Harnessing Yarrowia metabolism to generate high value terpenoids

Lutein, a pigmented carotenoid with antioxidant effects, is extensively used in food and animal feed. Naturally, it is produced in different plants and algae. Animals, including humans, do not synthetase its own lutein, however, this compound is essential for health. At the moment lutein for food & feed or pharmaceutical usage is extracted from marigold flowers (Tagetes spp.), where it is accumulated in low amounts and which requires a vast amount of land and fertilisers, making it a very expensive product. So far, an efficient biotechnological production process does not exist. The aim of the project is to develop, for the first time, the production of lutein using engineered microorganisms. Using state of the art synthetic biology methods for genome engineering we will transfer the capacity to synthesise lutein from plants to the in industrial yeast Yarrowia lipolytica. The production will be further optimised by screening different combination of plant genes in order to find the right balance of them that leads to high amounts of this valuable compound. The strains harbouring the best combinations, therefore the best producers, will be studied in industrial-like conditions using bioreactors, where the production can be optimised and maximised. In addition, we will explore the capacity to produce lutein using waste streams from other industries (i. e. Glycerol from biodiesel production), which will help to develop a circular process and to reduce generated waste.

The project will be done in close collaboration between the group of Dr Rodrigo Ledesma-Amaro from Imperial College London and Acies Bio, one of the leading industrial biotech companies specialised in the development of new strains and bioprocesses.

In the course of the project "Harnessing Yarrowia metabolism to generate high-value terpenoids," we will develop new strains and bioprocesses with Yarrowia lipolytica for the production of high value, non-conventional terpenoids, with importance as antioxidant, food & feed, pharmaceutical or nutraceutical industries. This will be facilitated by the use of cutting edge synthetic biology tools.

The project will begin with the screening of new enzymes, identified by a bioinformatic approach, in a high-throughput manner using the automation at the London DNA Foundry. The characterisation of the activities and substrate preferences of these enzymes will allow us to create engineered strains of Y. lipolytica with the ability to produce the compounds of interest.

Then, several strategies will be carried out in order to maximise the production of the compounds of interest. Firstly, we will engineer the terpenoid pathway by balancing the ratio of the enzymes involved in the synthesis, engineering the localisation of those enzymes and screening new variants of the identified enzymes by error-prone PCR. Secondly, we will focus our efforts in the upstream pathways, leading to the formation of the precursors of the pathway such as Acetyl-CoA.

Most promising combinations of genetic interventions will be combined in best-producer strains. The performance of these strains will be optimised in benchtop bioreactors first. Then, the efficient scale-up to 100L will be able to demonstrate the capability of the engineered Y. lipolytica strains to produce high-value terpenes at high titers.

The proposed research project is expected to have high impact and provide the basis for the development of next stage Y. lipolytica as microbial host for the production of carotenoids and other isoprenoid molecules. We will build on the UK`s recent investments into the field of synthetic biology by using existing infrastructure, such as the London DNA Foundry. This will allow us to work with state of the art equipment and methods in synthetic biology and provide new knowledge to advance the academic success of UK`s institutions further and create new companies or support SMEs leading the new cycle in biotechnological development. Apart from the economic and scientific excellence and progress, we will encourage the dissemination of results to the general public.

Acies Bio, a biotech company supporting this proposal, will have a direct impact with results obtained from the proposed project. They will benefit from generated knowledge from the field of synthetic biology and process development, as they state in their letter of support. We have been discussing the impact of the project already before, and we believe there is a strong match between scientific progress and the development of commercially viable biotech solutions. Apart from Acies Bio, developed tools and knowledge will be of interest of other strain development companies, many of them working with Yarrowia lipolytica (i.e. Novogy in the US) or other yeast species (i.e. Amyris and Ginkgo Bioworks). With this in mind, knowledge developed in this project will have a significant economic impact in many areas of industrial biotech, e.g. nutraceuticals, food&feed production, and agricultural biotech. The new technology developed will attract new investment to the biotech sector and provide means for the development of new start-up companies, and new high value-added jobs.

The proposed project is conceived to have a high social impact in addition to scientific and economic aspects. Media is becoming increasingly aware and interested in synthetic biology and we expect to have high coverage of project results, similarly to our previous published papers, patents, and reviews. We will promote the results in general media as well as in specialised science-oriented editions both in UK and Slovenia. All in all, this will bring a better understanding of the science behind this project. It will shed light on the usage of synthetic biology for the production of value-added chemicals and how synthetic biology and genetic engineering in the industrial biotechnology sector can improve our quality of life.

The proposed project will have a strong impact on the career development of those directly involved in it. Both PDRAs will learn about different aspects of industrial biotechnology, from strain design to media development and scale-up. Additionally, the PDRAs will have a strong and tight connection with top-level research institutions as well as with industry (where part of the research in this proposal will be done). Besides, the PDRAs will be able to attend the most important conferences and trade shows on industrial biotechnology and synthetic biology in the world. The PDRAs will be able to supervise master students working on topics associated with this proposal. At the same time, master students working on the topic will benefit from the project. Last, but not the least getting funds for this BBSRC project will help me as an early career PI to carry out translational research at ICL on industrial biotechnology.