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Tailored chemical complexity through evolution-inspired synthetic biology

Periodic Reporting for period 4 - SynPlex (Tailored chemical complexity through evolution-inspired synthetic biology)

Reporting period: 2022-02-01 to 2022-07-31

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.
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. 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 (http://transator.ethz.ch/) which is widely used by the natural product research community. TransATor and transPACT greatly accelerate the identification of structurally novel bioactive molecules, as demonstrated by the rapid isolation of 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. 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.

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 six 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, alpha-hydroxylations, and branched esters. We performed extensive PKS resurrection experiments in Bacillus and Serratia to test the compatibility of foreign termination modules with the bacillaene PKS to determine optimal fusion sites resulting in >20 hybrid PKSs. After initial failures, we conducted an analysis of coevolving residues across PKS domains by statistical coupling analysis, which suggested that the fusion sites had to be slightly shifted. Gratifyingly, a range of hybrid PKS constructs now showed productivity, yielding the predicted polyketides. Useful for rapid engineering and polyketide detection was a Serratia platform involving the previously identified chlorination module, which introduced a chlorine atom into the target polyketide that could be used as isotope tag for mass spectrometry.

A second important result in biosynthetic engineering came from the unexpected discovery of a remarkable non-canonical splicing reaction that installs beta-ketoamides (alpha-keto-beta-amino acids) into ribosomal peptides. The same moiety was known before as an oxidative polyketide synthase-based modification in nonribosomal peptides. We showed that this modification is widespread among bacteria, can be utilized to rationally introduce at least 15 different ketoamide moieties into gene-encoded peptides and even proteins, can be exploited to introduce an autonomously generated unique reaction site for in vivo and in vitro protein labeling, and generates potent protease inhibitors. Two patent applications resulted from these findings.
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, including janustatins with low-nanomolar activity.
-Discovery of several novel rich producers of polyketides.
-Rationale for creating biosynthetically functional hybrid trans-AT PKSs

Progress on peptide and protein synthetic biology
-First method to introduce a wide range (at least 15) of beta-amino acids into gene encoded peptides and proteins in vitro and in vico.
-First in vivo protein labeling method utilizing an autonomously generated unique labeling site.
-Discovery of a new, widespread class of potent proteinase-inhibting natural products.
A multifunctional modular polyketide synthase