Project

Project

- Design of microalgal strains optimized for diterpenoid production

- Implementation of strain engineering and synthetic biology

- Enzymatic oxyfunctionalization of diterpene backbones

- Scale-up cultivation & industrial process modelling

- Product extraction

Fact Sheet

Funding ‚ā¨ 1.650.000

Start June 2018

Duration 36 Months

ERA CoBioTech is an ERA-Net Cofund Action under H2020 Grant agreement No. 722361

The MERIT Project

An international team of experts in algal synthetic biology, cultivation, and industrial process modelling, joins forces to design optimized microalgal strains, accompanying industrial production and product extraction processes for light-driven conversion of CO2 into high-value, medically and industrially relevant diterpenoid products.

Multiple levels of strain engineering and synthetic biology will be implemented to create green-cell factories with enhanced carbon flow from CO2 to terpenoids. Various strategies will be combined to produce novel diterpenoid products with high potential for a broad range of applications. Optimized strains will be grown to scale and processes for diterpene product extraction will be designed.

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Background

Twenty carbon containing (C20) diterpenes are complex, often oxyfunctionalized secondary metabolites found largely in plants. Their variety and complexity have made them highly interesting for numerous applications as medicines, antimicrobial agents, and high-value chemicals. The complex structures of diterpenoids are difficult and costly to chemically synthesize and can be expensive or inefficient to purify from their native host organisms.

All organisms produce the same 5-carbon building blocks used in terpenoid production pathways. Heterologous expression of modular terpene synthase pathways can be used to produce non-native terpenoids in engineered hosts. Production of terpenoid products in fermentative hosts has become a mature technology, however, relies on unsustainable use of organic carbon sources such as glucose and inherently competes for agricultural resources. Fermentative microbial hosts are additionally not optimized for the production of the C20 diterpenoid precursor geranylgeranyl pyrophosphate (GGPP).

 Microalgae are naturally optimized to produce GGPP as the precursor for light harvesting and photoprotective pigments in the cell. Over-expression of diterpene synthases (DiTPS) allows the conversion of this precursor into the numerous carbon skeletons of diterpenoid products. Algal cells also represent ideal chassis for the expression of cytochrome P450 monooxygenases (CYPs), which are needed for oxyfunctionalization of diterpene backbones to specialized structures. In photosynthetic cells, CYPs can be coupled to photosynthetic electron transport chains for efficient redox potential, an otherwise limiting factor in non-photosynthetic hosts.

Combination of DiTPS and CYPs in an algal chloroplast is the ideal chassis for complex oxyfunctionalized diterpenoid production, where both high concentrations of precursor and the redox electron flow are abundant. Algae hold the additional benefit of rapid growth rates in simple mineral salt solutions using only light and CO2 as energy and carbon inputs, respectively. These organisms are ideal hosts for the production of diterpenes and are inherently sustainable production chassis.

To date, engineering of microalgae for robust expression of transgenes has been a major limiting factor to their widespread application as green-cell factories for complex biotechnological targets. Through combined efforts of strain domestication and synthetic biology, the development of synthetic eukaryotic algal transgenes have recently been demonstrated to facilitate advanced engineering of these highly promising organisms.


Objectives

The project is divided into four work packages (WP1-4).


WP1: Development of a standardised synthetic biology platform for algal green cell factories producing diterpenoids

Aim: To develop robust synthetic biology platforms for C. reinhardtii and P. tricornutum by establishing versatile kits of standard parts with verified characteristics, recorded in a digital registry, as well as optimized workflows.

Objectives of WP1

-To generate kits of standard parts for the two microalgal species, which will enable interchangeable assembly of different permutations of parts in a high-throughput manner via the application of modular cloning principles.

-To develop a standardized workflow using one or more easily measurable reporter genes to assess the efficacy of the different parts in each microalgal host.

-To establish a searchable registry of parts and constructs using an online software platform to facilitate sharing information.

 

WP2: Engineering green cell factories for the conversion of carbon dioxide and organic carbon derived from waste material into high value and novel diterpenoids

Aim: To engineer heterologous diterpenoid production at lab-scale in P. tricornutum and C. reinhardtii.

 Objectives of WP2

-To apply the principles of synthetic biology by analysing distinct combinations of diterpene synthases (diTPS) to produce a variety of diterpene skeletons in P. tricornutum and C. reinhardtii

-To further modify these diterpenes using cytochrome P450 (CYP) enzymes for diterpene oxyfunctionalization

-To generate ‚Äúphototrophic platform strains‚ÄĚ for the efficient conversion of carbon dioxide and waste organic carbon sources into various diterpenoids

 

WP3 Scale-up, product extraction, and modelling of microalgae-based diterpenoid production

Aim: To develop processes for production and in situ extraction of diterpenoids from microalgae and to validate the process in an industrially-relevant environment. Results are integrated into LCAs and techno-economic models aiming at the identification of technological and economic bottlenecks for commercial scale processes as well as to assess its sustainability.

Objectives of WP3

-To select an optimal solvent which can retain cell viability, allows efficient diterpenoid extraction, and is suitable for further separation as well as for refinement of diterpenoid products.

-To design an algal diterpenoid production process at laboratory scale in which terpenoid production and integrated product recovery are optimized

-To compare a process without integrated product recovery and a process with integrated product recovery

-To scale-up the production process and transfer of the process into an industrially relevant environment

-To perform techno-economic modelling and sustainability analysis of the process

 

WP4 Communication of MERIT results

Aim: Communication activities within MERIT aim at increasing the stakeholder`s awareness for the great innovation potential of microalgae as green cell factories, which should foster the development of a sustainable European bio-based economy by including microalgae as production hosts.

Objectives of WP4

-To communicate with stakeholders via various communication channels, following a detailed communication & dissemination plan. Communication activities within MERIT aim at increasing awareness of the values of synthetic biology and algal biotechnology for identified stakeholders. The aim of this work package is to engage all consortium partners with identified stakeholders in order to promote the great innovation potential of microalgae as green cell factories, while promoting the development of a sustainable European bio-based economy.