Omics-Based Expression Cassette for Heterologous Protein Production in Y. lipolytica

Abstract

The nonconventional yeast, Yarrowia lipolytica, is a promising protein expression host, having achieved recombinant protein expression yield on par with the commonly used host, Komagatella phaffii (Pichia pastoris). However, strong, fully constitutive genetic elements and expression cassettes for protein expression in Y. lipolytica remain limited. In this study, we leveraged genome-wide transcriptomics to uncover five strong promoters and four terminators. Among these, the promoter of ribosomal protein L41 demonstrated superior activity to the strongest previously reported promoters. We further demonstrated the functionality of pL41 across different media conditions and by using it to express diverse heterologous proteins. Similarly, we showed that the terminator of glutathione-S-transferase (tGST) supported higher protein expression and low transcriptional readthrough compared to commonly used terminators. To support protein secretion efforts, we utilized a secretomics-guided signal peptide screen to unveil three signal peptides, demonstrating broad applicability to different proteins. Integrating these genetic elements into a new expression cassette (YALI-pSTOmics1) resulted in a 3-fold increase in secretory expression of bovine fibroblast growth factor 2 compared to a combination of the best available state-of-the-art genetic tools for gene expression in Y. lipolytica. This expression cassette represents an open-source alternative to expensive commercial ones. Furthermore, the novel promoters and terminators provide options for metabolic engineering, where reuse of existing genetic parts is often a limitation.

Introduction

Access to recombinant proteins is crucial for a wide range of applications. Heterologously produced enzymes underpin many industrial and food production processes, while recombinantly produced biologics constitute the majority of commercially available therapeutic proteins. , Thousands of heterologously derived proteins also remain indispensable to both basic and applied research.

The high-yield expression of recombinant proteins, with their native conformation and functionality, is necessary to meet the demand for their various applications. However, effective heterologous protein expression can be hindered by limitations such as poor codon usage efficiency, mRNA instability, protein misfolding, and absent or incorrect post-translational modifications (PTMs). − Protein expression in eukaryotic hosts can be beneficial in alleviating folding and PTM bottlenecks due to their ability to handle complex proteins and perform various protein modifications. − Compartmentalized protein processing in eukaryotes ensures that folding of recombinant proteins predominantly occurs in the chaperone-rich/low redox environment of the endoplasmic reticulum (ER), thus reducing the likelihood of incorrect folding.

Yeast expression systems uniquely combine the PTM capabilities of a eukaryotic host with the ease of handling bacterial expression systems. Saccharomyces cerevisiae and Komagatella phaffii (formerly Pichia pastoris) are the two most widely used yeasts in industrial recombinant protein production. , However, S. cerevisiae often suffers from low yields due to cytoplasmic misfolding associated with its predominant post-translational secretion pathway. , Its tendency to hypermannosylate recombinant proteins can also result in inactive or hyperallergenic proteins. While K. phaffii offers higher secretion and less mannosylation, it presents drawbacks, such as clonal variability and protein folding limitations. − These challenges necessitate the need for alternative yeast expression platforms.

Yarrowia lipolytica is a nonconventional dimorphic yeast known for its robust natural lipase and protease secretion. Its high proteome similarity to mammals makes it a promising host for expressing complex proteins from higher organisms. Additionally, its predominant secretion of proteins through the cotranslational pathway has enabled recombinant protein yields comparable to K. phafii, , while its low mannosylation tendency also enables the production of more active proteins. Consequently, over 130 recombinant proteins have been successfully produced in Y. lipolytica. , The yeast has also received GRAS (generally regarded as safe) status for various biotechnological applications, including the production of single-cell oil/protein, organic acids, enzymes, and recombinant proteins. − Its ability to grow on a wide variety of complex substrates, including hydrocarbons and diverse agro-industrial side streams, has further broadened its applications. ,

Exploration of genetic elements, such as promoters, signal peptides, and terminators, have enabled the use of Y. lipolytica for protein production and other biotechnological applications. Initial genetic engineering efforts in Yarrowia lipolytica utilized the alkaline extracellular protease (XPR2) promoter; however, tight pH regulation and dependency on high peptone concentrations for full induction limited its industrial scale adoption. The subsequent development of upstream activating sequence (UAS)-based hybrid promoters, such as hp4d, resulted in improved protein expression, albeit with growth-phase dependency. , To achieve full constitutive expression, the promoter of translation elongation factor alpha (pTEF) was adopted and further enhanced by incorporating tandem UAS sequences or intronic units. , However, repetitive UAS sequences introduced challenges to DNA manipulation and genetic stability, while the intronic unit could introduce splicing complexities. ,, Although recent screenings have identified novel native and hybrid promoters superior to pTEF, the number of strong promoters capable of consistently achieving high gene expression across various conditions remains limited. Consequently, metabolic engineering efforts often reuse the same strong promoters across multiple genomic locations, increasing the risk of genetic instability through homologous recombination. Thus, there is a need for more well-characterized Y. lipolytica promoters with strong activity and fewer caveats.

While the effort to uncover new promoters and improve the activity of existing ones has continued, terminators in Y. lipolytica have received little attention. Currently, only a few native terminators (tXPR2, tLip2, tTEF), a S. cerevisiae cytochrome C terminator (tCyC), and short synthetic terminators are predominantly used. However, terminators significantly influence protein expression by facilitating rapid RNA pol II dissociation from nascent mRNA and providing 3′-downstream elements critical for mRNA stability and half-life. , Hence, the availability of well-characterized terminators is also desirable for improved and tunable gene expression in Y. lipolytica.

Similarly, the evolution of signal peptides for heterologous protein secretion inY. lipolytica has followed the same trends as those of the promoters and terminators. XPR2 and lipase2 (Lip2) signal peptides or their corresponding modified or hybrid versions are the most predominantly used to target recombinant proteins for secretion. Recently, 5 new signal peptides were unveiled by a combination of genomic and secretomics data mining, bioinformatic prediction, and experimental assessment of secretion capacity. , However, these newly identified peptides have seen limited adoption. Moreover, bioinformatics predictions often rely on generalized characteristics, potentially overlooking effective signal peptides that deviate from the established consensus sequences. Therefore, a more extensive characterization of signal peptides would be beneficial to achieving higher recombinant protein yields in Y. lipolytica.

Thus far, the identification and characterization of genetic elements in Yarrowia lipolytica have relied primarily on heuristic predictions based on the species’ characteristics, followed by incremental, trial-and-error refinement. This approach barely covers the landscape of potential genetic elements and limits the opportunity to discover superior elements outside of obvious phenotypic traits. Consequently, there is a need for a more systematic and data-driven approach to the exploration of genetic elements on a genome-wide scale.

In this study, we employed a systematic, omics-based approach to identify novel genetic tools for protein expression in Y. lipolytica. Leveraging transcriptomics data, we uncovered previously unknown promoters and terminators that significantly enhance protein expression. Furthermore, by characterizing signal peptides from secretomics data, we revealed their secretion efficiencies to be context-dependent. Finally, we integrated these optimal genetic elements into a highly standardized, open-source expression unit for recombinant protein production in Y. lipolytica.

Results

Genome-Wide Transcriptome Profiling Reveals Previously Unreported Y. lipolytica Promoters

Genome-wide exploration of genetic elements offers the opportunity to identify novel genetic engineering tools with no readily identifiable visual phenotypes. We applied this approach to the identification of promoters capable of driving strong heterologous protein expression in Y. lipolytica. To do so, we leveraged 5′-Cap analysis of gene expression (5′-CAGE) datasets to infer genome-wide promoter strength from transcript abundance. ,

To rank Y. lipolytica promoters based on transcript abundance, we analyzed publicly available Y. lipolytica transcription start site (TSS) datasets obtained from the YeastTSS database (http://www.yeastss.org/). The provided data sets represent the yeast’s transcriptional landscape in YPD media during the early exponential growth phase (OD600 = 0.5). The dataset utilized only uniquely mapped tags in TSS identification and a preset tag cluster threshold distance to define putative core promoters. To aid visualization, we presented transcript abundance from core promoters across the six Y. lipolytica chromosomes and mitochondrial genome as average tags per million (TPM) of replicate samples. TPM levels of all genes in each chromosome are presented with respect to their genomic positions (Figure A). Subsequently, we identified nine genes with the highest TPM levels and annotated them using UniprotDB and NCBI as follows: YALI0D24816p, ribosomal protein L41, TEFα, cytochrome C, YALI0E07832p (cell wall SED1-like protein), YALI0F25575p (glutathione transferase activity, hereafter referred to as GST), 40S ribosomal protein (RP s11), an unknown RNA, YALI0_D07535g (DDRA2 like protein), and YALI0E10549p.

1.

Exploration of Y. lipolytica’s transcriptional landscape reveals previously unknown promoters. (A) Genome-wide transcript abundance of Y. lipolytica grown on YPD at 30 °C. Transcript levels are presented as average tags per million reads (TPM) of each gene relative to the genomic position on each chromosome. The 10 genes with the highest TPM values were annotated using their gene names and homology to other yeast proteins. (B) 1.5 kb genomic region upstream of the start codon was designated as the ‘promoter’. Promoter strength was screened by assessing the expression of fluorescent reporter protein (DsRed) from a single genomic locus. (C) Fluorescence output from various native high TPM promoters was obtained using flow cytometry. Data are shown as mean of n = 3 biological replicates and symbols as individual data points. Error bars represent standard deviation.

We reasoned that while transcript abundance generally reflects promoter strength, a strong 5′-upstream region should not only contain a core promoter that promotes a high transcription rate but should also confer a strong 5′-UTR element that promotes high translation efficiency. − Therefore, we decided to test the strength of the promoters of these genes at the protein level using fluorescent reporter proteins (Figure B). To this end, 1.5 kb upstream of the start codon of each gene was subsequently designated as the ‘promoter’, potentially spanning the 5′-UTR sequences, core promoter region, and other upstream regulatory elements.

To mitigate yeast autofluorescence, we first assessed the suitability of humanized Renilla reniformis GFP (HrGFP) and Discosoma sp. Red (DsRed) to report promoter activity with a low signal-to-noise ratio by expressing both reporter proteins under the control of the low-expression asparagine synthase promoter, pASN2. Expressing fluorescent proteins at a low level allowed for a precise comparison of the fluorescence output to background yeast fluorescence. We hypothesized that the brighter reporter would offer a better distinguishing effect and allow us to select a clear winner among promoters with closely similar expression levels. Therefore, we cloned the reporter expression cassettes onto the standardized EasyCloneYALI vector, followed by integration into the D1 locus. We observed no increase in red fluorescence in wild-type Y. lipolytica, while green fluorescence increased between mid-to-late exponential phase as cell density peaked, narrowing its margin with the reporter strain (Figure S1). Although DsRed exhibited a longer maturation time, it produced the least variability between replicate samples, indicating a stronger signal-to-noise ratio. Therefore, we concluded that DsRed will offer a more refined distinction between promoters as well as between low-expressing promoters and yeast autofluorescence.

Next, we cloned the 1.5 kb promoter fragments onto DsRed and assessed their fluorescence output at the single-cell level using flow cytometry. Promoter activity was defined as the median intensity of single-cell populations. The tested promoters demonstrated variable expression levels (Figure C). Five of these promoters (pDDRA2, pCyC, pS11, pSED1, and pL41) showed higher activity than low TPM control promoters, pLip2 and pASN2. However, no further correlation between transcript abundance (TPM) and expression levels was observed among these 5 promoters at this point, suggesting the additional role of translational control elements in determining their final protein outputs. Strikingly, however, the L41 promoter (pL41) showed nearly 10-fold higher DsRed expression compared to the other 4 promoters. Therefore, we selected this as a candidate for further evaluation.

To further assess the strength of pL41, we compared its DsRed expression level with those of the strongest established Y. lipolytica promoterspTEF-in, pEXP, and hybrid promoters UASB8-TEF and 8UASB-LeuM (hp4d). Remarkably, pL41 resulted in a significantly higher fluorescence than all tested promoters (ANOVA, p < 0.0001), with a 2-fold higher activity than the strongest reported promoter pTEF-in (Figure A). This finding highlights pL41 as a potentially strong promoter of recombinant protein expression. To further test the robustness of pL41, we proceeded to characterize it under various conditions.

2.

Novel Y. lipolytica promoter drives robust protein production under various conditions. (A) Fluorescent output of novel promoter pL41 compared to commonly used Y. lipolytica promoters (pUASB8-TEF, pUASB8-TEF, pEXP1, and pTEFintron). W29 represents the background wild-type Y. lipolytica fluorescence. (B) Fluorescent output of the novel promoter pL41 compared to commonly used Y. lipolytica promoters (pUASB8-TEF, pUASB8-TEF, pEXP1, and pTEFintron) at different growth phases in cells grown in YPD versus YSC media at shake-flask scale and 28 °C. W29 represents background wild-type Y. lipolytica fluorescence. (C) Red fluorescence output of pL41 in agro-industrial side-stream-based media (rapeseed cake hydrolysate) and minimal Delft media compared to that of YPD. pL41-DsRed; Y. lipolytica expressing DsRed under the control of pL41, W29; wild-type Yarrowia lipolytica w29. (D,E) Split luciferase-based HiBiT blotting and HiBiT extracellular assay showing the sizes and expression levels of diverse proteins of different complexity under the control of pL41, respectively. FGF2-G3: thermostable fibroblast growth factor (18 kDa); albumin: serum albumin (66 kDa); fetuin A: asialofetuin A (55 kDa); TGF-beta3: transforming growth factor-beta3 (12 kDa). Data are shown as mean of n = 3 biological replicates, with bars as standard deviation and symbols as individual data points.

Novel Y. lipolytica Promoter Drives Robust, Constitutive Expression across Diverse Conditions

Since the transcription start site (CTSS) data sets show the transcriptional landscape of Y. lipolytica in YPD media, we wondered whether the activity of pL41 is media-dependent. Therefore, we assessed the activity of pL41 in different media, including a complex medium derived from an agro-industrial sidestreamrapeseed cake hydrolysate. We first showed that pL41 remained the highest-performing promoter at 3 different growth phases in YPD and the commonly used minimal media yeast synthetic dropout media (YSC) (Figure B). We then showed that pL41 exhibited no reduction in fluorescence in a second minimal medium (Delft) and rapeseed cake hydrolysates compared to rich media (ANOVA, p = 0.99), confirming its robust constitutive activity (Figure C).

Next, we evaluated the versatility of pL41 in driving the secretory expression of different heterologous proteins of diverse origin and varying PTMs. The target proteins were tagged with the N-terminal XPR2-prepro signal peptide for extracellular secretion and with C-terminal HiBiT tags to facilitate detection and quantification by split-luciferase-based HiBiT blotting and protein detection systems. Markedly, we observed that all target proteins were successfully secreted, albeit at different levels, likely owing to their distinct PTMs or stability (Figure D,E). Collectively, these results further demonstrate that pL41 effectively drives robust and versatile gene expression in Y. lipolytica and could be suitable for various biotechnological applications.

TSS Data Sets Enable the Discovery of Novel Low-Readthrough Terminators

Despite high TPM values, some promoters exhibited low protein expression levels. While differences in translation efficiency due to 5′-UTR regulatory elements could partly explain this discrepancy, strong terminator sequences and 3′-UTR elements could instead be responsible for their high TPMs by facilitating efficient RNA polymerase II dissociation and turnover or by conferring higher mRNA stability. , Therefore, we reasoned that high TPM genes with strong 3′-regions could be used as sources of novel terminators that enhance recombinant protein expression.

To investigate this, we defined terminator sequences as the 1 kb intergenic genomic region downstream of the stop codon of their respective genes and cloned these regions downstream of an HrGFP expression cassette under the control of pL41. We positioned a promoterless DsRed cassette downstream of each terminator to allow for the quantification of terminator leakiness using quantitative PCR (qPCR), as terminator sequences contain inherent multiple stop codons, making downstream fluorescent detection difficult without an internal ribosome entry site (iRES) (Figure A). A short DNA linker (fusion linker), allowing for easy detection of DsRed in this conformation, due to the lack of stop codon, was used as a control for basal readthrough level (Figure S2). Repeated attempts to clone YALI0_D24816g and unknown RNA terminators in this conformation proved unsuccessful, resulting in their exclusion from our list of potential terminators. Four of the 5 novel terminators tested, except for tDDRA2, resulted in a significantly higher HrGFP expression than the commonly used CyC (ANOVA, p < 0.0001) and TEF terminators (ANOVA, p < 0.001). Markedly, YALI0F25575p (GST-like protein) terminator resulted in the highest fluorescence, showing over 2-fold higher expression than other novel terminators (ANOVA, p < 0.0001) and 10- and 50-fold expression over tTEF and tCyC, respectively (Figure B). Further comparison with the recently reported Tpex20 also showed a 2-fold higher expression (ANOVA, p < 0.0001) than Tpex20, validating this terminator as superior to most commonly used terminators (Figure C).

3.

5′-CAGE emerged as a suitable technique for identifying strong terminators. (A) 1 kb region downstream of 7 genes with high TPM levels were investigated as potential strong terminator for protein expression. Flow cytometry quantification of the fluorescent output of an upstream reporter protein (HrGFP) and qPCR analysis of transcriptional readthrough (DsRed) were used as measures of terminator strength. Leaky terminators result in a high abundance of joint HrGFP and DsRed transcripts. (B) Histogram showing the activity of the highest-performing terminator (TGST) compared to those of commonly used Tpex20 and TCyC. (C) Relative HrGFP fluorescence output of high TPM terminators in YPD at the late exponential phase. A HrGFP-DsRed linker (fusion linker) prevalidated to allow readthrough transcription is used as a control. (D) qPCR analysis showing the ratio of HrGFP to DsRed mRNA transcript resulting from the tested terminators. Data in B and D are shown as mean of n = 3 biological replicates and symbol as individual data points, with bars representing standard deviation. Statistical significance was measured using one-way ANOVA, followed by Tukey's multiple comparison test. ns: not significant; ****: P < 0.0001; significant difference.

Furthermore, assessing terminator leakiness by qPCR quantification of HrGFP and DsRed transcripts reveals that the different terminators exhibit variable transcription and leakiness levels (Figure S3). However, the GFP expression-to-leakiness ratio correlates strongly with fluorescence outputs, further supporting our initial reflecting terminator hierarchy (Figure D). We observed that terminators s11 and SED’s low leakiness compensated for their relatively low HrGFP transcript abundance. Conversely, the higher leakiness observed with the tL41 terminator appeared to diminish its potential to achieve higher protein expression (Figure S3), likely due to higher amounts of the longer transcript (HrGFP-tL41-DsRed), which would have a longer translation time and a more complex transcript structure. The combination of the high HrGFP transcript and low leakiness likely contributed to the higher activity of tGST.

Collectively, these findings explain the high transcript abundance (TPM) observed for some of these genes and further highlight the significance of the terminator strength on protein expression.

Secretomics-Guided Signal Peptide Screen Reveals Context-Dependent Activity

Besides transcription level, secretion efficiency significantly influences the recombinant protein yield. Secretion signals are major drivers of protein secretion, dictating the choice of the secretion pathway and overall secretion efficiency. The availability of strong secretion signals is therefore important to achieving high protein yields.

To expand the toolbox of available signal peptides for protein secretion in Y. lipolytica, we set out to characterize novel signal peptides capable of driving high protein export in Y. lipolytica. Similar to our genome-wide transcriptomics approach, we first performed a label-free secretomic analysis of wild-type Y. lipolytica grown in YPD and rapeseed cake hydrolysates for 12 h. Gene ontology (GO) analysis of the 36 identified extracellularly secreted proteins revealed their predominant involvement in nutrient acquisition and cell wall synthesis (Figure A). Hydrolytic enzymes (such as glucosidases, nucleases, and ester bond hydrolases) and polysaccharide elongating enzymes were particularly enriched. Although with high GO FDR (p > 0.5), we also identified proteins involved in peptide cleavage and mineral acquisition pathways.

4.

Secretomics-guided signal peptide screen enables the identification of novel secretion tags. (A) Gene ontology analysis of proteins identified from Y. lipolytica secretome using PantherGO slimming data sets showing significantly enriched biological processes and molecular functions (FDR P < 0.05). (B) Luminescence intensity representing the secretion levels of 2 proteins, fibroblast growth factor 2 (FGF2), and napin3 (Nap3) from 13 novel signal peptides. Luminescence is normalized to OD600 nm, and the secretion level of each signal peptide is compared to the secretion level of the most commonly used signal peptide, XPR2-prepro. Best-performing signal peptides for each protein are identified by circles, while ovals show signal peptides exceeding the XPR2-prepro threshold for both proteins. Data are shown as the mean of n = 3 biological replicates and bars as the standard deviation.

Next, we selected 13 signal peptides spanning these biological processes to evaluate their secretion efficiency. The secretion efficiency of these signal peptides was tested on 2 similar-sized C-terminal HiBiT-tagged proteinsbovine fibroblast growth factor 2 (FGF2), having no complex PTM, and the disulfide bond-rich rapeseed storage protein, napin3 (Nap3). We reasoned that testing signal peptides on low PTM versus a disulfide-rich protein should allow the identification of signal peptides with strong translocation efficiency, regardless of protein complexity. We then expressed these proteins under the control of promoter L41 and terminator Tpex2 and quantified the secretion level from each signal peptide in YPD in 96 deep-well plates.

To establish a performance benchmark, we set a threshold (XPR2-prepro export) based on the secretion levels of each protein from the most commonly used XPR2-prepro signal peptide. Signal sequences exceeding this threshold in both proteins were considered strong signal peptides. On a global level, we observed that signal peptide efficiency strongly depends on the protein context. Signal peptides of YALI0D22396p resulted in the highest secretion of FGF2 (ANOVA, p < 0.0001), while YALI0F12067p and YALI0_A20350g (Lip2, a triacylglycerol lipase) were more effective for Nap3 (Figure B; circles) (ANOVA, p < 0.0001). Nevertheless, four signal peptidesYALI0_A20350g (Lip2) and YALI0_F12067, YALI0E00110p (phospholipid hydrolase), and YALI0D20680p (glucan-1-3 beta-glucosidase)exceeded the XPR2-prepro secretion threshold for both proteins (Figure B; oval shapes) (ANOVA, p > 0.9), validating the common use of Lip2 and introducing the latter 3 as new, potentially broadly applicable candidate signal peptides.

Integrated Expression Cassette Based on Novel Best-Performing Genetic Elements

Having identified novel high-performing promoters, terminators, and signal peptides, we next integrated these genetic elements into a robust and readily adoptable expression unit (YALI-pSTOmics1) and demonstrated their application in the secretory expression of FGF2, an important growth factor for cultivated meat production. We combined pL41, YALI0D22396p signal peptide, and tGST to express FGF2 from a single integration locus. We then compared the FGF2 expression level from this cassette with the state-of-the-art pTEFin, XPR2-prepro, and Tpex20 combination. Split luciferase quantification of FGF2 from culture supernatant shows that YALI-pSTOmics1 resulted in a 3-fold higher expression of FGF2 compared to the tested state-of-the-art combination (Figure ).

5.

Combination of omics-derived genetic elements enables higher FGF2 yields. Comparison of FGF2 yield using the YALI-pSTOmics1 expression cassette (composed of promoter L41, signal peptide from YaliOD22396p, and GST terminator) versus the best previously reported genetic elements in Y. lipolytica (comprising TEF-intron promoter, XPR2-prepro signal peptide, and Tpex20 terminator). Data are shown as the mean of n = 3 biological replicates and symbol as individual data points, with bars representing standard deviation. Statistical significance was measured using Welsh’s unpaired t-test. **: P < 0.01; significant difference.

Discussion

Yarrowia lipolytica, a nonconventional yeast, is emerging as a promising protein expression host, having demonstrated comparable yields to the commonly used host K. phaffii. However, well-characterized genetic elements and cassettes for high-level protein expression remain limited. In this study, we demonstrate a systematic, omics-driven approach for increasing the number of available genetic elements and expression cassettes for recombinant protein expression in Y. lipolytica.

Using genome-wide transcriptomics (5′-CAGE sequencing) data sets, we identified five novel promoters with high transcriptional activity, among which the ribosomal protein L41 promoter (pL41) exhibited an exceptionally strong activity, exceeding that of the strongest previously reported Y. lipolytica promoters. − This high activity likely reflects pL41’s natural role in promoting abundant ribosomal synthesis during rapid cell growth. Additionally, its consistent activity across various media conditions, including complex agro-industrial substrates, suggests its compatibility with existing bioprocesses and waste valorization applications. Notably, our promoter screening showed an unexpectedly low fluorescence output for the Hp4d (8UASB-Leum) promoter, which was previously reported as highly active, and is the basis of the commercial YLEX system. , This discrepancy likely stems from the timing of our measurement at late exponential phase, ∼16 h, whereas full hp4d induction occurs during the stationary phase. , This, however, further highlights the temporal limitation of the hp4d promoter.

Using the same transcriptomics-based approach as promoter exploration, we uncovered four previously uncharacterized terminators, among which tGST showed superior performance compared with commonly used terminators. The low transcriptional readthrough of tGST likely resulted in the high fluorescence output produced by the terminator. , Such efficient termination minimizes the formation of unnecessarily long transcripts, which can complicate mRNA folding and stability, while simultaneously ensuring effective RNA pol II recycling to drive recombinant protein expression. It is also noteworthy that, besides terminator leakiness, the observed readthrough (DsRed transcripts) in our tested terminators could also be due to the presence of cryptic sequences with promoter activity within the selected terminator sequences. − While it remains unknown to what extent these potential cryptic promoters contribute to our detected readthrough, terminators with low DsRed outputs, such as tGST, indicate minimal internal cryptic promoter sequences and would help conserve cellular resources needed for recombinant protein expression. Future efforts aimed at better understanding termination efficiency mechanisms, as well as the presence and impact of cryptic promoters within terminators on RNA pol II dissociation and readthrough, could benefit from our characterized terminator sets as foundational data sets.

A complementary secretomic-based signal peptide screen to enhance protein secretion revealed that signal peptide effectiveness in Y. lipolytica is protein-dependent. Variability in signal peptide efficiency may be attributed to differences in folding kinetics between the two different target proteins. Alternatively, the interaction between each signal peptide’s C-terminal and the target proteins’ N-terminal sequences could also be responsible for variable export efficiency. Differences in complex secondary structure formation between the C-terminal of signal peptides and the N-terminal of each target protein can influence ER translocation efficiency or impact efficient cleavage by signal peptidase. − Therefore, maintaining a diverse library of signal peptides is necessary to optimize the yields for distinct proteins. Predictive models could also be developed to identify efficient signal peptide matches for target recombinant proteins. The findings of our signal peptide screen could contribute to foundational data for future predictive models for signal peptide-recombinant protein matching in Y. lipolytica.

Integrating our identified genetic elementsthe pL41 promoter, YALI0D22396p signal peptide, and GST-like terminatorto a standardized expression cassette, YALI-pSTOmics1, resulted in robust secretory expression of bovine FGF2, surpassing the current state-of-the-art combination. These findings position the newly developed expression cassette as an open-source alternative to the existing expensive commercial expression systems. While this work reports a new expression cassette, the characterization of its constituent elements is predominantly based on the expression of a reporter protein and select proteins; therefore, their activity could differ from some target proteins. For instance, toxic or high-complexity proteins may be better expressed with low-strength promoters and terminators, as these would likely provide a more controlled expression rate, thereby mitigating toxicity and misfolding, respectively. − Therefore, applications involving more complex proteins may benefit from testing a few elements to unlock better compatibility and achieve higher yields. It is also noteworthy that while promoter L41 and terminator GST resulted in a combined 4-fold expression over existing promoters and terminators and YALI0D22396p signal peptide increased FGF2 export about 20-fold over XPR2 signal peptide, the overall protein secretion output was not cumulative. We hypothesize that this could be due to jamming or overloading of the translocation-folding machinery, resulting from the high mRNA output. − Optimizing secretion pathways by overexpression of chaperons or SRP-translocons, similar to what was done by Zahrl et al., could help consolidate gains in high intracellular expression. Alternatively, testing a combination of different genetic elements could unlock better complementarity and harmonize protein secretion.

Furthermore, our validation of these genetic elements and expression cassettes was limited to lab-scale shake flasks and microtiter scales. Future research would also benefit from validating the efficacy of these genetic elements on bioreactor scales, where parameters such as bioreactor conditions, nutrient availability, dissolved oxygen, and pH dynamics could alter final protein yields. Additionally, a genomic stability study under prolonged cultivation over hundreds of generations could shed light on whether this expression cassette meets the GMP standards for industrial protein production.

Nevertheless, this study increases the number of available genetic elements for protein expression in Y. lipolytica. Their characterization alongside existing tools provides data on the expression strength of these elements, enabling tunable gene expression for diverse applications. Our overall approach also represents an unbiased alternative to the traditional strategy of simply co-opting the genetic elements from metabolic pathway genes or nutrient acquisition proteins. Such a systematic, omics-guided discovery approach could also benefit efforts to identify the ‘perfect’ inducible Yarrowia promoter, free from limitations such as glucose repression or dependency on metals for induction, which is currently lacking in Y. lipolytica. ,, As partially demonstrated by the expression of select recombinant growth factors and food proteins, the open-source expression cassette reported in this study is well-timed to contribute to the alternative protein revolution in areas such as cultivated meat production and precision fermentation applications, where a robust host, such as Y. lipolytica, is required to convert cheap agrofood waste into valuable food proteins or ingredients.

Materials and Methods

Microbial Strains, Media, and Growth Conditions

Y. lipolytica W29 (ku70::cas9), described in the EasyClone Yali Toolkit, was used to measure the fluorescent output of the various genetic elements. The protease knockout strain, Y. lipolytica po1f (ku70::cas9), was used as the secretion strain. Both strains of Y. lipolytica were grown on agar/broth at 30 °C. Commercially available One Shot Top 10 E. coli competent cells (Invitrogen no. C404003) were used as cloning hosts. Selection of transformants was done on LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar) plates containing 100 μg/mL ampicillin at 37 °C. Successive selection of Y. lipolytica transformants was carried out on YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 15 g/L agar for solid media) containing 250 μg/mL nourseothricin and 300 μg/mL hygromycin.

Vector Construction and Transformation

Y. lipolytica genome, with accession number (GCF_000002525.2), was used as the reference genome. oligonucleotide primers (30 bp) from IDT were used to amplify the 1.5 kb upstream promoter and 1 kb terminator genomic region of high TPM genes. These genetic elements were cloned onto standardized EasyCloneYali vectors to express fluorescent proteins from the D1 genomic locus. The expressed protein sequences were retrieved from UniprotDB. Codon optimization based on Y. lipolytica codon usage was performed using the IDT codon optimization tool to obtain the respective coding sequences (CDS). The CDS, obtained as G-blocks from IDT, was cloned with a C-terminal HiBiT tag onto E1 integration vectors in a separate cloning campaign. Secretome analysis of Y. lipolytica grown in YPD was outsourced to DTU Proteomics Core. The CDS of the 13 signal peptides selected across this secretome were amplified from the genome and cloned onto the N-terminus of FGF2 and Nap3. Plasmid sequences were verified using the UnveilBio nanopore sequencing service. Linear integration cassettes were generated by NotI digestion of circular plasmids and integrated into Y. lipolytica using the LiAC method as described by Holkenbrink et al.

Reporter Protein Fluorescence and Transcript Quantification

To assess the fluorescence output of genetic elements, cells were inoculated in 10 mL of YPD media overnight. Overnight cultures were back-diluted to an OD600 nm of 0.1 per mL in YPD. 100 μL of cell suspension is transferred to a 96-well plate and grown at 30 °C for 16 h in a SynergyH1 plate reader, with intermittent OD and fluorescent measurements taken every 30 min. For single-cell fluorescent measurements, overnight cultures from 96-well flat or deep-well plates are diluted 10×, and the fluorescent output is measured using Novocyte Quanteon with a threshold of 10,000 events. Fluorescent data are subsequently analyzed using FlowJo software. Population gating was established as follows: one gate was created for the PBS diluent. Another gate was created for wild-type Y. lipolytica dissolved in PBS. Single-cell populations were gated by comparing the forward scatter area to the forward scatter height in order to exclude double or triplet cells that might exhibit higher fluorescence than single cells. Red and green fluorescence was acquired using the Y615::PE-Texas-Red and B525::FITC channels, respectively.

To quantify reporter protein transcripts, RNA extraction was performed using the Rneasy Mini Kit (Qiagen #74104). Postlysis RNA extraction steps were performed on the automated Qiacube Connect instrument (Qiagen no. 9002864) to minimize manual handling. 200 ng of total RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Biorad #1708890). qPCR assays were performed using custom-designed oligonucleotide probes and a TaqMan gene expression master mix. Gene expression was quantified using QuantStudio (Thermofisher # A43183).

Protein Expression and Quantification

To assess bovine and plant protein expression, a seed culture was prepared by inoculating a single loop of transformant Y. lipolytica strains into 10 mL of YPD and cultivating overnight at 30 °C with 225 rpm agitation. The seed culture was then transferred into a 250 mL baffled flask containing 40 mL of YPD broth and allowed to grow overnight at 30 °C and 225 rpm agitation. Subsequently, all cultures are normalized to an OD of 15 to allow for uniformity in protein quantification of growth factor or accessory protein expression. Cells were then harvested by centrifugation at 4500 g, and the supernatant was stored for protein quantification and HiBiT blot detection. Protein quantification was done using the HiBiT extracellular detection system (Promega #N2420), and luminescence reads were normalized to the cells’ optical density.

SDS-PAGE and HiBiT Blotting

SDS-PAGE was performed on culture supernatant samples using 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels. Separated protein gels were blotted onto nitrocellulose membranes (Thermofisher no. IB23001) using an Invitrogen iBlot 2 Gel Transfer Device at 20 V for 7 min. Subsequently, the membrane was washed briefly with 1× TBST (20 mM Tris-HCl, pH 7.6, 150 mM, 0.1% v/v Tween-20) and incubated in 10 mL of HiBiT Blotting reagent (consisting of 1:200 LgBit protein and 1× Nano-Glo blotting buffer) for 1 h under orbital shaking. 20 μL of Nano-Glo Luciferase assay substrate was introduced to the mixture and allowed to incubate for 2 min. CCD chemiluminescent imaging of the membrane was performed using an Amersham imager 680.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.5c00612.

• Additional experimental details and figures, list of plasmids, and sequences of novel promoters, terminators, and signal peptides (PDF)

R.E., L.J.J., and M.O.A.S. conceptualized the ideas in the manuscript. R.E. designed and carried out the transcriptomics-based promoter screening. R.E. and C.Z. designed and carried out the terminator experiments. R.E., A.K., J.A.A., and A.K. designed and carried out the signal peptide screen. R.E., A.K., and A.B. designed the final pST-Omics1 cassette. R.E., L.J.J., and M.O.A.S. wrote the manuscript. All authors contributed to the finalization of the manuscript.

The authors declare the following competing financial interest(s): MSOM is board member of Novonesis. MSOM and LJJ are founders of MATR Foods.