Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-ß-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

Construction and Regulation of the Abscisic Acid Biosynthesis Pathway in Yarrowia lipolytica.

Abscisic acid (ABA) is an important plant hormone with a variety of physiological functions such as regulating plant growth and helping plants to resist an adverse growth environment. However, at present, the ABA yield of heterologous biosynthesis by metabolic engineering is still low for industrial production. Therefore, five Botrytis cinerea genes (bcaba1, bcaba2, bcaba3, bcaba4, and bccpr1) related to ABA biosynthesis were expressed in Yarrowia lipolytica PO1h; its ABA production was 24.33 mg/L. By increasing the copy number of IDI and ERG12S, ERG20YMT, and bcaba3, bcaba1 genes, the yield of ABA was increased to 54.51 mg/L. By locating HMG-CoA reductase and HMG-CoA synthase in mitochondria, acetyl-CoA in mitochondria was converted into mevalonate; this increased the ABA yield to 102.12 mg/L. Finally, in the fed-batch fermentation process with the addition of dodecane, the ABA yield was up to 1212.57 mg/L, which is the highest yield of heterologous production of ABA by metabolic engineering.

Adaptive responses of erythritol-producing Yarrowia lipolytica to thermal stress after evolution.

Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains. KEY POINTS: • ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica • Genes encoding global regulators are mutated during thermal adaptive evolution • Ceramide and BCAA are critical molecules for cells to tolerate thermal stress.

Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.

Biosurfactants production by marine yeasts isolated from zoanthids and characterization of an emulsifier produced by Yarrowia lipolytica LMS 24B.

The present study investigates the potential for biosurfactant production of 19 marine yeast species obtained from zoanthids. Using the emulsification index test to screen the samples produced by the marine yeasts, we verified that five isolates exhibited an emulsification index ≥50%. Additional tests were performed on such isolates, including oil displacement, drop collapse, Parafilm M assay, and surface tension measurement. The tolerance of produced biosurfactants for environmental conditions was also analyzed, especially considering the media's temperature, pH, and salinity. Moreover, the surfactant's ability to emulsify different hydrocarbon sources and to metabolize kerosene as the sole carbon source was evaluated in vitro. Our results demonstrate that yeast biosurfactants can emulsify hydrocarbon sources under different physicochemical conditions and metabolize kerosene as a carbon source. Considering the Yarrowia lipolytica LMS 24B as the yeast model for biosurfactant production from the cell's wall biomass, emulsification indexes of 61.2% were obtained, even at a high temperature of 120 °C. Furthermore, the Fourier-transform middle infrared spectroscopy (FTIR) analysis of the biosurfactant's chemical composition revealed the presence of distinct functional groups assigned to a glycoprotein complex. Considering the status of developing new bioproducts and bioprocesses nowadays, our findings bring a new perspective to biosurfactant production by marine yeasts, especially Y. lipolytica LMS 24B. In particular, the presented results validate the relevance of marine environments as valuable sources of genetic resources, i.e., yeast strains capable of metabolizing and emulsifying petroleum derivatives.

[Advances in efficient biosynthesis of erythritol by metabolic engineering of Yarrowia lipolytica].

Erythritol is a novel 4-carbon sugar alcohol produced by microbes in the presence of hyper-osmotic stress. It has excellent potential to serve as an alternative sugar for people with diabetes and also a platform compound for synthesizing various C4 compounds, such as 1, 3-butadiene, 1, 4-butanediol, 2, 5-dihydrofuran and so on. Compared with other polyols, the fermentative production of erythritol is more challenging. Yarrowia lipolytica is the preferred chassis of erythritol biosynthesis for its high-titer and high-productivity. At present, there are still some bottlenecks in the production of erythritol by Y. lipolytica, such as weak metabolic activity, abundant by-products, and low industrial attributes. Progress has been made in tailoring high version strains according to industrial needs. For example, the highest titer of erythritol produced by the metabolically engineered Y. lipolytica reached 196 g/L and 150 g/L, respectively, by using glucose or glycerol as the carbon sources. However, further improving its production performance becomes challenging. This review summarizes the research progress in the synthesis of erythritol by Y. lipolytica from the perspectives of erythritol producing strains, metabolic pathways, modular modifications, and auxiliary strategies to enhance the industrial properties of the engineered strain. Key nodes in the metabolic pathway and their combination strategies are discussed to guide the research on promoting the production of erythritol by Y. lipolytica.

Transcriptome analysis reveals multiple targets of erythritol-related transcription factor EUF1 in unconventional yeast Yarrowia Lipolytica.

BACKGROUND: Erythritol is a four-carbon polyol with an unclear role in metabolism of some unconventional yeasts. Its production has been linked to the osmotic stress response, but the mechanism of stress protection remains unclear. Additionally, erythritol can be used as a carbon source. In the yeast Yarrowia lipolytica, its assimilation is activated by the transcription factor Euf1. The study investigates whether this factor can link erythritol to other processes in the cell. RESULTS: The research was performed on two closely related strains of Y. lipolytica: MK1 and K1, where strain K1 has no functional Euf1. Cultures were carried out in erythritol-containing and erythritol-free media. Transcriptome analysis revealed the effect of Euf1 on the regulation of more than 150 genes. Some of these could be easily connected with different aspects of erythritol assimilation, such as: utilization pathway, a new potential isoform of transketolase, or polyol transporters. However, many of the upregulated genes have never been linked to metabolism of erythritol. The most prominent examples are the degradation pathway of branched-chain amino acids and the glyoxylate cycle. The high transcription of genes affected by Euf1 is still dependent on the erythritol concentration in the medium. Moreover, almost all up-regulated genes have an ATGCA motif in the promoter sequence. CONCLUSIONS: These findings may be particularly relevant given the increasing use of erythritol-induced promoters in genetic engineering of Y. lipolytica. Moreover, use of this yeast in biotechnological processes often takes place under osmotic stress conditions. Erythritol might be produce as a by-product, thus better understanding of its influence on cell metabolism could facilitate processes optimization.

Engineering Yarrowia lipolytica as a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose.

De novo biosynthesis of high-value added food additive p-coumaric acid (p-CA) direct from cellulose/hemicellulose is a more sustainable route compared to the chemical route, considering the abundant cellulose/hemicellulose resources. In this study, a novel factory was constructed for the production of p-CA in Yarrowia lipolytica using cellulose/hemicellulose as the sole carbon source. Based on multicopy integration of the TAL gene and reprogramming the shikimic acid pathway, the engineered strain produced 1035.5 ± 67.8 mg/L p-CA using glucose as a carbon source. The strains with overexpression of cellulases and hemicellulases produced 84.3 ± 2.4 and 65.3 ± 4.6 mg/L p-CA, using cellulose (carboxymethyl-cellulose) or hemicellulose (xylan from bagasse) as the carbon source, respectively. This research demonstrated the feasibility of conversion of cost-effective cellulose/hemicellulose into a value-added product and provided a sustainable cellulolytic cell factory for the utilization of cellulose/hemicellulose.

Biosynthesis of the antioxidant γ-glutamyl-cysteine with engineered Yarrowia lipolytica.

The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

A strategy to reduce the byproduct glucose by simultaneously producing levan and single cell oil using an engineered Yarrowia lipolytica strain displaying levansucrase on the surface.

Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid.

Punicic acid is a conjugated linolenic acid with various biological activities including antiobesity, antioxidant, anticancer, and anti-inflammatory effects. It is often used as a nutraceutical, dietary additive, and animal feed. Currently, punicic acid is primarily extracted from pomegranate seed oil, but it is restricted due to the extended growth cycle, climatic limitations, and low recovery level. There have also been reports on the chemical synthesis of punicic acid, but it resulted in a mixture of structurally similar isomers, requiring additional purification/separation steps. In this study, a comprehensive strategy for the production of punicic acid in Yarrowia lipolytica was implemented by pushing the supply of linoleic acid precursors in a high-oleic oil strain, expressing multiple copies of the fatty acid conjugase gene from Punica granatum, engineering the acyl-editing pathway to improve the phosphatidylcholine pool, and promoting the assembly of punicic acid in the form of triglycerides. The optimal strain with high oil production capacity and a significantly increased punicic acid ratio accumulated 3072.72 mg/L punicic acid, accounting for 6.19% of total fatty acids in fed-batch fermentation, providing a viable, sustainable, and green approach for punicic acid production to substitute plant extraction and chemical synthesis production.

Efficient synthesis of squalene by cytoplasmic-peroxisomal engineering and regulating lipid metabolism in Yarrowia lipolytica.

Squalene, a high-value acyclic triterpenoid compound, is broadly used in the food and medical industries. Although the large acetyl-CoA pool and hydrophobic space of Yarrowia lipolytica are suitable for the accumulation of squalene, the current production level in Y. lipolytica is still not sufficient for industrial production. In this study, two rounds of multicopy integration of genes encoding key enzymes were performed to enhance squalene anabolic flux in the cytoplasm. Furthermore, the mevalonate pathway was imported into peroxisomes through the compartmentalization strategy, and the production of squalene was significantly increased. By augmenting the acetyl-CoA supply in peroxisomes and the cytoplasm, the squalene was boosted to 2549.1 mg/L. Finally, the squalene production reached 51.2 g/L by fed-batch fermentation in a 5-L bioreactor. This is the highest squalene production reported to date for microbial production, and this study lays the foundation for the synthesis of steroids and squalene derivatives.

One-pot process for the biotransformation of vegetable oils into natural deca- and dodecalactones.

Deca- and dodecalactones are highly desired natural compounds that are essential for creating flavor formulations with fruity, peachy, creamy, and floral notes. Although natural ingredients are preferred by consumers, these lactones cannot be extracted from natural sources. Therefore, the biotechnological processes that produce these compounds in their natural form are crucial for the flavor industry. Here, we report a study on the biotransformation of vegetable oils into natural deca- and dodecalactones. The proposed process is performed one-pot, through the sequential use of three different biotransformation steps, namely the lipase-mediated hydrolysis of the triglycerides, the use of probiotic bacteria for the hydration of the unsaturated fatty acids and the transformation of the obtained hydroxy-fatty acids into lactones derivatives employing Yarrowia lipolytica. By using a specific vegetable oil in combination with a selected bacterial strain, it is possible to obtain a preferred lactone derivative such as γ-dodecalactone, dairy lactone, tuberose lactone, or δ-decalactone in a concentration ranging from 0.9 to 1.5 g/L. Overall, our method is suitable for the industrial production of these lactones as it is easily scalable, it can be performed in only one bioreactor and it makes use of generally recognized as safe (GRAS) microorganisms.

YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica.

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

'Mother(Nature) knows best' - hijacking nature-designed transcriptional programs for enhancing stress resistance and protein production in Yarrowia lipolytica; presentation of YaliFunTome database.

BACKGROUND: In the era of rationally designed synthetic biology, heterologous metabolites production, and other counter-nature engineering of cellular metabolism, we took a step back and recalled that 'Mother(-Nature) knows best'. While still aiming at synthetic, non-natural outcomes of generating an 'over-production phenotype' we dug into the pre-designed transcriptional programs evolved in our host organism-Yarrowia lipolytica, hoping that some of these fine-tuned orchestrated programs could be hijacked and used. Having an interest in the practical outcomes of the research, we targeted industrially-relevant functionalities-stress resistance and enhanced synthesis of proteins, and gauged them over extensive experimental design's completion. RESULTS: Technically, the problem was addressed by screening a broad library of over 120 Y. lipolytica strains under 72 combinations of variables through a carefully pre-optimized high-throughput cultivation protocol, which enabled actual phenotype development. The abundance of the transcription program elicitors-transcription factors (TFs), was secured by their overexpression, while challenging the strains with the multitude of conditions was inflicted to impact their activation stratus. The data were subjected to mathematical modeling to increase their informativeness. The amount of the gathered data prompted us to present them in the form of a searchable catalog - the YaliFunTome database ( https://sparrow.up.poznan.pl/tsdatabase/ )-to facilitate the withdrawal of biological sense from numerical data. We succeeded in the identification of TFs that act as omni-boosters of protein synthesis, enhance resistance to limited oxygen availability, and improve protein synthesis capacity under inorganic nitrogen provision. CONCLUSIONS: All potential users are invited to browse YaliFunTome in the search for homologous TFs and the TF-driven phenotypes of interest.