
==== Front
FEMS Yeast Res
FEMS Yeast Res
femsyr
FEMS Yeast Research
1567-1356
1567-1364
Oxford University Press

39169472
10.1093/femsyr/foae027
foae027
Research Article
AcademicSubjects/SCI01150
Using Euf1 transcription factor as a titrator of erythritol-inducible promoters in Yarrowia lipolytica; insight into the structure, splicing, and regulation mechanism
https://orcid.org/0000-0001-8372-8459
Celińska Ewelina Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-637 Poznań, Poland

Korpys-Woźniak Paulina Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-637 Poznań, Poland

Gorczyca Maria Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-637 Poznań, Poland

https://orcid.org/0000-0002-6679-972X
Nicaud Jean-Marc Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France

Corresponding author. Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-637 Poznań, Poland. E-mail: ewelina.celinska@up.poznan.pl
2024
21 8 2024
21 8 2024
24 foae02727 3 2024
25 6 2024
20 8 2024
12 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Controllable regulatory elements, like inducible, titratable promoters, are highly desired in synthetic biology toolboxes. A set of previously developed erythritol-inducible promoters along with an engineered Yarrowia lipolytica host strain were shown to be a very potent expression platform. In this study, we push the previously encountered limits of the synthetic promoters’ titratability (by the number of upstream motifs) by using a compatible transcription factor, Euf1, as the promoter titrator. Overexpression of spliced EUF1 turned out to be very efficient in promoting expression from the compatible promoter, however, the erythritol-inducible character of the promoter was then lost. Analysis of the EUF1’s splicing pattern suggests that the intron removal is promoted in the presence of erythritol, but is not dependent on it. The 3D structures of spliced versus unspliced Euf1 were modeled, and ligand-binding strength was calculated and compared. Furthermore, the EUF1-dependent expression profile under different chemical stimulants was investigated. Depletion of carbon source was identified as the significant factor upregulating the expression from the Euf1-dependent promoter (2–10-fold). Considering these findings and transcriptomics data, a new mechanism of the Euf1-regulated promoter action is proposed, involving a ‘catabolite repression’ transcription factor—Adr1, both acting on the same ERY-inducible promoter.

Erythritol promotes but does not condition splicing and expression of EUF1 in Yarrowia lipolytica; Adr1 is inflicted in ERY-regulated gene expression.

recombinant protein
heterologous expression
synthetic promoter
inducible expression
yeast
splicing regulation
National Science Centre, Poland 10.13039/501100004442 2021/41/B/NZ9/00086
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pmcIntroduction

The current demand for custom-made yeast cell factories forces the need to expand the portfolio of tailored, preferably modular, synthetic biology tools. In this regard, inducible, titratable, tightly regulated promoters are in high demand. Knowing the physiology of Yarrowia lipolytica species, able to utilize lipids and proteins very efficiently, one could expect to find lipid- or organic nitrogen-inducible promoters present in its genome. Indeed, the first explored inducible promoters were: (i) pXPR2 (native for inducible alkaline extracellular protease) induced under a combination of pH, carbon:nitrogen, and peptones (Blanchin-Roland et al. 1994, Madzak et al. 1999, Ogrydziak 2013), and (ii) pPOX2 (Juretzek et al. 2000), activated in the presence of lipids i.a.oleic acid. Both these inducible promoters were later engineered to further customize their performance (Madzak et al. 2000, Shabbir Hussain et al. 2016). Still, due to the complexity of regulation and leakage (pXPR2), or difficulties with the hydrophobic inducer handling (pPOX2), their common use as tightly regulatable and titratable regulatory elements was limited.

Further research into the physiology and genomics of Y. lipolytica facilitated the finding of novel inducible promoters. Foremost, the pioneering studies on the characterization of the ‘erythritol utilization gene cluster’ (Carly et al. 2017, 2018, Rzechonek et al. 2017, Mirończuk et al. 2018) yielded the necessary molecular background. In that series of papers, the key genes deregulated in response to erythritol (ERY) supply were identified, including ERY dehydrogenase (EYD1, YALI0F01650g) (Carly et al. 2018), erythrulose kinase (EYK1, YALI0F01606g) (Carly et al. 2017), and a transcription factor (TF)—EUF1 (YALI0F01562g) governing the expression of the former two (Rzechonek et al. 2017, Mirończuk et al. 2018). Expression of all three genes was shown to be induced under ERY supply: for EUF1 10-fold upregulation was reported (Rzechonek et al. 2017, Mirończuk et al. 2018), for EYK1–41-fold (Carly et al. 2017), and 46-fold upregulation for EYD1 (Carly et al. 2018); holding a promise of very potent, inducible promoters governing their expression.

In-depth studies on the promoters’ architecture enabled the identification of the key DNA motives underlying their ERY-inducible/regulatable character. (Trassaert et al. 2017) studied a 300-bp fragment upstream from the EYK1 gene, and demonstrated that this element (pEYK300) governs ERY-inducible expression, quenched on glucose (GLUC) and glycerol (GLY) media. Two functional sequences were identified within pEYK300–motif A (GGAAAGCCGCY) and motif B (CNTGCATWATCCGAYGAC), followed by the TATA box. Interestingly, further studies, covering an expanded fragment pEYK450 showed that this regulatory element operates in two directions (Vidal et al. 2023) (is a bidirectional promoter), and the centrally located motives AB are flanked with two TATA boxes, operating divergently (towards EYK1 and EYL1 genes). Following studies by Park et al. (2019), revealed a similar architecture of the EYD1 promoter, encompassing motif A (ANTTNNNTTTCCNNATNNGG), a motif B (CGGNNCTNNATTGAGAANNC), and a putative TATA box (GATATAWA).

In the next step, these motives and different core promoters were shuffled to create a set of tuneable, titratable ERY-inducible promoters of various strengths and stringency of expression induction (Trassaert et al. 2017, Park et al. 2019, Vidal et al. 2023); including also—pEYK300 (300 bp of pEYK1, one A motif and one B motif), pEYK3AB (triple A motif), and pEYK5AB (quintuple A motif), used in this study. Their strength and titratability, by both the chemical inducer concentration and the number of upstream functional motives, were studied in detail. Strikingly, the transcriptional activity of the pEYK300-based promoters increased with the increasing number of the A motives, from 1 to 4, which was found to be the key element responsible for ERY-inducibility. Motif B was suggested to be more involved in GLUC repression. However, a further increase in the A motives number did not allow for further promoter titration (Park et al. 2019). Since the overall higher titers of a reporter protein could be reached in that study (when pTEF core was used), the most credible early hypothesis was that insufficient abundance of the ERY-inducible TF limited further titration. Hence, knowing from previous studies on EUF1 (Rzechonek et al. 2017, 2024) (discussed hereafter), and its direct effect on EYK1 expression (Mirończuk et al. 2018), our primary aim was to test if the ERY-inducible promoter could be further titrated by the increased abundance of a compatible TF—EUF1.

Following the former, pioneering study (Rzechonek et al. 2017), a very recent investigation into the EUF1’s regulome (Rzechonek et al. 2024) shed some new light on its operation mechanism. Apart from supporting previous notions on the ‘erythritol utilization cluster’ (EYI1, EYI2, EYK1, and EYD1–all upregulated in the presence of operable EUF1), it was demonstrated that sharp induction (>10-fold) of these genes requires three conditions to co-occur—(i) EUF1 must be operable, (ii) ERY must be present, and (iii) GLY must be absent from the culture medium. The latter suggests the existence of some kind of ‘GLY catabolite repression’ inflicted in the ERY-metabolism regulation in Y. lipolytica (the genes did not reach high upregulation until GLY was present). The requirement for ERY presence suggests that some other phenomena, activated in response to ERY, must occur. Moreover, having the sets of genes deregulated in response to EUF1 action, the authors run regulatory sequences mining in the search for conservative motives (presumably—EUF1-binding sites). The identified motives—the most common ATGCA/reverse TGCAT, followed by CGGAT/reverse ATCCG, and CGGCTT/reverse AAGCCG—were previously identified as elements of motif B (CNTGCATWATCCGAYGAC) and the central fragment of motif A (GGAAAGCCGCY). The previous studies identified the least frequent motif/motif A as the one required for promoter induction by ERY and erythrulose (Trassaert et al. 2017), and the most common motif/motif B as the GLUC repression element; which at the moment seems to stay in contrast to what has been inferred from the transcriptomics analysis. The question raised by the authors was whether EUF1’s biological function goes beyond orchestrating ERY utilization, as the scope of its regulome suggests a more extensive impact.

In our current research, we are interested in harnessing TFs as tools in engineering biotechnologically relevant traits in Y. lipolytica, like stress resistance and recombinant protein (rProt) synthesis (Gorczyca et al. 2023, Korpys-Woźniak and Celińska 2023, Gorczyca et al. 2024). From our high-throughput screens, we have learned that constitutive overexpression of EUF1 (along with a reporter rProt) contributes to significant growth cessation (YaliFunTome database: https://sparrow.up.poznan.pl/tsdatabase/?page=gene&name=TF054), and that the addition of GLUC and inorganic nitrogen source significantly contributed to shaping this phenotype (no ERY was added). In this study, we aimed to test EUF1 as the titrator of pEYK300-based promoters for rProt synthesis, using its native and spliced forms.

Materials and methods

Yeast strains and basic handling conditions

All the strains used in this study are listed in Table S1. Yarrowia lipolytica strains were constructed in the background of a double auxotrophic strain, deleted for major lipases, bearing zeta platform, and foremost—unable to utilize ERY– JMY7126 (here ECY_49) (Park et al. 2019). Complementation of auxotrophy was done by either transformation with a DNA construction or a solo cassette, as indicated in Table S1. If not reverted, auxotrophic strains were cultured in either rich or lysine-supplemented media.

The strains were routinely maintained as glycerol stocks in a −80°C ultrafreezer and freshly plated on YPD (yeast extract, peptone, dextrose) agar plates [(g/l): yeast extract, 10 (Biomaxima, Lublin, Poland), bactopeptone, 20 (BTL, Łódź, Poland), glucose, 20 POCh, Gliwice, Poland), and agar, 15 (BTL)] before experiments.

Molecular biology protocols

Standard molecular biology protocols were used in this study (Barth and Gaillardin 1996, Sambrook and Russell 2001). Restriction digestion of DNA fragments was done using BamHI, AvrII, ClaI, and NotI enzymes purchased from Thermo Fisher Scientific (Waltham, USA). DNA fragment amplification was run using Phire DNA polymerase (Thermo Fisher Scientific) and primers listed in Table S2. All the amplicons to be used as DNA construction parts were initially cloned in pCR Blunt II TOPO vector (Thermo Scientific, Waltham, USA) and restriction digested/sequenced (Genomed, Warsaw, Poland).

Cloning in JMP62 vectors was conducted using 200 U T4 DNA ligase (New England Biolabs, Ipswich, USA). DNA plasmids isolation, DNA fragment gel-extraction, and purification of DNA fragments were all conducted using an appropriate kit from A&A Biotechnology (Gdynia, Poland). All the reactions and protocols were used per the manufacturer’s recommendations.

Escherichia coli JM109 transformations were done according to a heat-shock method. Following transformation, the bacterial strains were plated on LB (Luria–Bertani) medium [(g/l): yeast extract (BTL), 5; bactopeptone (BTL), 10; NaCl (POCh), 5], supplemented with kanamycin or ampicillin [Merck-Millipore, Darmstadt, Germany; at 40 or 100 (µg/ml), respectively] and agar [Biomaxima; 15 (g/l)]. Yarrowia lipolytica was transformed using the lithium acetate heat-shock protocol (Barth and Gaillardin 1996). Yeast transformants were plated on YNB (yeast nitrogen base) medium [g/l: YNB (Sigma-Aldrich/Merck-Millipore), 1.7; (NH4)2SO4 (PoCh), 5; glucose (PoCh), 20; supplemented with lysine at 0.8 g/l, when required]. Clones were selected after 24 h at 37°C for E. coli, and 48 h at 28°C for Y. lipolytica.

Culture conditions and samples analysis

Batch cultivations—media composition and conditions

The precultures and the main cultures were each time conducted in the same medium, composed as follows (g/l): YNB, 5.1; glucose, 50; ammonium sulfate and glutamic acid, 7.5 each; buffered with 0.2 M maleate buffer at pH 5.0 (Gorczyca et al. 2024). Alternatively, where indicated, YPD medium was used as the main culturing medium (g/l): yeast extract, 10; bactopeptone, 20; and glucose, 20. The precultures and the main cultures were run in 24-well Duetz-System square plates in 2.5 ml working volume (EnzyScreen BV, Netherlands), agitated at 250 rpm, at 28°C. The precultures were developed for 18 h at 28°C. The main cultures were inoculated at 10% (v/v) and continued up to 60 h. Samples were collected, and the measurements of fluorescence (FL) and OD600 were done immediately, after applying appropriate dilutions (5–20-fold, in a sterile saline solution).

As indicated, the basic medium YNB could be supplemented with ERY at 5 g/l, to induce expression from the ERY-inducible promoters, or with osmoactive compounds sorbitol (SORB) and ERY (all purchased from POCh or Merck-Millipore) at higher concentrations, to induce an osmotic stress response. As assessed using an osmometer and previously described protocol (Kubiak-Szymendera et al. 2022), the basic medium’s osmolality was 1 Osm/kg. The osmoactive compounds were added in a concentration to reach an osmolarity of 3 or 1.2 Osm/kg, considered ‘high’ and ‘low’ osmolarity, respectively. The amounts of the osmoactive compounds for ‘high’ osmolarity concentration were as follows (g/l): SORB, 360 and ERY, 244. To reach the ‘low’ osmolarity, the loads were 10-fold lower.

Analytical methods

Samples were analysed for growth and FL from the reporter protein (inYFP or RedStar) following dilution in 0.75% NaCl (POCh) to match a linear range of the methods. Absorbance was measured at 600 nm in transparent 96-well plates (Costar; Merck). FL was determined at ex/em 510/550 and 550/595 nm for inYFP and RedStar, respectively, in black opaque plates (Thermo Fisher Scientific). Both measurements were done using a Tecan Spark automatic plate reader (Tecan Group Ltd., Mannedorf, Switzerland).

Determination of GLUC, ERY, SORB, citric acid, and MAN concentrations in the clarified postculturing medium was done by HPLC according to a previously described methodology (Korpys-Woźniak et al. 2020). Briefly, the analysis was run using an Agilent Technologies 1200 series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive-index detector (G1362A) and a Rezex ROA‐Organic Acid H + column (Phenomenex, Torrance, CA, USA). Operating conditions were as follows: 0.005 N H2SO4 as eluent at a flow rate of 0.6 (ml/min); the column temperature was set at 40°C. External standards (Sigma Aldrich) were used for the identification and quantification of the peak areas in chromatograms (analysed using ChemStation for LC 3D software; Agilent).

Transcriptomics data analysis

Transcriptomics datasets reanalysis

The transcriptomics datasets reanalysed in this study were originally deposited in SRA BioProject—NCBI (nih.gov) database under Bioproject number ID 893604 (PRJNA893604) by Rzechonek et al. (2024). The following BioSamples were considered in this reanalysis: BioSample: SAMN31428143; sample name: MK1_gluc_24; SRA: SRS15523360 versus BioSample: SAMN31428139; sample name: MK1_ery_24; SRA: SRS15523356; and BioSample: SAMN31428144; sample name: MK1_gluc_32; SRA: SRS15523361 versus BioSample: SAMN31428140; sample name: MK1_ery_32; SRA: SRS15523357. The two comparisons correspond to differential transcriptomes of Y. lipolytica strain MK1 (operable ERY pathway; functional EUF1) under ERY provision (YNB + 50 g/l GLY + 50 g/l erythritol—samples ‘ERY’) versus its lack (YNB + 50 g/l GLY + 50 g/l GLUC—samples ‘GLUC’) in the culture medium, at 24 and 32 h of culturing.

The datasets were reanalysed (Genomed). De novo intron mapping was conducted according to the previously described methodology (Korpys-Woźniak and Celińska 2023). Briefly, splice site detection was carried out using the SGSeq package (Goldstein et al. 2016) in the R programming language. Mapping of exons and introns was conducted in the IGV program (Robinson et al. 2011). Coverage depth was calculated by SamTools (Li et al. 2009).

The data were also analysed to identify differentially expressed genes (DEGs). Generally, this analysis was conducted according to the previously described protocol (Korpys-Woźniak and Celińska 2021). Briefly, the genes were mapped to a reference genome Y. lipolytica GCF_000002525.2 using Hisat2 software (Kim et al. 2019). Reads for a specific gene were counted using HTseq (Anders et al. 2015). Differential analysis of sequence read count data was conducted using edgeR (Chen et al. 2023). In addition, since only a single biological replication (single run per condition) was made available in Bioproject number ID 893604, extra restrictions to statistical analysis were implemented to improve the significance of the observations. The considered dispersion of biological variation was bcv = 0.4. Only genes showing logFC (FC–fold change between normalized counts) > |3|, at P-value and FDR < .05 were considered significant.

Detection of cis-regulatory elements within pEUF800

Cis-regulatory elements within pEUF800 were detected using MatrixCatch (Deyneko et al. 2013), using the default setting and, P-value correction: Bonferroni step-down, P-value threshold = .05.

Statistical analysis

Statistical analysis was performed using Statistica 13 software (Tibco, CA, USA). After confirming homogeneity of variance (ANOVA), Tukey’s HSD/RIR test was used to identify statistically homogenous groups of data at P < .05. Error bars indicate ± SD of biological triplicate, each analysed in technical duplicate.

Euf1 3D structure modelling and ligand docking

The Euf1 protein structure modelling was done in AlphaFold3 (Abramson et al. 2024), including basic 3D structure modelling, oligomerization, and DNA (motif A, 10 bp) binding. The accuracy of the entire structure was assessed using TM (template modelling) score and ipTM for the dimers (assess the accuracy of the predicted relative positions of the subunits within the complex). Structures were visualized and analysed using PyMol open-source version (Copyright (c) Schrodinger, LLC. Published under a BSD-like license. Version 3.0.3). Structures for further analyses were processed using Pdb tools (Rodrigues et al. 2018). Functional domain prediction was done using the InterPro tool (Paysan-Lafosse et al. 2023). Binding pockets were predicted, scored, and visualized using P2Rank (Krivák and Hoksza 2018, Jendele et al. 2019, Jakubec et al. 2022). The binding pockets prediction accuracy was assessed using: the P2Rank score, probability of pocket, and average conservation. The docking of ligand molecule (ERY/DNA) was executed and scored in Haddock (DNA and ERY) and Prodigy (PROtein binDIng enerGY prediction; only ERY) (Kurkcuoglu et al. 2018, Vangone et al. 2019, Honorato et al. 2021, 2024). Results were expressed in either Haddock score or Gibbs free energy (G). The former metric combines various energy contributions to provide a measure of the quality of the docked complexes (both ERY and DNA), designed to be an indicator of the overall strength and stability of the interaction between the protein and DNA. It combines multiple relevant energy terms, i.e. attractive or repulsive forces between atoms due to van der Waals interactions, electrostatic interactions between charged groups in the protein and DNA—ionic interactions/electrostatic energy, and the energy associated with the removal of solvent (water) molecules from the interaction interface—desolvation energy, combines enthalpy and entropy into a single value. Results of Prodigy analysis (ERY binding) are expressed as changes in Gibbs free energy, ΔG, expressed in kcal/mol. Results ΔG < −9 kcal/mol indicate very high affinity, between −7 and −9 kcal/mol: high affinity, between −5 and −7 kcal/mol: moderate affinity, and > −5 kcal/mol: low affinity.

Results and discussion

EUF1 as a titrator of ERY-induced promoters

To answer the principal question of this study, on further titratability of pEYK300-based ERY-inducible promoters using a compatible TF, the EUF1 gene was cloned and overexpressed under the control of a constitutive promoter pTEF. This way, its load per cell was increased. The effect of the increased abundance of EUF1 on the transcriptional activity of pEYK300-1AB, pEYK300-3AB, and pEYK300-5AB was investigated; which was gauged by FL from the reporter protein cloned under the pEYK300-based ERY-inducible promoters. Transformed strains were cultured with and without the inducer (ERY+/ERY−) in the basic (YNB) and rich medium (YPD). Results on normalized FL (sFL–specific FL) are shown in Fig. 1 and Table 1. Foremost, we observed that overexpression of unspliced (full) EUF1 yielded no significant differences when compared to the control variants (‘Lys5-pEYK300-xAB’; FC below or close to 1.0 in Table 1). On the other hand, the increased load of spEuf1 significantly enhanced transcription from the pEYK-based promoters, reaching over 3-fold higher rProt amounts, in specific cases (Table 1, Fig. 1). Notably, the results obtained in the YNB medium were more consistent than those from the YPD medium. In the former, a clear effect of spEUF1 overexpression could be seen, and a low background level from the control and fullEUF1 variants. This differentiation of the effects reached in different media highlights the importance of nutrient availability for the investigated phenomena; which were depleted from the YNB variant at the time of sample collection (HPLC results, not shown; elaborated on and experimentally verified in more detail hereafter).

Figure 1. Titration of ERY-inducible/Euf1-dependent promoters (pEYK300-1AB/3AB/5AB) under co-overexpression of full (fullEUF1) or spliced (spEUF1) TF and ERY provision (ERY+; dark blue, solid) or absence (ERY−; light blue, framed) in minimal (YNB; A) or rich (YPD; B). Variant ‘Lys5−’ indicates prototrophic control without the TF co-overexpression. Y-axis: protein synthesis capacity defined as raw fluorescence readouts normalized per biomass, expressed as sFL. The type of promoter regulating expression is indicated in the x-axis. Bars indicate mean values from at least four biological replicates (four different subclones), each read in technical duplicate ± SD.

Table 1. Normalized amounts of the reporter protein (RedStar) expressed under ERY-inducible/Euf1-dependent promoters (pEYK300-1AB/3AB/5AB) under co-overexpression of full (fullEUF1) or spliced (spEUF1) TF and ERY provision (Inducer+) or absence (Inducer−) in minimal (YNB) or rich (YPD) medium. The sFL values were calculated based on fluorescence readouts FL (sFL—specific FL) normalized per biomass. FC—fold change values for sFL read for corresponding strains and conditions, differing in the indicated measures: number of ‘A’ motives 3AB versus 1AB, or 5AB versus 3AB, or in the type of co-overexpressed TF EUF1–spliced (spEuf1) or its lack (‘control’).

Strain	Inducer ±	Medium	sFL	FC 3AB versus 1AB	FC 5AB versus 3AB	FC sp/fullEUF1 versus control	
fullEUF-pEYK300-1AB	ERY−	YNB	4922.3			0.83	
spEUF1-pEYK300-1AB	ERY−	YNB	6458.2			1.09	
Lys5-pEYK300-1AB	ERY−	YNB	5941.4				
fullEUF-pEYK300-3AB	ERY−	YNB	6371.6	1.29		0.74	
spEUF1-pEYK300-3AB	ERY−	YNB	28080.8	4.35		3.25	
Lys5-pEYK300-3AB	ERY−	YNB	8640.7	1.45			
fullEUF-pEYK300-5AB	ERY−	YNB	11605.1		1.82	0.82	
spEUF1-pEYK300-5AB	ERY−	YNB	41197.2		1.47	2.90	
Lys5-pEYK300-5AB	ERY−	YNB	14225.1		1.65		
fullEUF-pEYK300-1AB	ERY+	YNB	5466.2			0.97	
spEUF1-pEYK300-1AB	ERY+	YNB	15565.7			2.77	
Lys5-pEYK300-1AB	ERY+	YNB	5620.0				
fullEUF-pEYK300-3AB	ERY+	YNB	8356.5	1.53		0.67	
spEUF1-pEYK300-3AB	ERY+	YNB	41084.5	2.64		3.29	
Lys5-pEYK300-3AB	ERY+	YNB	12499.3	2.22			
fullEUF-pEYK300-5AB	ERY+	YNB	14970.8		1.79	0.86	
spEUF1-pEYK300-5AB	ERY+	YNB	46213.7		1.12	2.65	
Lys5-pEYK300-5AB	ERY+	YNB	17470.9		1.40		
fullEUF-pEYK300-1AB	ERY−	YPD	9652.6			1.09	
spEUF1-pEYK300-1AB	ERY−	YPD	27578.9			3.11	
Lys5-pEYK300-1AB	ERY−	YPD	8873.1				
fullEUF-pEYK300-3AB	ERY−	YPD	8132.6	0.84		0.54	
spEUF1-pEYK300-3AB	ERY−	YPD	45050.9	1.63		2.97	
Lys5-pEYK300-3AB	ERY−	YPD	15153.4	1.71			
fullEUF-pEYK300-5AB	ERY−	YPD	19186.0		2.36	0.93	
spEUF1-pEYK300-5AB	ERY−	YPD	34610.0		0.77	1.68	
Lys5-pEYK300-5AB	ERY−	YPD	20563.4		1.36		
fullEUF-pEYK300-1AB	ERY+	YPD	13823.1			1.37	
spEUF1-pEYK300-1AB	ERY+	YPD	21437.9			2.12	
Lys5-pEYK300-1AB	ERY+	YPD	10117.9				
fullEUF-pEYK300-3AB	ERY+	YPD	12318.4	0.89		0.68	
spEUF1-pEYK300-3AB	ERY+	YPD	34777.2	1.62		1.91	
Lys5-pEYK300-3AB	ERY+	YPD	18208.3	1.80			
fullEUF-pEYK300-5AB	ERY+	YPD	19264.7		1.56	1.23	
spEUF1-pEYK300-5AB	ERY+	YPD	36960.9		1.06	2.35	
Lys5-pEYK300-5AB	ERY+	YPD	15710.5		0.86		

Considering the better controllable variant (YNB), in all the analysed cases (overexpression of sp/fullEUF1 or its basic, native expression in Lys5 prototrophs) the amounts of rProt cloned under pEYK300-1/3/5AB increased with the increasing number of A motif (pEYK300 < pEYK300-3AB < pEYK300-5AB) (Table 1). This observation is consistent with previous notions by (Trassaert et al. 2017, Park et al. 2019). That effect was independent of the introduced genetic modification (sp/full EUF1 or sole auxotrophy complementation). The variant with 3A motives rendered from ∼30% to over 4-fold more of the sFL units than the basic variant (1A). The incorporation of two further A motives (the 5A variant) triggered ∼12% to ∼80% improvement over the 3A. This notion corresponds to the previously stated problem, that further promoter titration by engineering its architecture is not linear and some upper limit was reached. This study shows that even upon enhanced availability of the main titrator, Euf1, this effect cannot be overcome. It could be speculated that intrinsic limitations make the transcription from pEYK-5AB saturated, and further titration cannot be achieved. Our observation stays in sharp contrast to the results shown by Blazeck et al. (2011) demonstrating an unlimited, linear increase in the transcriptional activity of the promoters with 1 to (at least) 24 upstream activating sequences.

Some leakage of the pEYK300-3AB/5AB promoter was observed in the absence of ERY, especially in the case of the pEYK300-5AB promoter (light blue bars in Fig. 1). Strikingly, the promoter leakage was unexpectedly high when spEUF1 was overexpressed. This surprising observation was ‘titratable’—higher sFL amounts were reached with increasing promoter strength, in the absence of the chemical inducer (r = 0.99), indicating that now the number of A motives became limiting. Nevertheless, the new ERY-less system with spEUF1 as the inducer although it meets the requirements of being titratable by the number of A motives, is not inducible, so it is not useful as a controllable regulatory part for synthetic biology.

Altogether the above experiment showed that overexpression of spEUF1 allows for further titration of the pEYK300-based promoters; and indeed, some upper limit of the ERY-induced rProt synthesis is reached above 3A motives in the promoter structure. Strikingly, an increased abundance of the spliced variant of EUF1, spEUF1, is sufficient to activate expression from pEYK300-3AB/5AB promoters in the absence of the inducing chemical compound. Such a phenomenon suggests some association between the inducer and the splicing event. The important questions raised after these experiments were whether ERY is the actual factor inducing the splicing event, and what ERY-responsive molecular scissors catalyse this phenomenon, if any. But before investigating these, the peculiar effect of high-nutrient availability had to be addressed.

EUF1-dependent expression in relation to carbon source load and under different chemical inducers

To investigate in more detail the observed differences in the pEYK-based promoters’ behavior depending on the nutrient availability (YNB versus YPD media, Fig. 1A and B), and instructed by previous studies (Rzechonek et al. 2024) suggesting the importance of carbon source load on ERY-inducible phenomena, we designed an experiment in which the pEYK300-1AB promoter’s activity was tested in the presence or absence of carbon source (GLUC), and under supplementation with different concentrations of ERY as well as SORB. The former was used as a specific inducer, while SORB, to inflict osmotic stress. It was interesting for us to investigate if the EUF1-dependent expression is specific to ERY or is responsive to osmostress as well; since hyperosmolality is commonly used as an inducer of ERY synthesis (Yang et al. 2015, Rakicka-Pustułka et al. 2020). Two different fluorescent reporter proteins were used to gauge the pEYK300 transcriptional activity (RedStar and inYFP). In addition, promoter pEUF800 (800 bp upstream from EUF1’s ATG) was considered in this analysis, as we identified the key A and B motives in its sequence (sequence analysis is presented hereafter in this section). Of technical note relevant to this experiment, SORB was not consumed, nor produced; ERY was not consumed (due to Δeyk1 genotype) and not synthesized above 1 g/l (HPLC data not shown). The strains were initially grown in the YNB basic medium, and after reaching the stationary phase of growth they were washed, and inoculated in media with or without a carbon source, in the presence or absence of ERY and SORB at different concentrations. The results of this experiment in terms of specific rProt synthesis are shown in Fig. 2 and Table 2.

Figure 2. Normalized amounts of a reporter protein (inYFP/RedStar) expressed under different promoters (pTEF, pEYK300, and pEUF800) synthesized after 1 h (A), 3 h (B), 6 h (C), and 24 h (D) of incubation in the presence (GLUC+; dark blue, solid) or absence (GLUC−; light blue, framed) of glucose, in media supplemented with ERY at 5 g/l (ERY5), 24 g/l (ERY24), and 240 g/l (ERY240), or SORB at 36 g/l (SORB36), and 360 g/l (SORB360)—panels from left to right; indicated in the x-axis. Data are mean values from at least two (typically six) biological replicates (different subclones), each read in technical duplicate ± SD.

Table 2. FC in the fluorescence from a reporter protein (inYFP or RedStar expressed from pTEF, pEYK300, or pEUF800 promoters) read in media supplemented with chemical inducers (ERY/SORB at different concentrations: 5, 24, 240, 36, and 360 g/l, as indicated in the conditions’ name) without glucose (GLUC−) over the readout in the control medium with glucose (GLUC+) at 20 g/l. Values were colour-coded for easier evaluation (blue—the lowest; red—the highest).

Data presented in Table 2, showing FC of sFL read in media devoid of glucose over those with glucose at 20 g/l, confirmed our hypothesis. Indeed lack of carbon source acts as a strong inducer of expression from pEYK300 in the presence of ERY (all concentrations tested) and SORB (36 g/l). The increase in sFL was gradual along the incubation time, reaching a very high level of 8–10-fold higher sFL at 6 h of incubation (ERY5 and ERY24). It decreased afterwards at 24 h, because of glucose consumption in the control variant (with initial glucose at 20 g/l). The results of this experiment should be considered in industrial practice exploiting pEYK300-based Y. lipolytica expression platforms, by adopting the final starvation stage. Another relevant point would be removal of the motif B from the pEYK-based synthetic promoters to eliminate the ‘glucose/glycerol catabolite repression’ mechanism.

The highest, genotype-based induction of inYFP/RedStar synthesis was observed for pEYK300-driven expression under exposure to ERY at 5 and 24 g/l and, surprisingly, SORB at 36 g/l (Fig. 2C, Table 2), meaning that SORB can be considered an alternative chemical inducer of the pEYK-based systems in Y. lipolytica, without the need for Δeyk1 background. Yet, the concentration must be subjected to optimization. In general, higher concentrations of the chemical inducers (240 and 360 g/l) contributed to a decrease in the rProt levels in this strain. To our interpretation, the profile of sFL levels across increasing concentrations of the inducers resulted from the combination of the following: (i) under severe osmotic stress transcriptional activity of the promoters is quenched, as the cell promotes the osmolyte synthesis (and not consumption) to survive and (ii) under these conditions, the global cellular stress response drives silencing of rProts synthesis via multiple ways (experimentally defined at transcriptional and proteomics level previously; Kubiak-Szymendera et al. 2022). In this context, we would like to explain that the uniform enhancement in the fluorescence under the SORB360 condition (Fig. 2) is a result of enhanced background fluorescence rather than genotype-driven observation. Even though the excitation/emission wavelengths were carefully optimized for inYFP and RedStar readouts in our basic medium, under exposure to SORB360, some compounds interfering with the fluorescence readout at the wavelengths used are synthesized. It is known, that exposure to hyperosmolarity induces synthesis of i.a. proline or betaine, which could contribute to that interference (Thomas et al. 1994).

Trying to understand the unexpected lack of significant induction of pEUF800 under ERY provision, we analysed the promoter’s sequence in search of known ERY-related motifs. The whole intergenic space between ATGs of EUF1 (YALI0F01562g) and, located upstream, YALI0F01540 g is slightly more than 3100 bp. Previously, for ERY-inducible genes (EYK1, EYL1, and EYD1), typically 300–500 bp upstream from a gene’s ATG were considered sufficient to be used as a synthetic promoter (Trassaert et al. 2017, Park et al. 2019, Vidal et al. 2023). Within this pEUF800 region, a motif with a consensus sequence GGGAAAGTTTT was found seven times; and that was the most frequent cis-binding element within that region. Considering the cis-elements earlier identified as associated with ERY induction (box A, AAGCCG, or box B, ATGCA/ATCCG; Trassaert et al. 2017, Park et al. 2019, Rzechonek et al. 2024), a single motif A was found at position −276 upstream from ATG, while an element of box B (ATCCG) was found in its proximity—at position −230 (and beyond the selected fragment −2804). On the other hand, another piece of the core element of motif B (ATGCA that was found in the promoter region of all EUF1-regulated genes; Rzechonek et al. 2024), was identified in the promoter region of the other gene, sharing the intergenic region, YALI0F01540 g (−2206 and −3194 bp upstream from EUF1’s ATG), but not in pEUF800. A core consensus sequence of STRE motif (AGGGG; Estruch 2000) was identified at position −969, and its reverse (CCCCT) twice, at −563 and −1017. It is known that the STRE is functional in both orientations, and the core sequence is strictly conserved (with no restrictions on the neighbouring). The presence of a STRE-like sequence in the promoter does not imply the functionality of this element. It is known that the basic stress-induced activation is then observed, however, additional copies enhance that effect more than in an additive manner. Three copies of STRE in the pEUF800 suggest its strong upregulation in response to environmental stress (like SORB360). Interestingly, a single copy of the STRE motif was also found in the promoter region of pEYK300, approximately 100 bp upstream ATG, which could explain its upregulation in response to SORB36.

In summary, although pEUF800 contains all the necessary DNA motives to be regulated in response to ERY and, according to the just demonstrated glucose-repression mechanism, it remained transcriptionally inactive. We speculate that maybe the length of the promoter was for some reason inadequate, or some important regulatory element is contained in the EUF1’s intron, localized at the 5′ terminus of the gene. The intron was not cloned in our experiment, as immediately downstream from the pEUF800, the reporter gene was cloned.

In our auxiliary experiment (Fig. S1) on the expression level of native EUF1 from its native promoter, we observed very high (>10-fold) upregulation of expression under SORB240 condition; demonstrating the operability of the promoter, and its inducible character. Surprisingly, no such tremendous increase was observed under high ERY provision (to the same osmolality level) (Fig. S1; more data and discussion on EUF1’s expression under ERY supplementation will be given hereafter based on transcriptomics data). Previous studies have shown that ERY indeed has a very distinctive role in Y. lipolytica’s response to osmostress, as it provides resistance to hyperosmolality that is independent of the HOG pathway (Rzechonek et al. 2020). No other osmoprotectant (proline and trehalose) could account for such an effect. In our previous studies, the expression of EUF1 was also slightly upregulated (logFC ∼0.3; significant at P < .05) in response to oxidative stress, when ERY was not present in the culture medium (Korpys-Woźniak and Celińska 2021).

In summary, the above experiment demonstrated the key role of carbon source load on transcriptional activity from the EUF1-dependent, ERY-inducible promoters. The experimental data and the promoter sequence structure suggest that the ERY-inducible genes are, at the same time, GLUC/GLY-repressible genes. Further insight and a functional-mechanistic model of the promoter's activity are provided below.

Splicing pattern of EUF1

To answer the question on putative ‘ERY-induced splicing’ of EUF1 transcript, we set out for chemostat cultures under ERY + and ERY− conditions to analyse the associated transcriptomes and map the splicing events within the EUF1 transcript. In parallel, the other authors published datasets of transcriptomics analyses of Y. lipolytica strains maintained in batch bioreactor cultures in the presence of ERY or GLUC run in GLY-based medium (Rzechonek et al. 2024). RNAseq was conducted on samples collected at 24 and 32 h of the batch culture in a GLY-based medium (50 g/l of GLY) supplemented with ERY at 50 g/l (ERY24 and ERY32) and GLUC at 50 g/l (GLUC24 and GLUC32). The comparisons presented there, could not answer our questions, but the available raw datasets could be reanalysed to match our experimental plan.

Here, the splicing events in EUF1 were mapped de novo, based on the predicted junction sites and transcript coverage depth. The transcript coverage depth analysis mapped the junction sites corresponding to those identified previously using bioinformatics prediction (gene start: coordinate: 243 493, followed by 110 bp exon 1; junction site coordinates: 243 604 followed by 78 bp intron; junction site coordinates: 243 683 followed by 2744 bp exon 2; gene end coordinate: 246 427). The following analysis aimed at determining the ratio between the two forms (spliced/unspliced) in respective samples: GLUC 24 h of culturing, ERY 24 h, GLUC 32 h, and ERY 32 h. The results are shown in Fig. 3 and Table S3.

Figure 3. Splicing frequency of EUF1 in the transcriptomes of Y. lipolytica cells in batch cultivations in the presence of glucose or erythritol at two different time points (differing in the carbon source concentration). Datasets published by Rzechonek et al. (2024). Raw datasets were reanalysed. GLUC, glucose, ERY, erythritol, GLYC, glycerol, time of culturing indicated in the sample name (−24 h and 32 h), the concentration of a given chemical compound is marked as a number following a specific metabolite, e.g. ERY50—erythritol at 50 g/l. Data labels indicate a normalized count of spliced forms of EUF1 transcript.

The highest fraction of spliced EUF1 transcripts was detected in samples from the stationary phase of growth when ERY was contained in the medium at ∼50 g/l, while GLY was completely depleted [please refer to Fig. 2 in Rzechonek et al. (2024)]. Considering the normalized amounts of the transcript, the calculated ‘count’ of the spEUF1 transcript was 4.42 units in that sample. In the previous time point (end of exponential growth phase) the ERY concentration was the same, but GLY was still present at 20 g/l (Rzechonek et al. 2024). In that sample, the EUF1 splicing was more frequent, considering the percentage of spliced versus unspliced transcript, but the global counts of spEUF1 mRNA were nearly halved (2.37 units). The difference comes from i.a. the difference in the expression level of EUF1, which was 1.69-fold higher in the stationary phase of growth when ERY was the sole carbon source (logFC; induction is NS at P < .05; Table S3). To our understanding, the splicing machinery could operate at an equal pace, but the number of mRNA moieties was higher, so the splicing event could become the limiting step. The EUF1 gene was also expressed in GLUC samples when ERY concentration in the medium was close to 0–1 g/l [values read from Fig. 2. in Rzechonek et al. (2024)]. In our previous studies, we also observed its expression in the absence of ERY (Korpys-Woźniak and Celińska 2021, 2023). Contrary to our presumptions, EUF1 splicing occurred also in the GLUC32 sample (ERY at 1 g/l and GLUC close to 40 g/l); in that sample, 2.44% of EUF1 transcripts were spliced. The expression level and splicing frequency were the lowest in GLUC24 samples; still, both processes were operable also in the absence of ERY and high concentration of GLUC and GLY (Fig. 3).

Altogether these findings suggest that EUF1 is expressed at some basic level without ERY as a chemical inducer (more data provided hereafter). Some upregulation in expression takes place when: (i) ERY is present and (ii) other carbon sources are depleted. The EUF1 transcript’s splicing frequency is higher in the presence of ERY, but the process is also active in the absence of ERY. So, the splicing event cannot be considered as ‘ERY-induced’. Considering calculations of the spEUF1 transcript counts, it can be seen that the splicing events are promoted in the presence of ERY, but they are not strictly dependent on its presence.

Effect of EUF1 splicing on its 3D structure and ligand-binding affinity

If (as evidenced above) EUF1 is expressed and spliced either in the presence or absence of ERY and carbon source (GLUC/GLY) and the two forms of transcript coexist in the cell, it is highly probable that both forms are also translated (the intron lacks a stop codon) and hence full and spliced protein structures are present in the cell. So, we were interested in how the splicing event affects the protein structure, its DNA (motif A)-binding affinity, and if the ERY-binding domain could be identified. Therefore, we modelled full and spliced Euf1, docked the DNA and ERY ligands, and assessed the energy of binding (Fig. 4, Table 3; Supplementary Material Files). The modelled monomeric structures (fullEuf1 and spEuf1) received TM scores of 0.49 (Fig. S2, upper panels). Based on the primary sequence of the polypeptides we knew that Euf1 belongs to Zn(II)2Cys6 (Zn2C6) proteins. These are classified in class III (C6) zinc finger proteins, containing a DNA-binding domain that consists of six Cys residues bound to two Zn atoms. The following InterPro analysis enabled identification of the following three functional domains: (i) Zn2Cys6 DNA-binding domain (fullEuf1: 25–85 AA residues; spEuf1: 24–67 AA), (ii) Fungal TF domain (fullEuf1: 305–584 AA residues; spEuf1: 279–558 AA), and (iii) disordered regions (fullEuf1: 1–27, 112–220, 606–646, 757–789, 833–887, and 914–960 AA; spEuf1: 1–17, 86–194, 580–620, 739–763, 809–861, and 888–934 AA). Since the Zn2C6 proteins very frequently operate as dimers, after the initial modelling of monomers, we modelled homodimers as well (Fig. S2). The dimeric structures received TM scores of 0.52–0.54 for fullEuf1 and spEuf1, respectively (Fig. S2), indicating better ranked structures as dimers than monomers. The dimeric structures possess the double Zn-fingers, creating an elegant space for DNA binding, particularly visible for spEuf1 in (Fig. 4).

Figure 4. The 3D structure of fullEuf1 (upper row) and spEuf1 (bottom row) TF. The Euf1 structure is modelled as a dimer (monomers—light green and turquoise), Zn2Cys6 domains are shown in red. Docking of ERY (purple; panel B) to one of the two monomers, and DNA to double Zn-fingers (purple; panel C) is shown by a white arrow. The most conservative binding pocket is shown in panel D. in blue/red at the interface of the two monomers' interaction.

Table 3. The energy of interaction between sp/fullEuf1-modelled protein and ligands: ERY and ssDNA (10 bp of motif A). Prediction of binding pockets in sp/fullEuf1-modelled protein and corresponding scoring and parameters of the predicted binding pockets.

Energy of interaction	
	fullEuf1-dimer	spEuf1-dimer	
	+ssDNA	+ERY	+ssDNA	+ERY	
HADDOCK score  *	88.7 ± 4.8	−15.7 ± 0.8	6.0 ± 8.8	−12.0 ± 0.3	
Van der Waals energy	−99.2 ± 0.6	−10.6 ± 0.8	−81.0 ± 3.9	−7.7 ± 0.8	
Electrostatic energy	−156.9 ± 26.1	0.1 ± 0.7	−34.6 ± 11.4	−0.7 ± 0.3	
Desolvation energy	32.8 ± 4.4	−5.0 ± 0.2	19.1 ± 0.9	−4.2 ± 1.0	
Sum of energies	−223.3	−15.5	−96.5	−12.6	
Buried surface area	1651.7 ± 95.8	251.1 ± 4.7	1274.1 ± 55.5	200.6 ± 4.8	
Interaction domain	Targeted at Zn-finger	Disordered	Targeted at Zn-finger	Disordered	
Progidy ΔG**		−5.37		−5.26	
Binding pockets prediction	
P2Rank  ***	fullEuf1-dimer	spEuf1-dimer	
Rank	1		1		
Score	17.01	8.9	
Probability	0.792	0.523	
Number of residues	32	25	
Average conservation score	1.773	1.579	
* HADDOCK score is an overall energy term that combines various energy contributions to provide a measure of the quality of the docked complexes, designed to be an indicator of the overall strength and stability of the interaction between the protein and DNA. In general—lower HADDOCK scores indicate better binding affinity. It combines multiple relevant energy terms, i.e. attractive or repulsive forces between atoms due to van der Waals interactions, electrostatic interactions between charged groups in the protein and DNA—ionic interactions/electrostatic energy, and the energy associated with the removal of solvent (water) molecules from the interaction interface—desolvation energy. BSA refers to the surface area of the protein and DNA that becomes inaccessible to solvent (water) upon complex formation.

** Score expressed in changes in Gibbs free energy, ΔG, expressed in kcal/mol. ΔG between −5 and −7 kcal/mol indicates moderate affinity. Prodigy enables scoring protein–small ligand interaction. DNA is not accepted.

*** P2Rank score. Average conservation (scale 0–4; the range of the per-residue of information content with higher values corresponding to higher conservation).

Interestingly, substantial differences in the 3D structures between fullEuf1 and spEuf1 dimers were noted (Fig. 4, Table 3; Supplementary Material Files). In the former, the Zn2C6 domains are localized at high distances, on the opposite sites of the dimer; only after DNA motif A docking, do the Zn fingers come together in spatial proximity. In contrast, the 3D structure of spEuf1, even devoid of DNA ligand, presents the Zn2C6 domains immediately next to each other, resembling a ‘DNA-binding competent state’. Only a small spatial change happens when docking the DNA motif A to spEUF1. Furthermore, regarding the strength and stability of the interaction between the TF and DNA, the dimer of spEuf1 received much better scores in this matter than the one involving fullEuf1 (Table 3; Haddock score 88.7 ± 4.8 versus 6.0 ± 8.8 for full and spliced Euf1; the lower Haddock score, the better binding affinity); suggesting the indeed spEuf1 has better propensity for DNA binding.

In terms of ERY binding, it was predicted to dock on the surface of a disordered region in both dimers, fullEuf1 and spEuf1. While it is well-documented that the disordered regions possess an important regulatory role, here, ERY binding in such a region was weak and random (Table 3); rather forced by the user, than nature. The energy of interaction estimated by Prodigy was ΔG between −5.26 and −5.37 kcal/mol, indicating moderate/low affinity. Moreover, ERY docking was predicted to localize at the disordered regions, even though several binding pockets were predicted within sp/fullEuf1 (Fig. 4). Another interesting difference between structures fullEuf1 and spEuf1 was revealed regarding the parameters of the most-highly ranked predicted binding pockets (by P2Rank; Table 3). For both dimeric structures, the primary binding pocket was localized on the opposite sites of the 3D structure in relation to Zn-fingers, however, the overall score, probability, and span were all higher for the fullEuf1 structure, when compared to its spliced variant. Notably, this domain becomes substantially modified after Euf1 splicing—it is predicted with significantly lower probability, is smaller, and less conserved. Considering the very good ratings of the binding pocket predicted for fullEuf1, and the results presented in Fig. 1, we carefully suggest that this binding domain plays an important role in functional regulation of fullEuf1, by binding a regulatory molecule. Due to structural changes of the binding pocket in spEuf1, the spliced variant may lose its regulatable character, as shown in Fig. 1. Further research into the identification of a potential interaction partner is foreseen. Preliminary computational assessment of a potential interaction with another TF inflicted in pEYK1 promoter regulation, Adr1 (discussed hereafter), suggest that these two proteins do not interact with each other via the binding pocket identified (Fig. S3).

Altogether these data suggest that Euf1 operates as a dimer, forming a double Zn-finger domain. When in its spliced variant, it is in a constitutive ‘DNA-binding competent state’. ERY ligand most probably does not directly interact with the Euf1 dimer, either if formed by fullEuf1 or spEuf1; no domain for such an interaction was predicted. On the other hand, an interesting change to the predicted binding pocket (localized on the opposite side of the 3D structure to Zn fingers) happens when comparing the full and spliced Euf1. Based on functional studies, and the predicted structural changes we propose that this binding pocket plays an important role in functional regulation of fullEuf1, which is lost in spEuf1.

DEGs under spEUF1 abundance

To explain the unexpected lack of pEUF800-driven induction of inYFP synthesis in the presence of ERY (Fig. 2), we dug deeper into the previously obtained transcriptomics data (Rzechonek et al. 2024) in search of the EUF1 gene expression profile. In the current analysis, we compared samples ERY24 versus GLUC24 and ERY32 versus GLUC32 (such comparisons were not presented previously). To our surprise, EUF1’s expression level (logFC) in the ERY medium (50 g/l) was only 0.14-fold (logFC) higher at 24 h and 1.69-fold (logFC) higher at 32 h, and the increase was not statistically significant (NS at P and FDR < .05; Table S3). Just to double-check that observation, we conducted an auxiliary RTqPCR gene expression analysis of EUF1 gene in the cultures with high (244 g/l) and low (24 g/l) ERY load versus growth in sole GLUC medium. The results are shown in Fig. S1(B). Notably, at the sample collection time-point, the main carbon source (GLUC) was still present in the culture medium, as in ERY24 sample from Rzechonek et al. (2024). Our RTqPCR and (Rzechonek et al. 2024) transcriptomics assessment of EUF1 expression level well aligned, showing a lack of significant induction of EUF1 expression in the presence of ERY, at least until the alternative carbon source is still present in the culture media.

Such an observation well corresponds with the data presented in Table 2 and Fig. 2, where pEUF800-driven expression of inYFP was nonsignificant under ERY supplementation. It seems that the EUF1 expression level is only slightly induced under ERY provision, but its activation is a result of both—this slight induction and the splicing event (Fig. 1). In pioneering studies on EUF1 (Rzechonek et al. 2017), it was demonstrated the expression of EUF1 is over 10-fold higher in the presence of ERY (50 g/l), which seems to stay in contrast to what was found in our current research and the transcriptomics (Rzechonek et al. 2024). However, minor technical differences in the experimental setup could account for the apparent discrepancy. Foremost, the previously observed high upregulation was observed in Y. lipolytica cultures grown in ERY as the sole carbon source. The only sample corresponding to that former condition is ERY32, however, GLY depletion could have happened just before the sample collection, and the condition of sole ERY provision could have just been initiated. Secondly, the RTqPCR primers designed in that previous study flanked the 78 bp intron at the 5′ end rendering two amplicons—178 bp versus 256 bp (Fig. S1C) and the target gene counts were normalized per ACT1 (actin) gene. We used primers attached close to polyA tail (within 300 bp from the stop codon), and normalized the target transcript's counts on SEC61, as the internal calibrator, according to our previous results (Borkowska et al. 2020). All these small details could collectively contribute to the observed differences.

Finally, we looked for DEGs that exhibited differential expression under ERY addition versus GLUC variants at the corresponding time points. Only seven genes met our criteria and were considered significantly upregulated in ERY24 samples versus GLUC24. Three of them were the known ERY-utilization cluster members—EYK1—up by 6.99-fold, EYD1—5.14-fold, and EYI1—4.35-fold upregulation. The other upregulated DEGs were three members of the unknown family of secreted proteins YALI0A02783g, YALI0A13497g, and YALI0B06160g. A blastp similarity search indicated that the former is involved in the cell wall integrity pathway (Kamei et al. 2016). The last DEG upregulated at 24 h was identified as a TF YALI0F21923g, highly similar to Saccharomyces cerevisiae’s Adr1, a carbon-source-responsive zinc-finger TF, induces transcription of GLUC-repressed genes and promotes utilization of the alternative carbon sources (in S. cerevisiae—ethanol, GLY, and fatty acid). Here, the transcript of YALI0F21923 g was overrepresented in sample ERY24, where a mixture of GLY (20 g/l) and ERY (~50 g/l) was contained in the medium, versus control—GLUC24 (mix of GLY ∼25 g/l and GLUC ∼50 g/l). In the following time-point (ERY ∼50 g/l, no GLUC, no GLYC), its expression was elevated in ERY32 versus GLUC32, but not significantly different (logFC 2.76, but NS at P and FDR < .05). So, either the gene’s expression is repressed in the presence of GLUC, or—it is induced by ERY. Our previous studies involving YALI0F21923 g revealed that its overexpression supports efficient biomass formation and carbon utilization, but contributes to limited synthesis of recombinant protein (Gorczyca et al. 2024) (YaliFunTome database: TF116). It would be interesting to experimentally verify if this TF physically interacts with motif B, postulated to be involved in catabolite repression (Trassaert et al. 2017).

In the following time-point, altogether 41 DEGs were identified—35 upregulated and 6 downregulated (Table S3). Again, the ERY-utilization cluster genes EYK1, EYD1, and EYI1 (5.9-, 5.6-, and 5.1-fold upregulation) were found amongst the most upregulated genes. But still, even though the other carbon source was nearly completely depleted, the EUF1 gene upregulation level remained relatively low (1.64-fold; not statistically significant). A full list with assigned biological functions is provided in Table S3.

To answer the second question raised regarding EUF1’s operation in Y. lipolytica (about the ‘molecular scissors’ managing the EUF1’s splicing) we searched the deregulated genes for such that could be involved in the 78-bp intron removal. We expected the gene to be upregulated in ERY presence. None of the significantly deregulated genes had assigned or putative functions involving RNA splicing. As the significance criteria applied in this analysis were very stringent, we looked also at the remaining genes, identified but nonsignificantly deregulated. Directed by the pioneering study on splicing in Y. lipolytica (Mekouar et al. 2010), we specifically searched for U1 RNAs (YALI0B20936r and YALI0B14567r), and elements of mechanism degrading the intron-retaining transcripts UPF1/2 (YALI0E24629g and YALI0D23881g). In none of the ERY samples (24 or 32) the genes were significantly deregulated. The UPFs were slightly upregulated in ERY32 (∼0.5 logFC), as was U1 RNA’s component YALI0B20936r (to logFC 0.31), suggesting that the splicing-related mechanisms were intensified.

Altogether reanalysis of the transcriptomics data under here desired configuration showed that EUF1 expression is not immediately induced once ERY is provided, but requires an extra requirement to be fulfilled, namely the lack of another carbon source. In fact, its transcription is active, even in the absence of the compatible, chemical inducer; likewise, its splicing. Interestingly, apart from the typical downstream targets of Euf1 belonging to the ‘erythritol utilization cluster’, another TF, Adr1, was deregulated in the ERY/GLUC differential transcriptomes. We postulate that it may play a key role in the GLUC-dependent regulation of pEYK promoter, demonstrated in section 3.2. A putative mechanism of the two TFs’ interaction within the pEYK structure is provided below.

Summary and conclusions

In summary, this study shows that while overexpression of spEUF1 enabled higher transcriptional activity of the synthetic pEYK-based promoter, it practically eliminated its inducible character. Contrary to expectations, higher loads of spEUF1 could not further significantly titrate (by more than 50%) the pEYK300-5AB promoter, surpassing the rProts yields reached with pEYK300-3AB. It is plausible that the metabolic burden imposed by high-level overexpression of the reporter and the titrator contributed to overall lower protein yields. Such overloading-based limitations have been shown and investigated previously in our studies (Gorczyca et al. 2022). Furthermore, we demonstrated that native expression of EUF1 is not significantly induced by ERY—an increase in the expression level is observed, but is not statistically significant, as demonstrated by RTqPCR and RNAseq analyses. Likewise, the EUF1 splicing event, while promoted in the ERY presence, was also active when the chemical inducer was absent from the experimental system. No ERY-induced spliceosome element was identified in the omics data, suggesting either their constant presence in the cells or induction of the EUF1-specific splicing event by some other factor than ERY.

The most striking observation of this study is that EUF1-dependent, ERY-inducible genes are at the same time GLUC/GLY-repressed; and that lack of a carbon source (either GLUC or GLY) is a strong inducer of ERY-dependent expression in Y. lipolytica. Based on (i) our current results (Fig. 2), (ii) the promoter sequence of the ERY/EUF1-dependent deregulated genes (Trassaert et al. 2017, Rzechonek et al. 2024), and (iii) the reanalysed omics data we propose a mechanism for the ERY-inducible-GLUC/GLY-repressible genes regulation, involving combined activity of spEuf1 and Adr1 TFs. The mechanism involves two previously identified cis-regulatory motives, motif A localized upstream from motif B, both identified as important (Trassaert et al. 2017) and common (Rzechonek et al. 2024) for the ERY/EUF1-deregulated genes. The latter suggests a uniform character of the following model of regulation for the genes with motives A and B in their promoter regions. The model’s presumptions are: (i) spEuf1 exhibits affinity to a DNA motif A, while Adr1–affinity to motif B; (ii) fullEuf1 may also interact through an identified binding pocket with some inhibitor/activator; this binding pocket was predicted with significantly lower probability for spEuf1, and its structure was also changed (Table 3); it is thus probable that spEuf1 lost its regulatability via this domain; (iii) expression and splicing of EUF1 are promoted but not reliant on ERY presence; (iv) Adr1 is in a DNA-binding state when GLUC/GLY is present; and (v) when bound to motif B (another carbon source is present), Adr1 constitutes a mechanical hindrance disallowing transcription governed by Euf1. Schematically the model is presented in Fig. 5.

Figure 5. The model of ERY-inducible/carbon-source-repressible/Euf1-driven regulation of gene expression as an interplay between spEuf1 and Adr1 TFs within the ERY-inducible promoters. Functionally important motives and TFs are indicated in the legend and colour-coded. A regulation under three different conditions is presented: (i) top: ERY absent, carbon source (glucose, GLUC or glycerol, GLYC) is present—no substantial expression from the promoter, only leakage; (ii) ERY is present, carbon source is present—no substantial expression from the promoter, only leakage; (iii) ERY is present and the carbon source is exhausted—spEuf1-driven expression from the promoter is on. An additional regulation of the fullEuf1 via the predicted binding pocket is executed by an unidentified ligand (either inhibitor or activator).

Supplementary Material

foae027_Supplemental_Files

Acknowledgements

We would like to acknowledge Szymon Turek from Genomed for his work with RNAseq data reanalysis. Martyna Przybylak is acknowledged for helping with chromatography analysis. Graphical abstract was Created with BioRender.com.

Author contribution

EC conceived the study, designed all the experiments, constructed the strains, conducted part of the experiments, analysed and interpreted the data, secured funding, and designed and wrote the manuscript, and revision. PKW conducted a significant part of the experiments. MG conducted a part of the experiments, ran all the bioinformatic protein structure modelings, and studied the protein–ligand interactions. JMN inspired the promoter-titration experiment and provided the background strains for that experiment.

Conflict of interest

None declared.

Funding

This study was financially supported by the National Science Centre, Poland, grant number 2021/41/B/NZ9/00086.
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