Development and use of a synthetic biology toolkit to enhance high value lipid production in Nannochloropsis oculata

Abstract

Wild fish (sardines and anchovies) are the major source of the omega-3 fatty acid, eicosapentaenoic acid (EPA), but fish stocks are crashing all the while that global demand for EPA is increasing. The only sustainable option is to derive EPA products from sustainably grown feedstocks, foremost being microalgae. However, microalgae do not currently accumulate sufficient quantity of EPA to be commercially viable. Gene editing has shown significant potential to address this issue though cellular and specifically lipid metabolic engineering; however, the use of established CRIPSR systems is prohibitively expensive. This MRes thesis aimed, therefore, to develop use of BEC10, a novel CRIPSR nuclease (BRAIN-Biotech AG), to gene edit the stramenopile alga Nannochloropsis oculata CCAP 849/1 based on homology directed repair (HDR). The overall goal was to generate gene edited CCAP 894/1 clones that produce more EPA. To achieve this, a novel cell synchronization and electroporation protocol was developed using the Nepagene ELEPO21 electroporator, which yielded transformation efficiencies of up to ca. 25,000 CFU ug-1 (4 - 25-fold increase). Next, an episomal BEC10 expression vector was modified and used to test if the BEC10 nuclease could be stably expressed in CCAP 894/1. This was confirmed by a positive reporter gene luminescence assay and observation of the unique ‘clone reduction’ activity of the nuclease. In parallel to BEC10 nuclease development, a proof-of-concept episomal CRISPR-Cas9 vector was used to target and delete nitrate reductase (NR). The Cas9-based vector electroporations led to an overall HDR-mediated gene editing efficiency of 30% as shown by whole-gene deletion of NR. This is believed to be the first report of non-transgenic HDR-mediated gene editing in this Nannochloropsis species. HDR gene editing using the BEC10 system did not yield any mutant strains, however. Further development of BEC10 is required to address the timing of BEC10 expression and tools to increase the rate of HDR of the genome. Using the Cas9-based vector, the UDP-6-glucose dehydrogenase (UDGH) was targeted for HDR gene editing as a route to divert cellular resources away from cell wall synthesis to lipid biosynthesis and enhanced EPA production. Despite this, CRISPR-Cas9 HDR targeting vectors only yielded INDEL mutations in UGDH produced by the non-homologous end joining pathway (NHEJ). The failure to identify any HDR-derived transformants was possibly caused by UGDH gene knockouts being lethal. Nevertheless, several non-transgenic, marker-free UGDH-edited cell lines were generated which exhibit unique phenotypes. Ongoing investigation will elucidate the extent of the metabolic re-distribution in these strains and will characterize the total lipid content and fatty acid composition of the cell to determine if EPA accumulation has been increased. Overall, this thesis has demonstrated that N. oculata CCAP 849/1 can be gene edited by inducing either HDR or NHEJ repair pathways. This evidence is highly encouraging and establishes the basis of a synthetic biology toolkit to generate microalgal strains that may achieve the goal of producing lipids at a price that can supplants fish oil as a sustainable source of EPA.

Date of Award	9 Apr 2024
Original language	English
Awarding Institution	University of the Highlands and Islands
Sponsors	BRAIN Biotech AG (
Supervisor	David Green (Supervisor) & Nick Owens (Supervisor)