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Tailored chemical complexity through evolution-inspired synthetic biology

Periodic Reporting for period 3 - SynPlex (Tailored chemical complexity through evolution-inspired synthetic biology)

Reporting period: 2020-08-01 to 2022-01-31

1. In this project, we address significant gaps in our understanding of trans-acyltransferase polyketide synthases (trans-AT PKSs), complex multimodular biosynthetic enzymes that generate bioactive polyketide natural products in bacteria. These gaps include their natural evolution, biochemical features (substrate specificity), structural features, protein-protein interactions/recognition, and protein dynamics. Such understanding is crucial to realizing the overarching aim of the project, namely constructing functional artificial PKSs. The design of new trans-AT PKSs is a challenging task, requiring experimentation to determine whether the inherent catalytic flexibility is sufficient for generating hybrid “mosaic” enzyme systems and their corresponding products.
2. The project provides a discovery- and a bacterial production platform for novel bioactive substances for which no economic chemical syntheses are available due to their complex molecular structures. Access to such molecules is crucial for their further development as drug candidates to fight the imminent drug resistance crisis (antibiotics/anti-cancer therapeutics). As trans-AT PKSs incorporate unparalleled diversity in chemical space and naturally evolve by forming hybrid enzymes, harnessing their combinatorial potential presents an unprecedented opportunity for the discovery and engineering of new pharmaceutically-relevant chemical space.
3. In the framework of this project, methods for renewable production of complex bioactive natural products (both known and novel) will be developed. The SynPlex project aims to understand the principles of how modular enzyme reorganization during natural evolution resulted in metabolic complexity. These evolutionary principles will be applied to the development of general synthetic biology modules to enable future access to complex, synthetically challenging metabolites. Therefore, we aim to gain fundamental insights into the function of multi-domain enzymes and biosynthetic modules and to harness the mechanisms giving rise to their metabolic complexity. By utilizing the versatility of evolution-based enzyme design for large multifunctional proteins, one could generate an efficient, predictive, and adaptable engineering strategy for modular enzymes. To realize this goal, the project aims to (i) identify global patterns of PKS evolution across bacteria, (ii) identify PKS enzymes and modules with new functions to create a synthetic biology toolbox, (iii) develop a bacterial production host for hybrid PKSs, and (iv) to characterize the recombinant polyketides.
Initially, we performed a global analysis of evolutionary patterns for more than 1,700 trans-AT PKSs that revealed extensive recombination as the main mechanism for metabolic diversification, supporting one of the main hypotheses of this project (manuscript submitted). These data suggest that module block recombination between different pathways is a major mechanism of chemical diversification for these enzymes. This work has facilitated the generation of a computational algorithm to detect trans-AT hybrids (transPACT). In addition to transPACT, we developed an automated genome-to-structure prediction tool TransATor ( which is in use by the natural product research community, and successfully applied it to PKS discovery. TransATor and transPACT greatly accelerate the identification of structurally novel bioactive molecules, as demonstrated by the rapid isolation of five structurally diverse new polyketides based on genomic predictions. These initial discoveries also led us to identify Gynuella sunshinyii as a talented producer whose genome encodes six trans-AT PKS systems, and five distinct products have been characterized.
In an effort to prioritize small trans-AT PKSs as research model systems, candidate assembly lines were selected based on the global analysis described above. Of these minimalistic systems, the construction of the toblerol and legioliulin clusters on expression plasmids has been completed. We further identified the medium-sized oocydin PKS as a trans-AT system that is highly compatible with E. coli expression. Complementary work involved the development of non-E. coli bacterial strains as suitable expression hosts. We focused on Serratia plymuthica, the producer of oocydin and G. sunshinyii, a “superproducer” of six trans-AT PKS products. We succeeded in constructing genetic mutants carrying an inactivated PKS pathway in both bacteria.
Another highly successful tangent of the project resulted in the identification of 10 new modules with distinct architectures that serve as candidates for new enzymology, of which five were biochemically characterized in vitro. The characterized enzymatic domains are responsible for the introduction of the following moieties: mid-chain malonyl- and glycolyl-esters for polyketide chains with inserted oxygens, oximes, fatty acid starters, chlorine substituents, and branched esters.
We performed extensive PKS resurrection experiments to test the compatibility of foreign termination modules with the bacillaene PKS to determine optimal fusion sites resulting in 14 hybrid PKSs. The resultant polyketide stalling was evaluated through the characterization and quantification of prematurely released intermediates in MS-based assays. Termination modules from phylogenetically related hosts showed improved offloading of bacillaene and the analysis of fusion points indicated that joining PKSs between ACPs allowed for optimal communication between the modules. To enable the future exchange of larger cluster regions, further PKS resurrection experiments were conducted by replacing module 5 of the bacillaene PKS from Bacillus subtilis with the corresponding module from the orthologous cluster in Bacillus amyloliquefaciens. Wild type levels of bacillaene were detected by LC-MS and exchange experiments with modules from more distantly related clusters are underway.
The results obtained thus far have provided broad insights into the evolution, biochemistry, and biosynthetic products of trans-AT PKSs. Progress beyond the state of art includes:

-Identification of a general mechanism of PKS evolution that resembles engineering strategies that synthetic biologists aspire to, i.e. the formation of hybrid polyketides by merging components from different biosynthetic pathways.
-Computational community tools, including TransATor with widespread applications in drug discovery and the prediction of biosynthetic pathways and natural products.
-A biochemically characterized set of PKS modules introducing a wide range of non-canonical moieties into polyketides, including two modules with potential for polyketide tagging.
-Accelerated discovery of bioactive polyketides with unprecedented skeletons.
-Discovery of novel prolific producers of polyketides.

In the final project stage, we will shift our focus to the production of hybrid polyketides based on identified evolutionarily preferred recombination sites.

Excitingly, beyond the originally proposed work, we discovered a synthetic biology methodology to create peptides and proteins with highly coveted modifications. These now allow the introduction of polyketide-like extended-backbone building blocks into ribosomal peptides (published in Science). In addition, we accomplished the in vivo production of hypermodified peptides that contain the largest number of modifications known to date, including a ribosomal product with 24 D-amino acids (Nature Chemistry). Further results of the SynPlex project were published in many other high-ranking journals, including Nature Microbiology, Nature Chemical Biology, Proc. Natl. Acad. Sci. U. S. A., Angewandte Chemie (3x), and ACS Chemical Biology. In addition, we published an extensive review on biosynthetic enzymology in Nature Reviews Chemistry.