Chances are that if you have ever been sick with a bacterial infection, you have a bacterial metabolite called a ‘polyketide’ to thank for your restored good health. In addition to antibacterial properties, polyketides exhibit anti-fungal, anti-cancer, immunosuppressant and other useful activities, and so are often exploited as drugs to treat both people and animals¹. Examples you might know include the antibiotic erythromycin A, the anti-fungal amphotericin and the cytotoxic ixabepilone. Although in some cases it is possible to use polyketides directly from the bug (as with erythromycin A and amphotericin), in other instances, it is necessary to adapt the molecules’ physical and medicinal properties so they function better in our bodies (as with ixabepilone², a derivative of the natural product epothilone B). Remarkably, work on certain polyketides has even demonstrated that large portions of the structures are superfluous, and thus that simplified versions can be just as effective as the parent molecules³. This is perhaps not surprising when you consider that the molecules did not evolve to work in human and animal bodies, but rather for bacterial purposes such as defense and signaling⁴ in the soil and the sea.

The classical way to create new polyketides (such as ixabepilone) is to rework existing structures using synthetic chemistry, an approach called as semi-synthesis⁵. However, this strategy is both challenging and costly, and ill-adapted to altering the core skeleton of the structures. An attractive alternative was suggested as long ago as 1990 with the discovery of the first set of genes encoding the biosynthesis of erythromycin⁶. Remarkably, the gigantic multi-enzymes involved in assembling the polyketide – the polyketide synthases (PKSs) – exhibit a modular architecture, in which each task in building the molecule is carried out by a dedicated functional domain⁷. In essence, PKSs function like car assembly lines but on a molecular scale, each domain doing its job, and then handing the growing polyketide to the next domain in line. The domains are organized into units called modules (hence these systems are referred to as ‘modular PKSs’), such that each module is responsible for extending the chain with one building block, and its subsequent chemical tailoring to introduce diverse functional groups. In some cases, a single module constitutes a PKS protein (called a subunit), but more commonly, subunits contain multiple modules.

The modular organization of the PKSs offers the tantalizing prospect of creating bespoke polyketides by genetic engineering (now commonly referred to as ‘synthetic biology’); in principle, modification of domains, modules or even entire subunits within the PKS, should lead to predictable changes in the product structures⁸. Disappointingly, however, decades of efforts in laboratories around the world, which have largely targeted PKS domains, have met with limited success. In the majority of cases, the engineered PKS exhibited greatly reduced activity, or the assembly line broke down completely⁸. While the molecular explanations for these failures are not entirely clear, recent structural analysis of intact modules^9–11 has revealed that PKS domains are far from actually being modular, in the sense of existing as fully independent units. Instead, domains are highly interdependent entities, suggesting that we should rather employ whole modules, or even multi-modular subunits, as the basic ‘biobricks’ in synthetic biology in order to maintain these key relationships.

In our study, we aimed to show that a module-based engineering approach could be used to produce structurally-simplified polyketides, as a complement to synthetic chemistry. For this, we targeted a PKS system giving rise to some of the largest of all known polyketides – the 51-membered anti-cancer stambomycins – in the soil bacterium Streptomyces ambofaciens. In particular, we set out to remove 7 whole modules from the assembly line, to generate a series of ‘low-fat’ 37-membered mini-stambomycins. By testing various state-of-the-art strategies, including varying the domain composition of the modules, we ultimately succeeded in making the target mini-stambomycins, at yields only 8-fold reduced relative to the wild type stambomycins. Our results thus allowed us to identify factors that influence the efficiency of such engineering, as well as areas for further improvement. Finally, we directly compared two commonly-used genome editing techniques, PCR-targeting and CRISPR-Cas9, for PKS synthetic biology, showing that CRISPR-Cas9 is the more effective of the two. In conclusion, we hope that these results will be of utility for future efforts by the polyketide community to force other over-sized polyketides to lose weight.

For more detailed information on our work, please see the original article: “Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins” in Nature Communications (https://doi.org/10.1038/s41467-022-27955-z).

References

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