Refactoring monoterpenoid indole alkaloid production in microbial cell factories

Periodic Reporting for period 2 - MIAMi (Refactoring monoterpenoid indole alkaloid production in microbial cell factories)

Okres sprawozdawczy: 2020-07-01 do 2021-12-31

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.

We have made very good progress for this reporting period, and all work-package activities are on track, except for a few tasks for which we have applied for a minor extension of deliverable deadlines (see separate Covid-19 letter and ammenment). Below we report the main R&D, exploitation and dissemination activities and achievements for the second reporting period.

WP2:
The WP2 research activities for the second reporting period are on track and according to grant agreement. While delayed by the Covid pandemic, the functional validation of candidate genes is ongoing and will be achieved during the next six months. The main objective related to identification of gene encoding alstonine synthase and rauwolscine synthase has been achieved. The impossibility to study the genetic variation in Apocynaceae according to our initial plan has been mitigated by multi-plant genome sequencing and transcriptomics and offers new perspectives towards the identification of candidate genes.
W3:
In summary WP3 has characterized the products of enzymes that make a variety of yohimbine and heteroyohimbine stereoisomers and started characterizing products of enzymes that make related alkaloid ajmaline. Structural characterization of these enzymes provides insight into the mechanism of how they work, and provides insights into how sequence elements can be used to predict enzyme activity. Furthermore, this WP has successfully measured the localization of MIA in Rauvolfia using MS imaging, and these data are currently being prepared in a manuscript. Finally, which dovetails with the localization aspects reported, we have begun the first efforts in demonstrating protein-protein interactions among MIA enzymes
WP4:
The WP4 research activities for the second reporting period are on track and according to grant agreement. Partners carried out maintenance and enhancement of standard operating procedures (task 4.1) DNA parts (task 4.2) and databases (task 4.3). While significant progress was made in developing a second prototype of the BioCAD for the design of T-DNA vectors and EasyClone constructs. The main innovation achieved was the ability for users to perform bulk edits to up to 500 constructs simultaneously. While impacted by the Covid pandemic, activities have resumed as normal and no significant delay is foreseen at the time of reporting.
WP5:
WP5 has during the second report period leveraged the genome engineering pipeline established during the first reporting period, enabling the construction of an extra >1,000 strains. Likewise, capitalizing on the established genome engineering pipeline, combinatorial MIA biosynthetic pathway designs transformed into yeast, together with strictosidine quantification and genotyping of stochastically sampled yeast colonies has enabled the training of machine-learning algorithms to predict best-performing pathway designs and recommend further designs to explore. Moreover, while WP5 succeeded in growth-coupling LAMT activity in yeast during the first reporting period, no high-throughput screens have been prioritized with this assay during the second period as we since then have identified the most rate-limiting metabolic steps in MIA biosynthesis in yeast to be located downstream of LAMT. A new biosensor system has been developed and is envisioned to help us mitigate these reactions at higher throughput during 2022. In total all activities related to MS14-16 and deliverables D5.1-D5.2 have now been completed, while MS17 and deliverables D5.3-D5.4 are pending completion during the next 3-6 months
WP6:
Due to the close collaboration between WP5 and WP6, the WP representatives have met bi-weekly since August 2021. This communication secures the dialogue between the laboratory and the assessment teams, which is necessary for information to flow between the groups in an iterative process. The process also ensures that the assessment results give the laboratory team ideas and targets for their future optimization, for example, to focus on reducing utility required in an industrial setup from the environmental perspective, and titers from the economic perspective.
The next step of WP6 will be to find the optimum from both the economic and environmental perspective, where we identify the potential optimal yield where the return on investment is optimized, while simultaneously environmental impacts are minimized for the microbial production of tabersonine.
WP7:
Based on vindoline biosynthesis through a yeast fermentation process, vindoline having a chemical structure analogous to the MIA compounds, a DSP methodology was defined through cultivation broth centrifugation, sterilization, liquid-liquid extraction and chromatography purification to afford pure compound. The same methodology will be applied to tabersonine, alstonine and rauwolscine as soon as the cultivation broths are available. Once these DSP steps are demonstrated at small scale, the study will focus on parameter optimization for scale-up purposes and delivery of large quantities of pure MIAs compounds.
WP8:
In this reporting period (M19-M36) all project dissemination efforts were conducted as outlined in the dissemination plan (D8.11) and plan for the use and dissemination of the foreground (PUDF)(D8.13). This included a variety of dissemination activities, such as the publication of scientific results in renowned, peer-reviewed journals as well as project updates and communication on the dedicated project-website, social media, video content on Youtube, workshops and press releases. Due to ongoing Covid-19 restrictions offline events remained less frequent over the past 18 months. However, in order to adapt to these circumstances, a focus was laid on the production of additional online content, such as the science video series “Scientists explain!” and a dedicated animation movie.
In summary, all dissemination and exploitation activities so far aligned with the plan, and all deliverables were delivered within their respective deadlines.

The projects is on track, we are gathering data for meeting the deliverables listed under the GA. This section will be updated as we move forward.

Project logo

Periodic Reporting for period 2 - MIAMi (Refactoring monoterpenoid indole alkaloid production in microbial cell factories)

Udostępnij tę stronę

Pobierz