Publications - Cell and Metabolic Biology

Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level-1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.

A bottleneck in the development of new anti-cancer drugs is the recognition of their mode of action (MoA). We combined metabolomics and machine learning to predict MoAs of novel anti-proliferative drug candidates, focusing on human prostate cancer cells (PC-3). As proof of concept, we studied 38 drugs with known effects on 16 key processes of cancer metabolism, profiling low molecular weight intermediates of the central carbon and cellular energy metabolism (CCEM) by LC-MS/MS. These metabolic patterns unveiled distinct MoAs, enabling accurate MoA predictions for novel agents by machine learning. We validate the transferability of MoA predictions from PC-3 to two other cancer cell models and show that correct predictions are still possible, but at the expense of prediction quality. Furthermore, metabolic profiles of treated cells yield insights into intracellular processes, exemplified for drugs inducing different types of mitochondrial dysfunction. Specifically, we predict that pentacyclic triterpenes inhibit oxidative phosphorylation and affect phospholipid biosynthesis, as supported by respiration parameters, lipidomics, and molecular docking. Using biochemical insights from individual drug treatments, our approach offers new opportunities, including the optimization of combinatorial drug applications.

In addition to jasmonoyl-isoleucine (JA-Ile), a well-established signaling molecule for plant growth and defense, its precursor, cis-12-oxo-phytodienoic acid (OPDA), is thought to possess independent signaling functions. Its perception in vascular plants is still uncharacterized. Several OPDA functions in Arabidopsis were inferred from a mutant that is affected in the function of the OPDA REDUCTASE3 (OPR3), catalyzing the conversion of OPDA within peroxisomes. Recently, opr3 plants were found to accumulate JA-Ile via a cytosolic OPR2-mediated bypass. Given the uncoupling of OPDA and JA biosynthesis in the JA-deficient mutant opr2opr3, potential OPDA signaling was investigated by a transcriptome approach comparing wild type, opr2opr3 and the JA- and OPDA-deficient mutantallene oxide synthase. Dissecting the wound response of seedlings revealed that OPDA lacked a transcriptional signature, and that previously characterized OPDA-response genes were wound-induced independently of OPDA. Exogenous application of OPDA to opr2opr3 seedlings led to JA-Ile formation and signaling even in absence of OPR2 and OPR3 and resulted in activation of sulfur assimilation. These divergent responses to endogenously synthesized and applied OPDA suggest a compartmentalization of endogenous OPDA which was investigated by a trans-organellar complementation approach. OPR3 complemented the opr2opr3 mutant in terms of fertility and wound-induced JA-Ile production irrespective of its subcellular localization. In vitro enzymatic activity of OPR3, however, showed conversion of OPDA and 4,5-didehydro-JA (4,5-ddh-JA), therefore not allowing to conclude which compound is translocated. Dissecting the conversion of either OPDA or 4,5-ddh-JA by OPR2 and OPR1 organelle variants pointed to a strong OPDA compartmentalization supporting its lacking signaling capacity.

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In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

A bottleneck in the development of new anti‐cancer drugs is the recognition of their mode of action (MoA). Metabolomics combined with machine learning allowed to predict MoAs of novel anti‐proliferative drug candidates, focusing on human prostate cancer cells (PC‐3). As proof of concept, 38 drugs are studied with known effects on 16 key processes of cancer metabolism, profiling low molecular weight intermediates of the central carbon and cellular energy metabolism (CCEM) by LC‐MS/MS. These metabolic patterns unveiled distinct MoAs, enabling accurate MoA predictions for novel agents by machine learning. The transferability of MoA predictions based on PC‐3 cell treatments is validated with two other cancer cell models, i.e., breast cancer and Ewing\'s sarcoma, and show that correct MoA predictions for alternative cancer cells are possible, but still at some expense of prediction quality. Furthermore, metabolic profiles of treated cells yield insights into intracellular processes, exemplified for drugs inducing different types of mitochondrial dysfunction. Specifically, it is predicted that pentacyclic triterpenes inhibit oxidative phosphorylation and affect phospholipid biosynthesis, as confirmed by respiration parameters, lipidomics, and molecular docking. Using biochemical insights from individual drug treatments, this approach offers new opportunities, including the optimization of combinatorial drug applications.

Decades of research on the infamous antinutritional steroidal glycoalkaloids (SGAs) in Solanaceae plants have provided deep insights into their metabolism and roles. However, engineering SGAs in heterologous hosts has remained a challenge. We discovered that a protein evolved from the machinery involved in building plant cell walls is the crucial link in the biosynthesis of SGAs. We show that cellulose synthase–like M [GLYCOALKALOID METABOLISM15 (GAME15)] functions both as a cholesterol glucuronosyltransferase and a scaffold protein. Silencing GAME15 depletes SGAs, which makes plants more vulnerable to pests. Our findings illuminate plant evolutionary adaptations that balance chemical defense and self-toxicity and open possibilities for producing steroidal compounds in heterologous systems for food, cosmetics, and pharmaceuticals.

Methylerythritol cyclodiphosphate (MEcPP) is an intermediate in the biosynthesis of isoprenoids in plant plastids and in bacteria, and acts as a stress signal in plants. Here, we show that MEcPP regulates biofilm formation in Escherichia coli K-12 MG1655. Increased MEcPP levels, triggered by genetic manipulation or oxidative stress, inhibit biofilm development and production of fimbriae. Deletion of fimE, encoding a protein known to downregulate production of adhesive fimbriae, restores biofilm formation in cells with elevated MEcPP levels. Limited proteolysis-coupled mass spectrometry (LiP-MS) reveals that MEcPP interacts with the global regulatory protein H-NS, which is known to repress transcription of fimE. MEcPP prevents the binding of H-NS to the fimE promoter. Therefore, our results indicate that MEcPP can regulate biofilm formation by modulating H-NS activity and thus reducing fimbriae production. Further research is needed to test whether MEcPP plays similar regulatory roles in other bacteria.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.