17-ERACoBioTech: MicroalgaE as Renewable Innovative green cell facTories

This project [MERIT] sets out to leverage state of the art techniques to develop microalgae for the sustainable production of high-value, medically and industrially relevant molecules from carbon dioxide and light. Specifically, MERIT will focus on the production of twenty carbon containing (C20) molecules, known as diterpenoids. These are complex metabolites found largely in plants, and whose variety and complexity have made them incredibly interesting for numerous applications such as medicines, antimicrobial agents, and high-value chemicals. The complex structures of diterpenoids are difficult and costly to synthesize chemically and can be expensive or inefficient to purify from their native host organisms.

All organisms produce the same 5-carbon building blocks used in diterpenoid production pathways. By introducing these production pathways between species, we can produce non-native diterpenoids in engineered hosts (e.g. bacteria, yeast or microalgae). Production of diterpenoid products in fermentative hosts (e.g. yeast) has become a mature technology, however, relies on unsustainable use of organic carbon sources such as glucose and inherently competes for agricultural resources. Microalgae, however, are naturally optimized to produce precursor molecules needed to make diterpenoids, as these same molecules are used for harvesting light and for generation of antioxidant pigments in the cell. Algae hold the additional benefit of rapid growth rates in simple mineral salt solutions using only light and CO2 as energy inputs. These organisms are ideal hosts for the production of diterpenes and are inherently sustainable production chassis.

By introducing different enzymes that convert precursor molecules into the numerous carbon skeletons of diterpenoid products algal cells can be engineered to serve as diterpenoid production hosts. Additional engineering of these algal cells through introduction of enzymes that add oxygen molecules into the diterpene backbones will allow the algae to produce specialized diterpene structures, which often have medicinal properties. To date, engineering of microalgae to accumulate new non-native enzymes has been a major limiting factor to their widespread application as green-cell factories for complex biotechnological targets. Combining strain domestication with synthetic biology (an emerging discipline that uses engineering principles to design and assemble biological components) has recently been demonstrated to facilitate advanced engineering of these highly promising organisms. In this project, an international team representing global leaders in algal synthetic biology, outdoor algal cultivation, photobioreactor design, and process modelling, will join forces to drive forward the development of algal cell factories. The MERIT team already successfully engineered pathways for the production of several diterpenoids in microalgae. Multiple levels of strain engineering and synthetic biology will be implemented to create green-cell factories with enhanced carbon flow from CO2 to diterpenoids. Various enzymes will be combined to produce novel 'new-to-nature' diterpenoid products with potential for numerous applications. Optimized strains will be grown to scale and processes for extraction of the products will be designed. The project will generate many new avenues of commercialization potential and significantly contribute to the development of the European Bioeconomy.

The MERIT project will leverage state of the art synthetic biology techniques to engineer microalgae for the sustainable production of high-value, medically and industrially relevant novel diterpenoid products, branched hydrocarbons derived from the C20 isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP). The variety and complexity of these compounds, largely confined to plants, offer potential for numerous applications such as medicines, antimicrobial agents, and high-value chemicals, but their complexity also means they are difficult and costly to synthesize chemically, and can be expensive or inefficient to purify from their native host organisms. Heterologous expression of modular terpene synthase pathways can be used to produce non-native terpenoids in engineered bacteria and yeasts, but this relies on the use of fixed carbon from plants, and thus competes for agricultural resources. Microalgae, as photosynthetic organisms, are inherently more sustainable. Moreover, they are naturally optimized to produce GGPP for chlorophyll and carotenoid biosynthesis. In this project we will establish a synthetic biology platform of standard parts and workflows to facilitate expression of various permutations of diterpenoid biosynthesis enzymes in Chlamydomonas reinhardtii and Phaeodactylum tricornutum. Diterpene synthases and cytochrome P450s from various plant sources will be combined to produce novel 'new-to-nature' diterpenoid products and rates of production assessed in the laboratory. Optimized strains will be grown at pilot scale, and processes for diterpene product extraction designed. The data will be used to conduct life cycle and techno-economic assessments of the pipeline to identify potential bottlenecks for further optimization.

The outputs of the proposed research will be of major benefit to realizing the potential of microalgae as new industrial biotechnology platforms. There is considerable interest in Europe and globally in developing an algal biotech sector, both at the level of policy makers and research funders, but also industry. Microalgae offer the advantage of being photosynthetic, so by using sunlight to photosynthesise, their use as production systems means they may be more sustainable than conventional fermentation hosts such as bacteria and yeasts. However unlike plants, they can be grown on marginal land or in contained facilities, and so do not compete with food production.

In this project, an international team of experts in algal synthetic biology, cultivation, and industrial process modelling, will join forces to design a system for light-driven conversion of CO2 to high-value 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, including 'new-to-nature' compounds. These compounds have potential uses in the chemical as well as pharmaceutical industries, and are not accessible by classical chemical synthesis. Thus the project will contribute to a continuation and stimulation of research and development for a European Knowledge Based Bio Economy.
In parallel, work will be carried out to improve phototrophic productivity, which will increase the competitiveness of microalgal production schemes compared to those relying on the use of heterotrophic microorganisms, whose cultivation requires crop plant-derived organic carbon sources and competes for resources with food production. The involvement of industrial partners will facilitate moving through stages of technology readiness to the prospect of commercialization within 5-10 years.

The interdisciplinary nature of MERIT will ensure the training of highly skilled researchers and support personnel, who will provide the future workforce to underpin development of a bioeconomy in Europe. The involvement of industrial partners will ensure cross-sectoral integration, improving and enabling knowledge transfer.

All scientific results emerging from this project shall be published in international peer-reviewed journals as well as presented at international conferences, as soon as the partners have evaluated these and permission for dissemination has been given. Knowledge generated will be made available to the scientific community at conferences and dedicated workshops. Besides publication in peer-reviewed scientific journals, MERIT will also consult the project officer in charge in order to publish interesting results in journals published by the European commission (e.g. Horizon Magazine).
A project website will be jointly established by the scientific administration assistant so that all the universities involved can present the project and results, both for other scientists and professionals, including industry, and the general public (see Stakeholder table). This website will also contain information on the molecular tools and protocols developed in the project.

The MERIT communication plan aims to inform and initiate a dialogue with four distinct priority stakeholders including industrial stakeholders, EU citizens, University students and High school teachers about the MERIT project and its results (see Stakeholder table). Overall communication activities within MERIT aim at increasing the stakeholder`s awareness for the great innovation potential of microalgae as sustainable green cell factories. This increased awareness of stakeholders, especially of those belonging to the business sector, will foster the development of a sustainable European bio-based economy by including microalgae into the portfolio of established industrial microorganisms.