Plasmids were constructed using NEBuilder HiFi DNA assembly (New England Biolabs, Frankfurt am Main, Germany) and SLiCE cloning (Zhang et al., 2012). Plasmid and primer sequences are given in Supplementary Tables 1, 2, respectively. PCR amplification of DNA fragments was performed using high-fidelity polymerases: Phusion Polymerase (Thermo Fisher Scientific), Q5 DNA Polymerase (New England Biolabs, Frankfurt am Main, Germany), or PrimeSTAR GXL DNA Polymerase (Takara Bio, Saint-Germain-en-Laye, France) according to the manufacturer’s recommendations. All restriction enzymes were purchased from New England Biolabs (Frankfurt am Main, Germany). Amplified DNA fragments were gel-purified prior to further use. The primers were ordered from Biomers.net (Ulm, Germany). Escherichia coli ER2925, NEB 5α, or NEB 10β cells (New England Biolabs) were transformed with the plasmids. Strains were grown in Luria-Bertani medium containing an appropriate selection marker at 37°C (Ampicillin, 100 μg/mL or Kanamycin, 50 μg/mL). The plasmid constructs were confirmed by sequencing (Microsynth Seqlab, Goettingen, Germany).

Genetic transformation of the plasmids or linearized DNA fragments from S. cerevisiae was performed using the LiAc/SS carrier DNA/PEG method (Gietz and Schiestl, 2007). The adopted LiAc/SS carrier DNA/PEG method was used to transform P. pastoris with plasmids or linearized DNA fragments (Ito et al., 2018). The yeast strains were grown at 30°C in yeast extract peptone dextrose adenine (YPDA)-rich medium [1% (w/v) bacto yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 0.04% (w/v) adenine hemisulfate, and 2% (w/v) agar for solid medium] or in an appropriate synthetic complete (SC) media [0.67% (w/v) yeast nitrogen base with ammonium sulfate, 2% (w/v) glucose, and 2% (w/v) agar for solid medium] lacking amino acids to allow selection for transformed cells. When required, glucose was replaced by 2% (w/v) glucose. Single copy integration of each linearized plasmid into the GUT1 site of the P. pastoris genome was verified by colony PCR using primers GNPP001 and GNPP002.

To integrate the plasmid into the GUT1 locus of the P. pastoris genome, 1000-bp LHA (primers GNPP003/GNPP004) and 1,000-bp RHA (primers GNPP005/GNPP006) were PCR-amplified from the genomic DNA of the GS115 P. pastoris strain. A PmeI site was added to the 5′-end of GNPH004 and GNPH005. The expression plasmids, listed in Supplementary Table 3 (Naseri et al., 2017), were digested with NotI-HF and PmeI and assembled with the LHA and RHA fragments, resulting in the plasmids pGNPP1 to pGNPP7 listed in Supplementary Table 4. Subsequently, the pGNPP1 to pGNPP7 plasmids were digested with AatII. A synthetic promoter containing a plant TF binding site and a downstream GFP fragment (primers GNPP007/GNPP008) were PCR-amplified from the reporter plasmids listed in Supplementary Table 5 (Naseri et al., 2017). The PCR-amplified fragments were assembled in the digested plasmids to generate integration the plasmids pGNPP008 to pGNPP0024 listed in Supplementary Table 6. To construct a positive control plasmid, pGN005B was digested with NotI-HF and BamHI and subsequently gel-purified to remove a 650-bp fragment. The remaining 6,500-bp fragment was assembled with the PCR-amplified GUT1 integration locus (primers GNPP004/GNPP009, on pGNPP001) and the GAPDH1 promoter (primers GNPP010/GNPP011, on yeast GS115 DNA). The generated integration plasmid was named pGNPP025. pGNPP integration plasmids (see section “Results”) were linearized with PmeI (present within the GU1 homology buffer) and used to transform the JC308 P. pastoris strain (Lin Cereghinoa et al., 2001). Integration takes place at the GUT1 locus of the yeast genome. Positive clones were selected on SC-URA3 medium. The generated P. pastoris strains are listed in Table 1. Subsequently, expression of the chimeric TFs was controlled by an IPTG-inducible promoter, and their transactivation capacity was tested against one, two, or four TF binding sites placed upstream of the CYC1 minimal promoter that controls GFP expression.

Plasmid pL0A_0-1 (Hochrein et al., 2017) was digested with PmeI and SbfI and assembled with: (i) PCR-amplified ATAF1-derived ATF [primers GNPP012/GNPP013, on Entry vector X_mGAL1_NLS_GAL4 AD_ATAF1_4X (Naseri et al., 2019)] and ERG20 fused to its native terminator (primers GNPP014/GNPP015, on yeast BY4741 DNA) to generate pGNSC001; (ii) PCR-amplified JUB1-derived ATF [primers GNPP012/GNPP016, on Entry vector X_mGAL1_NLS_ GAL4 AD_ JUB1_2X (Naseri et al., 2019)] and GDH2 fused to its native terminator (primers GNPP017_1/GNPP017_2, on yeast YPH500 DNA) to generate pGNSC002; and (iii) PCR-amplified ANAC102-derived ATF [primers GNPP012/GNPP018, on Entry vector X_mGAL1_NLS_GAL4 AD_ANAC102_2X (Naseri et al., 2019)] and tHMG1 fused to its native terminator (primers GNPP019_1/GNPP019_2, on yeast YPH500 DNA) to generate pGNSC003. Donors containing plant-derived ATF-fused GDH2-, tHMG1-, and ERG20-encoding DNA (see section “Results”) were amplified from pGNSC001, pGNSC002, and pGNSC003 using primers GNSC020/GNSC021, GNSC022/GNSC023, and GNSC024/GNSC025, respectively. Transformation of strain Gen 0.2 with the three donors and plasmid pTAJAK105 (Bao et al., 2015; Table 1) allowed integration of the GDH2, tHMG1, and ERG20 donors into the ADE2.a (Bao et al., 2015), his3D1 (Bao et al., 2015), and ura3-52 (Bao et al., 2015) sites of the genome, respectively. Selection for integration was carried out on a medium lacking yeast auxotrophic marker LEU (SC-LEU), since the LEU2 gene is encoded on pTAJAK105. When colonies appeared, the transformation plates were replicated on non-selective plates (YPDA media) to remove the pTAJAK105 plasmid.

The PCR-amplified SNR52 promoter (primers GNPP026/GNPP027, on pCRCT) and PDC1 terminator (primers GNPP028/GNPP029, on yeast Gen 0.1 DNA; Naseri et al., 2019) were assembled in XhoI/AgeI-digested pCRCT (Bao et al., 2015). A sequence containing gRNA targeting the DPP1 gene (392-bp downstream of the start codon), tracrRNA, and the SUP4 terminator was introduced after the SNR52 promoter through the primer sequences. The generated plasmid was named pGNSC004.

The PCR-amplified CYC1 terminator (primers GNPP030/GNPP031, on p426-SNR52p-gRNA.CAN1.Y-SUP4t; Addgene #43803) was assembled in SmaI/SalI-digested pGNSC004, introducing a gRNA targeting LPP1 (429-bp downstream of the start codon) and tracrRNA after the SNR52 promoter. The resulting plasmid was named pGNSC005.

SalI/AscI-digested pGNSC004 was assembled with double-stranded oligonucleotides (primers GNPP032/GNPP033), introducing a gRNA targeting the GDH1 gene (658-bp downstream of the start codon). The generated plasmid was named pGNSC006.

Subsequently, NotI-digested pGNSC004 was assembled with the PCR-amplified LPP1 gRNA encoding cassette (primers GNPP034/GNPP035, on pGNSC005). The generated plasmid was called pGNSC007.

Subsequently, NotI/PacI-digested pGNSC007 was assembled with the PCR-amplified GDH1-gRNA encoding cassette (primers GNPP036/GNPP037, on pGNSC006). The generated plasmid was called pGNSC008.

Moreover, three donor DNAs, allowing a frameshift mutation in DPP1, LPP1, and GDH1, were constructed by annealing single-strand oligonucleotides (primers GNPP038/GNPP039, GNPP040/GNPP041, and GNPP042/GNPP043, respectively). Each donor contains a 50-bp homology arm in the up- and downstream targeting sequence of the gene. Three donors, together with pGNSC008 (expressing gRNAs, tracer RNAs, and iCas9 protein), were used to transform yeast cells. Selection for integration was carried out on a medium lacking the yeast auxotrophic marker URA (SC-URA), since the URA3 gene is encoded on pGNSC008. When colonies appeared, the transformation plates were replicated on non-selective plates (YPDA media) to remove the pGNSC008 plasmid.

We combined the previously reported HI-CRISPR system (Bao et al., 2015) and three plant-derived ATF/BSs, namely NLS-GAL4AD-JUB1-2X (Naseri et al., 2019), NLS-GAL4AD-ANAC102-2X (Naseri et al., 2019), and NLS-GAL4AD-ATAF1-4X (Naseri et al., 2019), to overexpress yeast genes, GDH2, tHMG1, and ERG20, respectively. To construct a strain overexpressing GDH2, tHMG1, and ERG20, previously characterized integration sites, shown to exhibit a high integration efficiency, were targeted (Bao et al., 2015). The GDH2, tHMG1, and ERG20 donors were integrated, respectively, at genomic ADE2.a, his3D1, and ura3-52 loci of yeast Gen 0.2, where each donor is integrated into a single locus (Figure 5A). Each donor contains an IPTG-inducible ATF/BS upstream of a CDS and ends with a yeast terminator. The 50-bp overhang sequences up- and downstream of each donor allow its integration into pre-designed genomic loci.

For inactivation of the GDH1, DPP1, and LPP1 genes, three donors, together with pCRCT-GDH1-DPP1-LPP1 (expressing gRNAs, tracer RNAs, and iCas9 protein), were used to transform yeast cells, where each donor is integrated into a single locus. Each donor contains the 50-bp overhang sequences up- and downstream of its integration into target genomic sites, and incorporates an 8-bp deletion included within the target gene, thus introducing a frameshift mutation. The ySCGN0.1 or ySCGN0.2 strains were obtained by integrating overexpression or inactivation donors into the yeast genome, respectively. The ySCGN1.2 strain harbors inactivated genes, lpp1, dpp1, and gdh1, in addition to overexpressed GDH2, tHMG1, and ERG20.

To determine the effect of plant-derived ATF/BS on GFP fluorescence output, single colonies of yeast reporter strains were inoculated into 250 μl SC-URA medium containing 2% glucose in 96-well deep-well plates. Plates were incubated for 16 h at 30°C in a rotary shaker at 230 rpm. The precultures were used to inoculate main cultures in 250 μl YPDA containing 2% glucose (non-induction medium) or YPDA containing 2% galactose and 20 mM IPTG (induction medium) to an OD₆₀₀ ∼0.1. Each strain was inoculated in three technical replicates per experiment. Cells were grown at 30°C for 12 h in a rotary shaker at 230 rpm. Samples were harvested after 4, 6, 8, 10, 12, 24, 36, and 48 h (see section “Results”) and analyzed using a BD FACSCalibur Flow Cytometer (BD Biosciences). At each time point, the medium was replaced with the fresh induction medium. GFP fluorescence values were obtained from a minimum of 10,000 cells in each sample. The geometric mean of the GFP fluorescence per cell was calculated using FlowJo Software.

Growth assays were done similarly to induction experiments, except that experimental cultures were inoculated to an OD₆₀₀ of ∼0.05 and grown at 30°C and 230 rpm in a rotary shaker. OD₆₀₀ was measured after 6, 8, 10, 12, 24, and 36 h. At each time point, the medium was removed and the fresh induction medium was added to each well.

The β-carotene pathway genes McrtI-, McrtYB-, and BTS1-CDSs under the control of inducible ATFs NLS-GAL4AD-GRF7/4X, NLS-GAL4AD-ANAC102/2X, and NLS-GAL4AD-GRF9/4X were previously assembled in Destination vector I to generate plasmid pCAROTENE (see COMPASS protocol; Naseri et al., 2019). Destination vector I was digested with EcoRI/SalI and used in a two-way assembly reaction using PCR-amplified Pro_GAPDH sequence (PROGAPMCRTI_for/PROGAPMCRTI_rev, on genomic DNA of the GS115 P. pastoris) and McrtI CDS fussed to Ter_FBA1 (MCRTIPOS_for/MCRTIPOS_rev, on Entry vector X-McrtI; Naseri et al., 2019) to generate pMCRTI_PGAP. Next, NotI-digested pCOMPASS32 was used in an assembly reaction using PCR-amplified Pro_GAPDH sequence (PROGAPMCRTI_for/PROGAPMCRTI_rev, on genomic DNA of the GS115 P. pastoris) and BTS1 CDS fussed to Ter_TDH3 (BTSPOS_for/BTSPOS_rev, on Entry vector X-BTS1; Naseri et al., 2019). NotI-digested pCOMPASS33 was used in an assembly reaction using PCR-amplified Pro_GAPDH sequence (PROGAPMCRTI_for/PROGAPMCRTI_rev, on genomic DNA of the GS115 P. pastoris) and McrtYB CDS fussed to Ter_SYN3 (MCRTYBPOS_for/MCRTYBPOS_rev, on Entry vector X-McrtYB; Naseri et al., 2019) to generate pMCRTYB_PGAP. I-CeuI- digested pMCRTI_PGAP was used in an assembly reaction using PCR-amplified Pro_GAPDH-BTS1-Ter_TDH3 (PGAP_BTS1_for/PGAP_BTS1_rev, on pBTS1-PGAP). The resulting plasmid was called pMCRTI-BTS1_PGAP. Then, FseI/I-SceI-digested pMCRTI-BTS1_PGAP was used in an assembly reaction using PCR-amplified Pro_GAPDH–McrtYB-Ter_SYN3 (PGAP_MCRTYB_for/PGAP_MCRTYB_rev, on pMCRTYB-PGAP). The resulting plasmid was called pCAROTENE-PGAP.

The yeast strains transformed with pCAROTENE plasmid (Naseri et al., 2019) were plated on SC-URA [supplemented with 2% (w/v) glucose]. Cells were grown at 30°C for 3–4 days. The cells were then plated on induction SC-URA medium plates containing 20 mM IPTG and 2% (w/v) galactose and 1 M sorbitol. Cells were grown at 30°C for 3–4 days. Subsequently, colony PCR was performed, followed by sequencing. Three independent colonies were chosen for β-carotene analysis by SFC. Yeast colonies were inoculated into 4 mL non-induction medium SC-URA [supplemented with 2% (w/v) glucose] and grown for 18–24 h at 30°C and 230 rpm in a rotary shaker. Subsequently, the pre-cultures were used to inoculate main cultures (50 mL) in induction medium [YPDA with the 20 mM IPTG, 2% (w/v) galactose, and 1 M sorbitol]. All shake-flask cultures were inoculated from pre-cultures to an initial OD₆₀₀ of 0.1. Cells were then grown in a 500 mL shake flask for 3 days at 30°C and 230 rpm to saturation.

Carotenoid extraction from saturated culture was carried out according to the acetone extraction method with some modifications (Lian et al., 2017). The stationary phase yeast cells were collected by centrifugation at 13,000 × g for 1 min and cell precipitates were resuspended in 1 mL of 3N HCl, boiled for 5 min, and then cooled in an ice-bath for 5 min. The lysed cells were washed with ddH₂O and resuspended in 400 μL acetone to extract β-carotene. The cell debris was removed by centrifugation. The acetone supernatant was collected in 2-mL microcentrifuge tubes. This extraction procedure was repeated until the cell pellet was white.

SFC analyses were carried out in triplicates. Carotenoids were separated by SFC Waters^TM in 5 min using packed Silica column 4.6 × 150 mm (5 μm) with carbon dioxide modified with 2% (v/v) Tetrahydrofuran (THF) as a mobile phase. The column temperature was set to 40°C and the backpressure was 100 bar, at a flow rate of 1 mL/min. The results were detected using a photodiode array (PDA) detector at 510 nm and analyzed with ChromScope Software.

A central aim of our work was to establish inducible, heterologous regulators for future use in synthetic biology dealing with P. pastoris. To this end, 17 ATF/BS combinations from two Arabidopsis TF families, namely: Growth-Regulating Factor 7 (GRF7) (Kim et al., 2012), GRF9 (Kim et al., 2003), the NAC TFs JUNGBRUNNEN1 (JUB1) (Wu et al., 2012), and ANAC102 (Christianson et al., 2009), were selected from our library of plant-derived ATF/BSs developed for S. cerevisiae (Naseri et al., 2017). The selected ATF/BS combinations resulted in weak (NLS-EDLLAD-GRF9/1x, NLS-JUB1_DBD-EDLLAD/1x, NLS-J UB1_DBD-EDLLAD/2x, NLS-GAL4AD-GRF7/1x, NLS-EDLLAD-GRF9/2x, NLS-GAL4AD-GRF9/1x, NLS-GAL4AD-GRF9/2x; 0–400 AU), medium (NLS-GAL4AD-GRF9/4x, NLS-GAL4AD-ANAC102/1x, NLS-EDLLAD-GRF9/4x, NLS-GAL4AD-GR F7/2x, NLS-JUB1_DBD-EDLLAD/1x, NLS-GAL4AD-GRF7/4x; 4 00–1,200 AU), and strong (NLS-GAL4AD-ANAC102/2x, N LS-GAL4AD-ANAC102/4x, NLS-EDLLAD-ANAC102/2x, NLS-EDLL-GAL4AD-ANAC102/4x, NLS-EDLLAD-ANAC102/2x, NLS-EDLLAD-ANAC102/4x; 1,200–8,000 AU) transcriptional outputs and low basal expression, upon integration into the ura3-52 locus of the genome for transcriptional level characterization (Naseri et al., 2017). The selected ATF/BSs spanned a transcriptional activity (determined by GFP expression levels) ranging from ∼0.1- to ∼9-fold as compared with the strong TDH3 promoter in S. cerevisiae (Supplementary Figure 1; Naseri et al., 2017). To ensure a fair comparison of the ATF/BSs in P. pastoris, each of the regulator cassettes was inserted into the GUT1 locus, which leads to high specific integration rates by native homologous recombination and low random genomic integration frequency (Schwarzhans et al., 2016; Vogl et al., 2018). Following selection of a single GFP-transgene, the transcriptional output of plant-derived ATF/BSs was assessed using flow cytometry. The characterized ATF/BSs were subsequently used for metabolic engineering applications by producing β-carotene in P. pastoris (Figure 1).

Plant-derived ATFs, consisting of different combinations of full-length plant TFs or their DBDs, heterologous ADs, and different numbers of the respective cis-regulatory motifs located upstream of the yeast CYC1 minimal promoter, were integrated into the GUT1 locus in the P. pastoris genome. GUT1 is an essential enzyme for glycerol metabolism. Strains with deleted gut1 (due to integration of ATF/BS cassettes within GUT1 locus) cannot grow on glycerol (Vogl et al., 2018), while growth on glucose or galactose is not reduced. Following the addition of inducer (20 mM IPTG and 2% galactose), ATFs are expressed and target their BSs within the synthetic promoter (upstream of the yeast CYC1 minimal promoter), resulting in GFP expression (Figure 2A). As shown in Figure 2B, we achieved a wide spectrum of transcriptional outputs for plant-derived ATF/BSs. Of note, five of the ATFs (30%) resulted in up to a ∼3.13-fold higher GFP reporter output than the yPPGN021 control strain expressing the native GAPDH promoter (Figure 2B, strains yPPGN016, yPPGN008, yPPGN011, and yPPGN010). We observed the strongest transcriptional activation capacity for NAC TF-derived ATF NLS- GAL4AD-ANAC102 in combination with 2 or 4 copies of its BS (Figure 2B, yPPGN005 and yPPGN004). Surprisingly, the EDLLAD-ANAC102 combination resulted in a low transcriptional output in P. pastoris despite being categorized as a strong regulator in S. cerevisiae (Naseri et al., 2017). A combination of EDLLAD and plant TFs typically results in a high expression level of the reporter gene in S. cerevisiae (Naseri et al., 2017); however, EDLLAD-derived ATFs (yPPGN04, yPPGN04, yPPGN18-20) resulted in low-to-medium expression of the reporter gene in all our tested drivers/reporters in P. pastoris.

We previously showed that by employing only nine inducible plant-derived ATF/BSs, a library of S. cerevisiae variants with a million members was established, allowing the fine-tuning of naringenin and β-carotene over a wide range (Naseri et al., 2019). Hence, here we aimed to further prove the transcriptional activation capacity of our plant-derived ATF/BSs for metabolic engineering purposes in P. pastoris, rather than extending the size of our library. For full exploitation of the plant-derived ATF/BSs, we further characterized the transcriptional output of a weak GRF9-, medium JUB1_DBD-, and strong ANAC102-derived ATF/BS (yPPGN06, yPPGN13, and yPPGN10, respectively) in comparison with the constitutive GAPDH promoter (Waterham et al., 1997) in induction medium over time (Figure 3). Cells were grown in 20 mL induction medium in 100-mL flasks. The output of GFP was measured by flow cytometry over a 48-h time-course.

JUB1_DBD- and ANAC102-derived ATF/BS allow tuning GFP expression from low to high responses, compared the GAPDH promoter of P. pastoris. The GFP fluorescent output of the JUB1_DBD-derived ATF/BS (Figure 3, green line) reached to the level of GFP was expressed from the GAPDH promoter (Figure 3, gray line) 12 h post-induction and was stable for at least 24 h. In contrast, the strong ANAC102-derived ATF/BS (Figure 3, black line) mediated a much higher expression output 12 h post-induction as compared with the GAPDH promoter. However, the reporter expression decreased dramatically and reached the level derived from the GAPDH promoter after 48 h. Growing P. pastoris to a high density is of great benefit when producing a heterologous protein (Karbalaei et al., 2020); however, a high level of oxygen is required to achieve a high yield, which demands a culture vessel to deliver a sufficient amount of oxygen to the yeast (Eck et al., 2018). Due to the inadequate essential nutrients (amino acids, carbohydrates, minerals, vitamins), hormones, growth factors, and change of the physicochemical environment (pH, osmotic pressure), as well as limited oxygen availability (Krause et al., 2016), the reporter expression derived from plant-derived ATFs could be negatively affected in our study. This effect might be more severe in the case of strong ANAC102-derived ATF In contrast, we observed that the weak GRF9-derived ATF/BS (Figure 3, light green line) resulted in a stable expression level of the reporter over time.

We studied the effect of a weak GRF9-, medium JUB1_DBD-, and strong ANAC102-derived driver/reporter (yPPGN06, yPPGN13, and yPPGN10, respectively) on cell growth. Following integration of the ATF/BS-GFP modules into the GUT1 locus of the genome, cell growth in induction (Figure 4A, solid orange line) and non-induction (Figure 4A, solid gray line) medium was assessed over 36 h. Cells expressing chromosomally integrated GFP under the control of the constitutive GAPDH promoter were used as a positive control. The growth curves of wild-type JC308 cells (not containing ATF) in induction (dashed orange line) and non-induction (dashed gray line) medium are included in all growth plots. The growth of wild-type JC308 was reduced in the induction medium as compared with that in the non-induction medium, demonstrating a negative effect of galactose on cell growth. The strain expressing the strong driver/reporter GAL4AD-ANAC102-2X-GFP (in induction medium) showed similar growth defects to the positive control strain (constitutively expressing the GFP reporter under the control of the GAPDH promoter). In contrast, the strains expressing the medium JUB1_DBD-EDLLAD-4X-GFP and weak GAL4AD-GRF9-1X-GFP drivers/reporters displayed only minor growth reduction as compared with the wild-type control strain. Moreover, our data reveal that when the ATFs are present within the genome but are not expressed (non-induction medium), yeast growth was only slightly affected. To assess whether the growth penalty is related to the high level of GFP production, strains expressing only the driver modules GAL4AD-ANAC102. JUB1_DBD-EDLLAD, or GAL4AD-GRF9 (yPPGN01, yPPGN02, and yPPGN03 strains, respectively) (no reporter module) were generated (Figure 4B). JUB1_DBD-EDLLAD strain showed similar growth defects to the JUB1_DBD-EDLLAD-4X-GFP expressing both the driver and reporter, suggesting that cell growth was slightly affected by the expression of fluorescent reporter protein derived from the binding sites of medium JUB1-derived ATF/BS. We observed that the expression of the strong ANAC102-derived ATF driver slightly affects the cell growth (Figure 4B, orange line), while the presence of the ANAC102 driver/reporter module resulted in a growth defect similar to constitutive Pro_GAPDH (Figure 4A, orange line). Moreover, the cells expressing ATFs showed a minor growth defect in the non-induction medium, compared to the cells expressing Pro_GAPDH (Figure 4B, gray line), emphasizing that the use of inducible promoters for the expression of ATFs may minimize the consumption of energy and nutrient resources and maximize biomass production capacity prior to the production phase.

We sought to confirm the transcriptional ability of plant-derived ATF/BSs for metabolic engineering applications in P. pastoris by testing a well-known phenotype. Although P. pastoris is a non-carotenogenic yeast, it has been used for the production of carotenoids (Araya-Garay et al., 2012). To convert FPP precursor to β-carotene, geranylgeranyl diphosphate synthase (BTS1), phytoene synthase/lycopene cyclase (McrtYB), and phytoene desaturaseare (McrtI) enzymes are needed (Figure 5A). Here, we transformed yeast P. pastoris cells (wild-type JC308 background) with the episomal pCAROTENE plasmid (Naseri et al., 2019; Figure 5B) encoding the McrtI-, McrtYB-, and BTS1-CDS of the β-carotene pathway under the control of the inducible ATF/BSs, NLS-GAL4AD-GRF9-4X, NLS-GAL4AD-GRF7-4X, and NLS-GAL4AD-ANAC102-4X (Naseri et al., 2017, ∼1.27—∼3.13-fold stronger than Pro_GAPDH) to generate the yPPGN022 strain (Table 2). Selection for the assembly was carried out on a medium lacking the yeast auxotrophic marker, URA3 (the selection marker gene is encoded on the plasmid carrying β-carotene pathway genes). Following induction of ATF expression, orange colonies were observed on the yeast plate (Figure 5C for yPPGN022). After transformation with the pCAROTENE plasmid, we observed stronger β-carotene accumulation in the yPPGN022 P. pastoris strain than that in the S. cerevisiae strain (Table 1 and Figure 5C for ySCGN01) [ySCGN01 has wild-type Gen 0.2 S. cerevisiae background (Naseri et al., 2019)] in induction medium (production phase). To further compare the β-carotene productivity of the two hosts, P. pastoris and S. cerevisiae, we constructed an optimized S. cerevisiae cell factory for isoprenoid production by redirecting carbon flux toward the production of the isoprenoid precursor, FPP (Figure 5D; Scalcinati et al., 2012; Lopez et al., 2015). Using CRISPR/Cas9-mediated one-step double-strand breaks (Bao et al., 2015): (i) three modules expressing IPTG-inducible super-strong plant-derived ATF/BSs were integrated into the genome to upregulate the expression of GDH2, tHMG1, and ERG20 (Figure 5E and Table 2 for ySCGN0.1); and (ii) DPP1, LPP1, and GDH1 were inactivated (Table 2 for ySCGN0.2). Transformation of the ySCGN1.2 strain, carrying both upregulation and inactivation groups (Table 2), with the pCAROTENE plasmid produced yeast colonies with a more intense color (Figure 5F and Table 2 for ySCGN02) as compared with the P. pastoris yPPGN022 strain. We selected three colonies from each strain for the quantitative determination of β-carotene content by supercritical fluid chromatography (SFC).

The SFC data (Figure 5G) demonstrate a ∼4.8-fold higher β-carotene level in the yPPGN022 P. pastoris strain than that in the ySCGN01 S. cerevisiae strain. As a further control, the episomal pCAROTENE-PGAP plasmid expressing the three genes from the GAPDH promoter was transformed into J308 P. pastoris strain to generate yPPGN023 strain. The SFC data (Figure 5G) demonstrate that the strain with ATF/BS control modules produced ∼3.4-fold more β-carotene than the strain with GAPDH promoters. We observed a relatively huge variation of β-carotene production levels in different replicates in the case of yPPGN23 strain. A common issue often encountered with the episomal expression systems is an unstable product output because of the limited control of copy number, and segregational instability even in selective medium (Futcher and Carbon, 1986). In the case of pCAROTENE-PGAP, the metabolic burden raised from the constitutive expression of the pathway enzymes may cause this effect to be more severe. In contrast, presence of the inducible promoter upstream of plant-derived ATFs allows β-carotene production after adding IPTG and galactose (in the production phase). Therefore, a considerable production of β-carotene was achieved in the case of pCAROTENE, compared to pCAROTENE-PGAP (Figure 5G). Although the production of β-carotene in the ySCGN02 S. cerevisiae strain was markedly improved as compared with that in the ySCGN01 wild-type strain (by ∼8.2-fold), it was only ∼1.7-fold higher than the level of β-carotene accumulation in the yPPGN022 strain, confirming the high transcriptional capability of plant-derived ATF/BSs for metabolic engineering applications in P. pastoris.