Diatoms are very promising organisms for biofuel production since they can produce high quantities of lipids when nutrient depleted. We hypothesize that diatom lncRNAs expressed under nutrient stress are likely to be major players in the pathways regulating energy and biomass production which is supported by preliminary data.
The project is addressed by 3 research objectives which are interconnected, complementary and reflect the synergetic collaborative nature of the present consortium. <br />Objective 1 is to identify and characterize lipid production and cell cycle regulatory lincRNAs. On the one hand our goal is to find the lincRNAs that stand as checkpoints to cell cycle progression without interfering with lipid accumulation. The working hypothesis is that mutating these checkpoints may allow faster cell cycle progression with concomitant lipid production. On the other hand we want to identify the lincRNAs responsible for shifting cellular metabolism towards lipid accumulation, so that these may be triggered without the presence of nutrient stress. Based on our previous results, we will use the following rationale: A positive regulator of lipid accumulation would be up-regulated under nutrient depletion and then down-regulated when the depleted nutrient is re-supplied to the medium and lipid production stops. Furthermore we can also hypothesize that a negative regulator of cell cycle progression would also fall in this category. On the other hand, a positive regulator of diatom cell cycle is likely to be down-regulated under nutrient depletion and up-regulated when the depleted nutrient is added to the medium and cell division resumes and, so would be considered a negative regulator of lipid accumulation. Objective 2 is to identify the major protein partners of the previously selected lincRNAs in P. tricornutum. The best one to two strains developed will be grown in pilot-scale reactors at A4F’s GMO compliant pilot-plant in Lisbon, Portugal. The primary goal of Objective 3 is to define their potential for the production of commodities such as lipids for biofuels and high-value products for specialty markets as well as un-refined biomass for feed/food industries.
In total 20 candidate lincRNAs which could be involved in the regulation of the molecular processes leading to lipid accumulation and cell cycle progression in P. tricornutum will be screened by the coordinator team.
In order to generate full functional knockouts of the candidate lincRNAs, the CRISPR-Cas9 technology is used to induce very large deletions in the lincRNA loci, through the use of a double RNA guide CRISPR-Cas9 strategy. Once the plasmids harboring all the parts necessary for the CRISPR-Cas9 machinery to be effective in P. tricornutum are generated, these will be used to transform the diatoms (by biolistic and/or conjugation). The diatom mutant strains will be genotyped, by PCR and sequencing, in order to verify that the presence of the full deletion of the targeted lincRNA loci. Homozygous lines will then be selected and purified and cultivated in liquid media for phenotyping. The phenotyping consists in evaluating different parameters, under control and nutrient depleted conditions, such as cell division, cell morphology, lipid production and differential expression of nutrient stress marker genes (phosphate transporters and alkaline phosphatases). A global transcriptomic study by ssRNA-Seq will be undertaken in the most promising (with the most marked phenotype) KO mutant lines. This will allow to identify the pathways, and putative protein partners which are under the regulation of the targeted lincRNAs.
Total biomass production will be measured over the growth period in complete industrial medium on the most promising diatom mutant lines by the partner team (A4F, Portugal). Biomass composition will be analyzed chemically in order to quantify major components, and compared to control cultures. Overall productivities of total biomass, protein, lipids (particularly TAGs), carbohydrates and selected pigments will be calculated and compared to those of WT strains. The potential for industrialization of these strains will be assessed according to the results obtained and by comparison to industrial standards.
The first task of the project involved the generation of mutant lines of the diatom Phaeodactylym tricornutum by the knockout of candidate lincRNAs by the coordinator team. In order to optimize the transformation rate and efficiency for the generation of the diatom mutant lines, the coordinator team optimized the use of a combination of different technics to generate CRISPR-Cas9 conjugation-based mutagenesis plasmids. The protocol developed was based on the uLoop system (Pollak et al., 2019), in which 2 plasmids corresponding to, 1) the proteins Cas9 and, 2) the “essential” elements to generate CRISPR-Cas9 gRNA (Promoter; gRNA scaffolding and targeting sequence; Terminator) were domesticated in a single plasmid with a very high rate of success. This also allows for a quick change of the gRNAs by PCR. Furthermore, a bacterial conjugation approach to generate cleaner mutant strains was also employed. After generating all the mutant strains using these two combined methods (single uLoop plasmid and bacterial conjugation) PCR screening on isolated colonies showed that very clean deletions between the gRNAs were obtained for all the mutant strains. However, even through the use of successive selection media, both on plate and in liquid cultures, we found that most of our diatom cultures were not 100% pure (a mix of mutant and wild type and/or heterozygous lines) since we were still able to detect the wild type allele by PCR amplification (wild type contamination and heterozygous strains was easy to distinguish). To optimize the purification of the cultures we decided to make successive dilutions on plates in order to obtain isolated colonies emerging from only one cell. We succeed to obtain a protocol allowing us to generate pure homozygous mutant lines for our candidate lncRNAs, in which, by removing the selection pressure we will be able to remove the plasmid (episome) containing the CRISPR-Cas9 system.
So far 15 lincRNAs candidates with a putative regulatory function have been targeted by CRIPRR-Cas9 through the conjugation system. At least two genetically independent homozygous lines have been obtained for each gene. The deletion of the full locus has been confirmed by thorough genotyping even after removal of the pressure selection (which allowed for the loss of the episome and hence all transgenic material) for several generations. They are currently being phenotyped (as described in the methods section).
In collaboration with M. and C. Boccara, at the Museum of Natural History and Institut Langevin, the project coordinating team participated in the study of cell dynamics in Phaeodactylum under different experimental constraints. Thanks to a non-invasive and non-destructive microscopy method called « Dynamic Cell Imaging « (DCI), this study allowed to follow in vivo the dynamics of lipid vesicles during phosphate deficiency. This collaboration resulted in a paper published in European Journal of Phycology.
The coordinator team is currently phenotyping the recently generated mutant bank of lincRNAs KO lines of Phaeodactylum tricornutum with a putative regulatory role in nutrient stress responses for cell growth, lipid production, morphology as well as the differential expression analysis of nutrient stress markers genes.
The next step, once the phenotyping is completed, is to 1) do a transcriptomic study of the most promising 1 to 2 KO lines and to 2) transfer them to our industrial partner, A4F, in Portugal, as planned.The latter will allow assessing total biomass production in an industrial setting. Biomass composition will be analyzed chemically in order to quantify major components, and compared to control cultures. Similar analysis of biomass and components will be done after shifting to nutrient depleted medium. Overall productivities of total biomass, protein, lipids (particularly TAGs), carbohydrates and selected pigments will be calculated and compared to those of WT strains. The potential for industrialization of these strains will be assessed according to the results obtained and by comparison to industrial standards.
1. Bey H, Charton F, Cruz de Carvalho H, Liu S, Dorrell RG, Bowler C, Boccara M, Boccara C (2021). Dynamic Cell Imaging: application to the diatom Phaeodactylum tricornutum under environmental stresses. European Journal of Phycology.
2. Cruz de Carvalho MH*, Bowler C (2020). Global identification of a marine diatom long noncoding natural antisense transcripts (NATs) and their response to phosphate fluctuations. Scientific Reports, 10; 14110. (*corresponding author)
Diatoms are very promising organisms for biofuel production since they can produce high quantities of lipids when nutrient depleted. This nonetheless comes at the cost of cell division. Preliminary results revealed that in P. tricornutum over 40% of the processed transcripts under stress can be classified as long noncoding (lnc)RNAs. LncRNA are currently gaining momentum as major regulators of biological processes and recent evidence has shown their regulatory roles in cell division and lipid metabolism in mammalian cells. We hypothesize that diatom lncRNAs expressed under nutrient stress are likely to be major players in the pathways regulating energy and biomass production which is supported by preliminary data. In this proposal we partner groundbreaking systems biology research, aiming to enhance biomass accumulation through lncRNAs gene editing in diatoms, with industrial expertise, aiming to generate a portfolio of uses for the diatom biomass feedstock, in a biorefienery approach.
Madame Helena CRUZ DE CARVALHO (Institut de biologie de l'Ecole Normale Supérieure)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IBENS Institut de biologie de l'Ecole Normale Supérieure
A4F A4F, Algafuel, SA
Help of the ANR 319,781 euros
Beginning and duration of the scientific project: February 2020 - 42 Months