Unveiling the enzymatic machinery driving an atypical cyclization of monoterpene indole alkaloids for metabolic engineering purposes – MIACYC

The MIACYC project is based on the development of an optimized pipeline for the elucidation of biosynthetic pathways of plant natural products, combining spatial metabolic analyses with transcriptomic analyses conducted at the cellular level and supported by the use of artificial intelligence. As such, the project integrated approaches of:

- in-depth annotation of the metabolome using LC-MS/MS and in silico annotation tools of plant extracts and enzymatic bioconversion media to ensure a fine characterization of AIMs. - histolocalization of AIMs by MALDI-MSI followed by cell recovery by laser capture microdissection (LCM) to determine their preferential accumulation site

- structural and stereochemical elucidations of AIMs by NMR and quantum chemistry (DFT, TD-DFT-ECD, HF/DFT-NMR).

- genome sequencing of AIM-producing plant species to support transcriptomic analyses and study the possible formation of genomic gene clusters

- transcriptomic analyses conducted at the organ, tissue (after capture) and cell (single cell RNA seq) scale to correlate AIM accumulation and gene expression

- overexpression of heterologous transcription factors regulating AIM synthesis coupled with RNA seq analysis to stimulate the expression of biosynthesis genes

- prediction of candidate genes by combining previous analyses and supported by machine learning.

- functional tests of candidate genes by overexpression in Nicotiana benthamiana or Saccharomyces cerevisiae, microsome tests or using recombinant proteins

- purification of AIM products by preparative HPLC. The results from this first series of analyses also allowed the development of AIM-producing cell factories. This bioproduction approach leverages the following approaches:

- integration of plant genes into the yeast genome using CRISPR/Cas9 to create cell factories . - feeding yeast with halogenated biosynthetic precursors to ensure the synthesis of "new-to-nature" AIMs.

- modifications of the subcellular localization of enzymes and the formation of metabolons to optimize synthesis flows and circumvent the operational limitations associated with protein expression in a different environment.

- bioreactor culture and process engineering to optimize AIM production rates by yeast.

The MIACYC project resulted in the development of a particularly efficient AIM biosynthesis pathway gene identification pipeline, based on the most recent cellular analysis techniques. This has enabled significant advances in the understanding of AIM metabolism, including:

- the identification of a cytochrome P450 (CYP71) ensuring multiple AIM cyclization reactions, particularly of the C-C and C-N type. These reactions are thus at the basis of the formation of strychnane (akuammicine), akuammilane (strictamine) and mavacuranes (epi-pleiocarpamine, main target of the project) skeletons in A. scholaris and Catharanthus roseus (published)

- the evolutionary study of the CYP71 clade explaining the appearance of C-N cyclization activities in gentianales (published)

- the demonstration of the capacity of the enzyme tabersonine 3-oxidase (T3O) to catalyze the C-N cyclization of vincadifformine to form vincamine (second target of MIACYC) (in progress)

- the scRNAseq analysis of A. scholaris combined with spatial metabolomics which led to the identification of enzymes ensuring the synthesis of major AIMs of A. scholaris (in progress) - genome sequencing of AIM-producing Apocynaceae (Vinca minor, C. roseus, Tabernaemontana elegans, Voacanga thouarsii, Rauvolfia tetraphylla) (5 published articles)

- identification of the last ajmaline synthesis enzyme (AIM of major pharmacological interest) (published) - identification of a CYP71 ensuring the epoxidation of tabersonine forming pachysiphine, precursor of the anti-Alzheimer AIM conophylline (published)

- identification of a regulatory mechanism of the central enzyme of AIM biosynthesis (strictosidine glucosidase, SGD) (unpublished)

- combination of machine learning and empirical calculation for the structural validation of the first natural trimer isolated from C. roseus (published)

- discovery of the first example of hybrid AIM associating a vobasane unit and a pyrraline unit (published) and first vobasane-aspidospermane-aspidospermane trimers isolated from Voacanga africana (published)

- the revision of the structure of melonin and identification of a new AIM (meloninane) skeleton (published) - the update of the largest spectral database dedicated to AIM (422 MS/MS spectra) and the design of a chemoinformatics tool generating specific spectral signatures of a natural product skeleton. In a second step, this knowledge was exploited to develop cell factories producing AIM.

Our main results include:

- the creation of factory cells for the bioproduction of alstonine and rauwolscine as well as their halogenated derivatives (2 published articles)

- the impact of subcellular relocation of enzymes on the AIM synthesis flux in yeast (unpublished)

From a scientific point of view, the MIACYC project has enabled the understanding of C-N cyclization mechanisms in the synthesis of alkaloids of pharmacological interest. This knowledge can be transferred to the characterization of the synthesis of other natural metabolites with similar structural characteristics. More generally, the work pipeline developed during MIACYC will also be valuable for the characterization of other key steps in the biogenesis of alkaloids or natural products of interest. In this context, BBV and BioCIS have once again joined forces to identify new unknown mechanisms of alkaloids that are crucial for generating molecules with very high added value. These mechanisms have questioned the scientific community for more than 50 years and their understanding would open the door to new strategies for the bioproduction of anticancer compounds. This work has therefore been the subject of a new funding request to the ANR and has also allowed the expansion of our network of collaborators participating in this project. The development of the pipeline for elucidating natural product biosynthesis pathways already has concrete applications since it is one of the central elements of the Horizon COMBO project (No. 101135438) which began in 2024. With the collection of samples (sponges, algae) now complete, the generation of associated multi-omic data will enable the exploitation of the MIACYC pipeline to predict and then characterize genes associated with the synthesis of marine metabolites of interest from the fall of 2025. In parallel, the progress made in the development of cell factories s producing metabolites of interest will also serve as a basis for the creation and application of bioproduction strategies for alkaloids of interest. In this context, BBV has strengthened its partnership with an industrial group based in the Centre region and requested additional funding from the Centre-Val de Loire region for the acquisition of a new 50-litre bioreactor and a dedicated centrifuge that will enable the scale-up of the production of these compounds classified in the CMR category (carcinogenic, mutagenic, toxic for reproduction). The acquisition of such equipment will therefore enable the increase in culture volume in a context of total risk control for operators and will accelerate the possibilities of transfer to the industrial partner.

Plant alkaloid diversity relies on a complex enzymatic machinery leading to unique backbones conferring specific pharmaceutical properties for humans. For a few years and in particular during this period, the medicinal use of these alkaloids has been seriously affected by supply limits caused by their poor synthesis in planta and by the overexploitation of the natural resources from which they are extracted. Unveiling the plant enzymatic cocktail synthesizing alkaloids of interest may solve these problems by allowing transferring plant pathways into heterologous platforms for an alternative supply of alkaloids through metabolic engineering. While the main alkaloid synthetic routes have been identified, it is now essential to focus on other and more original reactions ensuring the formation of less emblematic alkaloids but whose potential is high. MIACYC proposes (i) to develop an efficient pipeline for biosynthetic pathway elucidation through the identification of enzymes involved in an atypical cyclization of three potent alkaloids (vincamine, pleiocarpamine and strychnine) and (ii) to turn their production in yeast and that of their derivatives. This original elucidation procedure is based on the combination of single-cell level technical approaches studying the accumulation of both alkaloids and transcripts specifically located in different plant cell types. This will allow taking advantage of the high spatial compartmentation existing in plants accumulating alkaloids and in particular Alstonia sp. The transfer of the identified genes into yeasts will be achieved using CRISPR / Cas9 to target genome hot-spots, resulting in the creation of yeast cell factories easily cultivable in bioreactors alkaloid production. Finally, widening the synthetic biology toolbox in MIACYC will also allow combining these new reactions with known ones for production of new-to-nature alkaloids with enriched properties.