enzyme engineers swap domains or modify preferences for building blocks, protein structures may be disrupted or individual steps slow down and peptide formation stalls. We analyse engineered NRPSs in order to identify kinetic bottlenecks and learn from failure. For this purpose, we employ spectrophotometric assays and UPLC-MS to identify reaction products, side products and intermediates bound to the enzyme. We have developed the HAMA assay to record specificity profiles of NRPS for dozens of substrates in parallel. Kinetic profiles are analysed in light of structural models. In the next step, kinetic bottlenecks can perhaps be removed by site-directed mutagenesis or directed evolution to generate more efficient designer NRPSs.
Evolutionary protocols can solve immensely complex problems, such as the redesign of an assembly line natural product synthetase, as long as few good solutions can be effortlessly distinguished from a large number of bad solutions. In a directed evolution experiment, incremental improvements accumulate in iterative rounds of mutagenesis and screening until a desired property has been achieved. The ability to screen large numbers of mutants is crucial for this approach. We explore innovative high throughput screens for NRPS activity based on lab-on-a-chip devices, cell surface display and fluorescence activated sorting as tools for directed evolution experiments.
We aim to perform combinatorial biosynthesis of peptides by constructing a DNA-programmable, biocatalytic peptide synthesizer that we call the NONRIBOSOME. DNA has been used to arrange enzymes in catalytic cascades where proximity enhances catalytic throughput. This effect is particularly pronounced in enzymatic assembly lines such as nonribosomal peptide synthetases, where intermediates of the peptide formation reaction are covalently tethered to the protein (Huang et al., 2020). We have split apart a nonribosomal enzyme and reassembled the functional complex on a DNA strand. By using DNA as a template, we aim to program NRPS modules for the synthesis of novel peptides which may, compared to nonribosomal natural products, have better activity, fewer side effects, or avoid antimicrobial resistance.
Contact
Hajo Kries
Prof. Dr.Head of the Technical Biochemistry Department