Periodic Reporting for period 3 - MIAMi (Refactoring monoterpenoid indole alkaloid production in microbial cell factories)
Reporting period: 2022-01-01 to 2023-06-30
Plants produce some of the most potent human therapeutics and have been used for millennia to treat illnesses. Despite the huge repertoire of plant-derived pharmaceuticals, most of these products do not make it to the market because they are found in minute quantities in plants, they are difficult to extract, and there is limited knowledge about their biosynthetic pathways, making them underrepresented in recently introduced medicines. This is the case for monoterpenoid indole alkaloids (MIAs), including potent anti-cancer drugs vinblastine and vincristine, and anti-Alzheimer tabersonine exemplifying established MIA therapeutics on market.
The mission MIAMi is to develop new tools and methodologies to discover complex biosynthetic pathways in plants and optimize their production in microbes. MIAMi consortium will implement a new integrated omics-based platform technology to uncover MIA biosynthetic pathways and tools to enable the refactoring of > 1,000 biosynthetic pathways of promising new anti-cancer and antipsychotic drug leads in yeast, ultimately enabling technologies for both discovery of new natural product biosynthetic pathways, and sustainable production, testing and development of new plant-derived pharmaceuticals.
The mission MIAMi is to develop new tools and methodologies to discover complex biosynthetic pathways in plants and optimize their production in microbes. MIAMi consortium will implement a new integrated omics-based platform technology to uncover MIA biosynthetic pathways and tools to enable the refactoring of > 1,000 biosynthetic pathways of promising new anti-cancer and antipsychotic drug leads in yeast, ultimately enabling technologies for both discovery of new natural product biosynthetic pathways, and sustainable production, testing and development of new plant-derived pharmaceuticals.
We have made very good progress for this reporting period, and all work-package activities are on track. Below we report the main R&D, exploitation and dissemination activities and achievements for the third reporting period.
WP2:
The WP2 research activities led to the discovery of new MIA biosynthetic enzymes. The main objective related to identification of gene encoding alstonine synthase and rauwolscine synthase (yohimbine synthase more specifically) has been achieved by combining multiple omics approaches in Rauwolfia tetraphylla and candidate gene prediction though “guilt by association” and machine learning based approaches. While we did not engage the GWAS on R. tetraphylla due to low access to the genetic diversity of this plant we have mitigated this issue by sequencing multiple genomes and transcriptomes of Apocyanaceae and other MIA producing plants. During the last 18 months, this has enabled us to identify orthologues of already known MIA biosynthetic potentially displaying distinct catalytic efficiency that could be valorized in the microbial cell factories developed in the MIA project to increase the biosynthetic fluxes. In addition, we have also identified other enzyme candidates with putative yohimbane biosynthetic activities. All these new omics resources, together with upcoming metabolomics data and machine learning based gene prediction, pave the way for the identification of new enzymes involved in other MIA biosynthesis.
W3:
In collaboration between UCPH and UT involving a short-term exchange of PhD student, the possible effects on metabolite production and localization in R. tetraphylla as consequence of caterpillar attack was investigated. Plants at UT were exposed to caterpillars, leaves were harvested and imprinted, shipped to UCPH where they were analyzed by DESI-MSI. No noticeable effect was observed in the distributions of MIAs in the areas that were attacked by caterpillars.
WP4:
During the last reporting period, DLX has completed all the data management related to depositing sequencing data for Vinca minor, Catharanthus roseus v.2.1 Voacanga thouarsii, Rauvolfia tetraphylla v.1.0 Pachypodium lamerei, Stemmadenia litoralis and the 18 French Guiana Apocynaceae species.
WP5:
During the last reporting period MIAMi partner DTU has worked with partner UT to complete the identification of pathway enzymes, from which libraries (>1,00 designs) of strain designs have been constructed. Based on stochastic sampling of colonies with different designs, machine learning models have been trained to tested for prediction of MIA production based on genotypic designs. This has been successfully demonstrated in yields models which can predict production from genotypes with >80% correlation coefficient between observed and predicted values. The study has been submitted for peer-review and is also available from BioRXiv (https://www.biorxiv.org/content/10.1101/2023.06.18.545451v1). In summary all deliverables and milestones pertaining to WP4 have been completed.
WP6:
Based on the close collaboration between WP5 and WP6, the WP representatives have met bi-weekly since August 2021. From the beginning of WP7, AXT, UT and DTU teams have also met monthly or bi-weekly since November 2021. This communication has secured the dialogue between the laboratory and the assessment teams, which was necessary for information to flow between the groups in an iterative process.
For this last period, scale-up of production at 2L scale has been performed at UT, guided by results from DTU at small scale. The work done at 2L bioreactors was extensive to first overcome the unexpected huge loss of productivity and secondly characterize the numerous strains for feedback on molecular engineering. This led to a scale-up at pilot scale by UT and DTU, producing few grams of alstonine and vindoline sent to AXT for WP7 issues, as 2L broths. Rauwolscine production at pilot scale need further optimization of strains and 2L process on the same pattern of alstonine. Halo-MIA production scale-up is ongoing.
Based on the experiments performed at a 30L scale, a maximum titer of 20-25 mg/L was obtained, and an impact assessment was performed as a part of deliverable D6.4. To accomplish the deliverable, two scenario’s and corresponding process schemes were simulated. The base case of 25 mg/L and an optimistic case (hypothetical) of 1 g/L. This range was picked to be consistent with the impact assessment of the bench scale data D6.2.
WP7:
During the last 18 months, MIAMi consortium has performed in silico analysis of potential MIA drug targets with a focus on GPCR from humans - a major class of drug targets of approved small-molecule drugs. From this we have identified >7 GPCRs to be responding to MIAs. Next we have expressed MIA drug targets in yeast and mammalian cells and performed bioactivity testing of natural alstonine and rauwolscine produced in MIAMi cell factories, identifying ADRA2A and 5HT2C receptors as drug targets for alstonine. These results are currently under peer-review. Due to delays on cell factory design and scale-up, MIAMi consortium did not complete purification and shipment of >10 mg natural and new-to-nature MIAs to subcontractors for further in vitro and in vivo bioactivity testing. These assays will be conducted during Q3-Q4 2023. a. ½ page of what have you contributed with and what was achieved in the last 18 months. Please also write what wasn’t achieved and what is the reason for that.
WP8:
During the last 18 months, MIAMi consortium partners have met for workshop and final reporting meetings, as well as had numerous (50+) virtual meetings to coordinate WP activities. Furthermore, MIAMi partners have presented our EU-financed research in oral and poster formats at numerous conferences, as well as published the research in open-access high-impact journals in full compliance with the grant agreement. Last but not least, a start-up called Biomia (www.biomia.com) has been incorporated to commercialize the intellectual property filed by MIAMi partner DTU.
WP2:
The WP2 research activities led to the discovery of new MIA biosynthetic enzymes. The main objective related to identification of gene encoding alstonine synthase and rauwolscine synthase (yohimbine synthase more specifically) has been achieved by combining multiple omics approaches in Rauwolfia tetraphylla and candidate gene prediction though “guilt by association” and machine learning based approaches. While we did not engage the GWAS on R. tetraphylla due to low access to the genetic diversity of this plant we have mitigated this issue by sequencing multiple genomes and transcriptomes of Apocyanaceae and other MIA producing plants. During the last 18 months, this has enabled us to identify orthologues of already known MIA biosynthetic potentially displaying distinct catalytic efficiency that could be valorized in the microbial cell factories developed in the MIA project to increase the biosynthetic fluxes. In addition, we have also identified other enzyme candidates with putative yohimbane biosynthetic activities. All these new omics resources, together with upcoming metabolomics data and machine learning based gene prediction, pave the way for the identification of new enzymes involved in other MIA biosynthesis.
W3:
In collaboration between UCPH and UT involving a short-term exchange of PhD student, the possible effects on metabolite production and localization in R. tetraphylla as consequence of caterpillar attack was investigated. Plants at UT were exposed to caterpillars, leaves were harvested and imprinted, shipped to UCPH where they were analyzed by DESI-MSI. No noticeable effect was observed in the distributions of MIAs in the areas that were attacked by caterpillars.
WP4:
During the last reporting period, DLX has completed all the data management related to depositing sequencing data for Vinca minor, Catharanthus roseus v.2.1 Voacanga thouarsii, Rauvolfia tetraphylla v.1.0 Pachypodium lamerei, Stemmadenia litoralis and the 18 French Guiana Apocynaceae species.
WP5:
During the last reporting period MIAMi partner DTU has worked with partner UT to complete the identification of pathway enzymes, from which libraries (>1,00 designs) of strain designs have been constructed. Based on stochastic sampling of colonies with different designs, machine learning models have been trained to tested for prediction of MIA production based on genotypic designs. This has been successfully demonstrated in yields models which can predict production from genotypes with >80% correlation coefficient between observed and predicted values. The study has been submitted for peer-review and is also available from BioRXiv (https://www.biorxiv.org/content/10.1101/2023.06.18.545451v1). In summary all deliverables and milestones pertaining to WP4 have been completed.
WP6:
Based on the close collaboration between WP5 and WP6, the WP representatives have met bi-weekly since August 2021. From the beginning of WP7, AXT, UT and DTU teams have also met monthly or bi-weekly since November 2021. This communication has secured the dialogue between the laboratory and the assessment teams, which was necessary for information to flow between the groups in an iterative process.
For this last period, scale-up of production at 2L scale has been performed at UT, guided by results from DTU at small scale. The work done at 2L bioreactors was extensive to first overcome the unexpected huge loss of productivity and secondly characterize the numerous strains for feedback on molecular engineering. This led to a scale-up at pilot scale by UT and DTU, producing few grams of alstonine and vindoline sent to AXT for WP7 issues, as 2L broths. Rauwolscine production at pilot scale need further optimization of strains and 2L process on the same pattern of alstonine. Halo-MIA production scale-up is ongoing.
Based on the experiments performed at a 30L scale, a maximum titer of 20-25 mg/L was obtained, and an impact assessment was performed as a part of deliverable D6.4. To accomplish the deliverable, two scenario’s and corresponding process schemes were simulated. The base case of 25 mg/L and an optimistic case (hypothetical) of 1 g/L. This range was picked to be consistent with the impact assessment of the bench scale data D6.2.
WP7:
During the last 18 months, MIAMi consortium has performed in silico analysis of potential MIA drug targets with a focus on GPCR from humans - a major class of drug targets of approved small-molecule drugs. From this we have identified >7 GPCRs to be responding to MIAs. Next we have expressed MIA drug targets in yeast and mammalian cells and performed bioactivity testing of natural alstonine and rauwolscine produced in MIAMi cell factories, identifying ADRA2A and 5HT2C receptors as drug targets for alstonine. These results are currently under peer-review. Due to delays on cell factory design and scale-up, MIAMi consortium did not complete purification and shipment of >10 mg natural and new-to-nature MIAs to subcontractors for further in vitro and in vivo bioactivity testing. These assays will be conducted during Q3-Q4 2023. a. ½ page of what have you contributed with and what was achieved in the last 18 months. Please also write what wasn’t achieved and what is the reason for that.
WP8:
During the last 18 months, MIAMi consortium partners have met for workshop and final reporting meetings, as well as had numerous (50+) virtual meetings to coordinate WP activities. Furthermore, MIAMi partners have presented our EU-financed research in oral and poster formats at numerous conferences, as well as published the research in open-access high-impact journals in full compliance with the grant agreement. Last but not least, a start-up called Biomia (www.biomia.com) has been incorporated to commercialize the intellectual property filed by MIAMi partner DTU.
The projects is on track, we gathered the data needed to meet the deliverables listed under the GA.