January – March2024
At this stage, the preliminary experimental pipeline was carried out, which involved establishing: the stages of monitoring lipid accumulation; the cultivation time and incubation conditions, respectively, the selection of specific media for lipid accumulation. The Y. lipolytica ACA-DC 50109 parental strain was included in the Department’s collection and the preliminary morphological and physiological characteristics of the strain were determined by Biolog YT MicroPlates. These are 96 wells plates that alow determination of assimilation or oxidation profile on a variaty of carbon sources; Finantial and planing documents for contracting stage of the project were prepared, as well as documents for consumables and equipment acquisitions. A selective bibliography was initiated, which includes 20 bibliographic titles regarding: the EAL technique; analyses associated with the EAL experiment (biochemical techniques that allow monitoring the stages necessary for completing the oleaginous growth phases of Y. lipolytica on specific media for EAL). This list is being selected because both the experience of the project director and the analysis of the latest publications on the project topic are required.
April – June 2024
Optimization of Adaptative Laboratory Evolution-ALE culture media and working conditions for experiments carried out in small volumes. Elements of the working flow during growth and lipid production curve of the Y. lipolytica ACA-DC-50109 strain were established and the growth curve was monitored by sampling after 11 days of cultivation; determination of the specific parameters: pH variation, cell density, biomass, lipid content (qualitative and quantitative methods), microscopic appearance of the cells. As a result, it was necessary to evaluate the obtained parameters in order to establish control points for implementation of the EAL technique on limiting culture medium. Establishing the protocol for the quantitative determination of total lipids in the biomass from production cultivation of Y. lipolytica under EAL conditions; Establishing a method for citric acid and glycerol determination in the culture medium; Establishing the protocol for the qualitative assessment of the accumulation of neutral intracellular lipids in Y. lipolytica cells by epifluorescence microscopy; Annotation of reference genome for Y. lipolytica and a genome used in the literature for the transcriptomic analysis of the ACA-DC 50109 strain in order to identify orthologous genes associated with the lipogenesis process. BLAST alignments of some coding sequences having a central role in the process; Identification of nucleotide sequences from Y. lipolytica strains existing in the NCBI GenBank database, based on similarity to the annotated and translated coding sequences. Generation of a database of 534 nucleotide sequences associated with the lipogenesis process in Yarrowia lipolytica.
July – September 2024
Alternative methods for selecting evolved clones obtained from cultivation on 7% carbon source medium were tested; Streamlining protocols for characterizing evolved clones using biochemical and microscopy techniques by increasing the quality and reproducibility of the results obtained; Literature analysis in order to establish a DNA and RNA isolation workflow of suitable quality for sequencing on Illumina and Nanopore platforms. Identification of recommended programs in literature, for processing raw sequencing data and hybrid flow assembly; Correlation of experimental data obtained from cultivating Y. lipolytica in the presence of 7% glycerol regarding the content of lipids, proteins, polysaccharides and biomass, as well as the assimilation of the carbon source; Identification of cultivation parameters that may influence the ability of the R. toruloides strain to simultaneously accumulate triacylglycerols and carotenoid compounds; Morpho-physiological characterization of the parental strain of R. toruloides NRRL-Y-27012 from the perspective of the ability to tolerate osmotic and ionic stress, as well as to assimilate different carbon sources; Identification of methods for quantifying carotenoids content, compatible with the determinations used to monitor intracellular lipids content; Databases analysis in order to identify existing genomic R. toruloides data, as well as best practices for analyzing sequencing data; literature Identification of studies on the ability genus Cunninghamella to assimilate carbon sources derived from agro-industrial waste and to produce polyunsaturated fatty acids; literature identification of appropriate and most frequently used methods for isolation and purification of nucleic acids from filamentous fungal cultures, using both commercial kits and in-house protocols; Establishing methodological benchmarks regarding the main strategies for mitigating the difficulties associated with processing of filamentous fungal mycelia samples during staining techniques specific for epifluorescence microscopy for lipid bodies highlighting.
October – December 2024
The Evosco team get bigger: 2 PhD positions were filled by PhD student Lecu Ana-Maria Alexandra and PhD student Georgescu Ana-Maria, as well as 2 partial postdoctoral positions by dr. Avram Ionela and dr. Irina Gheorghe-Barbu. Ten evolved clones obtained following an EAL process using glucose as a carbon source and as a selection method, cultivation on carbon free media were preserved long-term at -80 °C. A characterization of the evolved clones obtained was performed, using microscopy techniques, as well as chemical and biochemical analyses of the biomass and the culture medium. A workflow was developed for obtaining and processing genomic and transcriptomic sequencing data for the parental strain of Y. lipolytica. Adaptation of a protocol for Nile Red fluorochrome staining and qualitative analysis of lipid accumulation in R. toruloides by epifluorescence microscopy. Adaptation of the method for extraction and purification of lipids accumulated by R. toruloides following cultivation on EAL medium. Validation of methods for assililation monitoring of the carbon source from the EAL medium. Implementation of the workflow for the annotation of sequencing data. Establishment of an experimental plan for determining the parameters necessary for the implementation of the EAL technique using the C. echinulata CCF 2591 strain in the presence of substrates selected from the scientific literature and juidance from the Prof Aggelis.
January – March 2025
A series of samples representing the evolved clone populations obtained at the end and beginning of the EAL rounds in which phenotypic changes were observed compared to the parental strain were preserved at -80 °C. Genomic DNA isolation protocols from the parental strain Y. lipolytica were tested, with attention paid to the pretreatment steps prior to cell lysis, in order to obtain samples with increased purity. Design of qRT-PCR primers for gene expression profile analyses for the parental strain vs evolved clones of Y. lipolytica ACA-DC 50109 The working protocol for monitoring and perpetuating the EAL rounds for R. toruloides was established, based on the chosen carbon source and the selected selective pressure method. Choice of a preservation method for the evolved clones obtained from the EAL process. Preservation of samples representing the evolved clone populations existing at the time of observing an increase in the proportion of cells that sediment with difficulty after centrifugation. Identification of phenotypic changes that are indicative of a functional EAL process, increasing the proportion of cells that do not sediment following centrifugation; estimating losses that cause errors when characterizing evolved clones in terms of dry biomass and lipid content due to sedimentation difficulties.Testing EAL conditions in accordance with the particularities of the C. echinulata CCF2591 strain and determining the optimal methods for selecting spores with high lipid content. Testing multiple variants of pre-processing of C. echinulata biomass for genomic DNA isolation, in combination with several kits to establish an efficient isolation and purification method. Selecting an optimal method for obtaining the dry biomass necessary for the quantification of neutral intracellular lipids and the main competing compounds, respectively highlighting by qualitative methods the heterogeneity of spore suspensions obtained following the implementation of the EAL technique. Initiating preliminary cultivations of R. toruloides at laboratory bioreactor scale, using pure carbon sources, to establish the biotic and abiotic parameters necessary for the transition from batch shake-flask cultivations. Selection and preliminary processing of agro-industrial residues that can be converted with maximum yield into neutral intracellular lipids. Preparation of progress reports for ongoing activities, reception, installation and training, reorganization of the workspace. The working visit of Mr. Prof. Georgios Angelis took place between February 3-6 and was materialized through discussions and analysis of some results. Submission and acceptance of three abstracts for poster presentations at international scientific conferences. Establishment of methods for searching for economic agents with relevant activity profiles at national and European level, as well as the creation of a preliminary list of economic agents with activity in microbial biotechnology or the production of agro-industrial residues.
April – June 2025
Isolation during the EAL experiment applied to Y. lipolytica ACA-DC 50109 of 3 evolved clones, preserved at -80°C, which following characterization demonstrated increased lipogenesis capacity. Preliminary determination of the lipid profile and cellular ultrastructure of the parental Y. lipolytica by chromatographic analyses and transmission electron microscopy, respectively. Isolation of DNA from samples of the parental Y. lipolytica strain and its sequencing using the Illumina short-reads technology. Assembly and analysis of the obtained data set. Testing of different variants of an RNA isolation protocol and choosing the optimal protocol to be used in subsequent analyses. Annotation of the assembled genome of Y. lipolytica ACA-DC 50109 for the identification of coding sequences. Annotation of coding sequences using gene ontology databases (KEGG, EggNOG), and identification of genes involved in lipogenesis, the Krebs cycle and glycolysis. Establishing the adapted work protocol for EAL experiments on R. toruloides, by adapting the parameters according to the strain-specific oleaginous phenotype. Characterization of clone populations obtained at the end of eight rounds of EAL on R. toruloides and preservation by selection on carbon-free medium of three clones that were preserved at -80 °C. Characterization of three evolved clones in terms of lipogenesis phenotype and biochemical profile. Testing of RNA isolation protocol variants. DNA isolation, sequencing and assembly of the R. toruloides NRRL-Y 27012 genome. Initiation and completion of the first 3 evolutionary rounds of an EAL experiment on a C. echinulata strain in two evolutionary lines differentiated by the C/N ratio. Obtaining experimental data on DNA and RNA isolation protocols tested on the parental C. echinulata strain and selecting optimal protocols for obtaining DNA and RNA samples of suitable quality for sequencing. Determination of the induced changes on the characteristics of external cellular structures for evolved clones preserved at -80°C. Sequencing of the parental strain of C. echinulata using Illumina technology, quality control and assembly of the obtained data. Determination of the biomass accumulation capacity in the presence of alternative carbon sources that represent the majority components of agro-industrial residues. Evaluation of the capacity to convert selected alternative carbon sources into neutral intracellular lipids. Development of progress reports for the activities currently underway. Reception, installation and training in the use of equipment. Presentation of the results at the ASM Microbes 2025 conference, Los Angeles, USA and at the Student Session of Scientific Communication by PhD and Master students involved in the project activities. Creation of a detailed list of economic agents at national and European level, including information on the activity profile, website and country of origin.