Novel tools and technologies to optimise metabolic engineering in diatoms

PhD project strategic theme: Bioscience for renewable resources and clean growth

In recent years, interest in microalgae as biotechnological hosts has increased rapidly. Compared to the classical model organisms for metabolic engineering, bacteria and yeast, microalgae present a sustainable alternative for producing high-value compounds, such as terpenoids. Because of their enormous structural complexity and limited availability in nature it is ineffective to chemically synthesise terpenoids on an industrial scale, and biotechnological approaches to produce terpenoids are widely explored as a commercially feasible method. With potable water becoming a restricted resource, marine microalgae are particularly attractive as they are able to grow on salt and waste water. Engineering of terpenoids in microalgae such as the diatom Phaeodactylum tricornutum requires the introduction of terpene synthases and cytochrome P450 enzymes. Current challenges seen in metabolic engineering of P. tricornutum are the overall low levels of transgene transcription as well as the eventual loss of expression over time. We hypothesise that both metabolic and epigenetic effects may contribute to decreasing transgene expression levels. The aim of this project will be to enhance and stabilise transgene expression in P. tricornutum and thus improve its quality as a biotechnological host. Using strains producing the diterpenoid manoyl oxide as a model for monitoring transgene activity, we will explore the molecular causes underlying the loss of transgene expression.

Current strains available in the lab have been generated with the transgene expression cassette being inserted randomly into the genome. The exact genomic locations of the transgenes in these strains will be identified to correlate landing sites with expression levels. Some genomic locations may be preferable over others for inserting heterologous sequences, due to chromatin properties. If ideal euchromatic locations were found, we would use CRISPR/Cas9 technology to target additional transgenes at those exact locations and test whether this improves expression levels and/or stability.

In addition to genomic locations, the impact of epigenetic marks on transgene expression will be assessed by mutating genes encoding epigenetic modifiers, such as DNA and histone methyl transferases, and histone deacetylases. Strains with reduced epigenetic variance and gene silencing might prove favourable for transgene expression. Based on literature research and work done in other species, we will identify a list of potential targets that will then be modified using CRISPR/Cas9.

Besides genetic and epigenetic effects in transformants, metabolic engineering may impose a strain on the cells, thus resulting in reprogramming of the cells' metabolism and altering the carbon flux. We will characterise the metabolic status of transformed cells by measuring important metabolites, such as carotenoid and chlorophyll pigments, which utilise the same precursor as diterpenoids. Investigating the impact of heterologous terpenoid production in P. tricornutum on cell homeostasis may help focus the carbon flux for terpenoid production without affecting cell growth and viability negatively.

Lastly, we will aim to expand the biotechnological toolkit for P. tricornutum by designing a promoter which allows precise activation of transgene transcription. The vitamin B12-repressible promoter of the methionine synthase (METE) gene will be the starting template for the generation of an 'on-switch' promoter. By scrambling the motifs of the METE promoter we hope to be able to create a reliable B12-independent switch for transcriptional activation of a heterologous expression cassette.