Designing strong inducible synthetic promoters in yeasts

Ethical statement

All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Pharma Foods International Co. Ltd. Experimental protocols and studies were reviewed and approved by the Animal Experimentation Management Committee of Pharma Foods International Co., Ltd.).

Media and buffers

YPD medium (10 g/L yeast extract [Nacalai Tesque, Kyoto, Japan], 20 g/L Bacto Peptone [BD Biosciences, San Jose, CA, USA], and 20 g/L glucose), SD + Met medium (6.7 g/L BD Difco yeast nitrogen base (YNB) without amino acids [BD Biosciences], 20 µg/mL l-methionine, and 20 g/L glucose), and SCSM-His-Leu-Ura medium (6.7 g/L YNB, 1.656 mg/L SCSM-His-Leu-Ura mixture [Cat. No. DSCS251, ForMedium, Norfolk, UK)], and 20 g/L glucose) were used to culture yeast cells. For biological fermentation, buffered-minimal glycerol yeast extract (BMGY), buffered-minimal methanol yeast extract (BMMY), and BMDY media were prepared by adding 20 g/L of glucose, methanol, and glycerol, respectively, to a basal medium composed of 10 g/L yeast extract, 20 g/L hipolypeptone [Nihon Pharmaceutical, Tokyo, Japan], 13.4 g/L YNB, 0.4 mg/L biotin [Nacalai Tesque], and 100 mM potassium phosphate buffer [pH 6.0]. Next, Luria-Bertani (LB) medium (25 g/L LB Broth, Miller [Nacalai Tesque]) containing 100 μg/mL ampicillin was used to culture Escherichia coli. Antibiotics (500 μg/mL G418 [Fujifilm Wako Pure Chemical, Osaka, Japan], 100 µg/mL Zeocin^TM [InvivoGen, San Diego, CA, USA], 50 µg/mL nourseothricin [Werner BioAgents, Jena, Germany], 500 μg/mL blasticidin [Fujifilm Wako Pure Chemical] and 300 μg/mL hygromycin [Fujifilm Wako Pure Chemical]) were used to select stably transformed yeast strains. Appropriate amounts of HSL (Cat. No. FK29472, Carbosynth, San Diego, CA, USA) and Dox (Cat. No. 631311, Clontech Laboratories, Mountain View, CA, USA) were dissolved in dimethyl sulfoxide (DMSO) (Cat. No. 13445-74, Nacalai Tesque) and water, respectively, to prepare stock solutions. DAPG (Cat. No. Sc-206518, Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in ethanol or DMSO to prepare a stock solution. Because DAPG has been previously found to be light-sensitive⁴², the stock DAPG solution was added to the media just before use, and all resultant media were stored in the dark. Pichia trace metal 1 (PTM1) solution (containing 6.0 g/L CuSO₄·5H₂O, 0.08 g/L NaI, 3.0 g/L MnSO₄·H₂O, 0.2 g/L Na₂MoO₄ ·2H₂O, 0.02 g/L H₃BO₃, 0.5 g/L CoCl₂, 20 g/L ZnCl, 65 g/L FeSO₄·7H₂O, 0.2 g/L biotin, and 5.0 mL H₂SO₄) was used for 5-L fed-batch fermentation. PBS buffer was prepared by diluting a 10 × PBS buffer stock (Cat. No. 7575-31, Nacalai Tesque). PBS-T buffer (containing 137 mM NaCl, 8.9 mM Na₂HPO₄·12H₂O, 2.7 mM KCl, 1.5 mM KH₂PO₄, and 0.05% Tween-20) and TBS-T buffer (50 mM Tris-HCl [pH 7.6] and 0.05% Tween-20) were used for ELISA and western blotting, respectively.

Strains, primers, and plasmids

The E. coli strain DH5α was used for plasmid subcloning. Here, the K. phaffii strain CBS 7435 (NRRL-Y11430, ATCC [Manassas, VA, USA]) and the two S. cerevisie strain BY4741-Phl_2-1E–Lux_2-4F¹³ and BY4741-hENT1¹³ were used as a parental yeast strain. The plasmids were individually linearized using appropriate restriction endonucleases and transformed into yeast cells by either the conventional chemical method⁴³ or electroporation⁴⁴. Successful integration of the plasmid in the desired configuration was verified through colony-direct PCR. Detailed information on all plasmids, yeast strains (K. phaffii and S. cerevisiae), primers, DNA parts, iSynPs, and insulators employed in this study are presented in Supplementary Data 1–7. All yeast strains except for those harboring centromeric plasmids were stored as glycerol stock and streaked onto the YPD plate before experiments. All experiments were performed using the resulting colonies, with at least three biological replicates unless otherwise noted. The following plasmids were used for plasmid construction: pGK415-phlTA¹³, pGK415-phlTA_1-2E¹³, pGK415-rphlTA_2-1E¹³, pGK401red-rtetTA_{K8N, L131L}¹³, pRS406red-p_phlO1 –TBG¹³, pGK416m-p_phlO6 –ymUkG1¹³, pGK416m-p_tetO7 –ymUkG1¹³, pGK416m-p_luxO5 –ymUkG1¹³, pGK416-ymUkG1⁴⁵, pUC19-MCS-Zeo³⁰, pPGP_EGFP⁴⁶, pNTI647⁴⁷, pYN169⁴⁸, and pYN186⁴⁸. pNTI647 dCas9-Mxi1 TetR KanMX was a gift from Dr. Nicholas Ingolia (Addgene plasmid # 139474)⁴⁷.

Fluorescence analysis with flow cytometry

Fluorescence analysis of yeast was performed using a previously described method⁴⁹ with slight modifications. Briefly, K. phaffii strains were grown in 500 µL/well YPD media on 96-well plates (Costar®, #3960 [Corning, Corning, NY, USA]) with different concentrations of inducers (10 µM for DAPG, 30 µg/mL for Dox, and 3 µM for HSL, unless otherwise noted) for 24 h (30 °C, 1000 rpm). For the KpDAPG-OFF system, 30 µM DAPG was added to the preculture media. The resulting yeast cells in the stationary phase were harvested and analyzed using a CytoFLEX (Beckman Colter, Brea, CA, USA) flow cytometer with a 488 nm laser and a bandpass filter for GFP (525/40 nm). Median fluorescence and forward scatter intensity measurements (i.e., FITC-A and FSC-A) were taken. Cell-size normalized fluorescence was then calculated by dividing the FITC-A signal by the FSC-A signal. Fold induction was calculated by dividing the normalized fluorescence of induced cells by that of uninduced cells. For S. cerevisiae, SCSM-His-Leu-Ura (Supplementary Fig. 7) or SD + Met (Supplementary Fig. 16) media were used, and mean fluorescence and forward scatter intensity were taken to calculate the cell-size normalized fluorescence.

SDS-PAGE analysis

An equivalent of 50 µL of the supernatant obtained by centrifugation of each yeast cell culture was mixed with 10 µL of 6 × sample buffer containing a reducing reagent (Cat. No. 09499-14, Nacalai Tesque). This was followed by incubation at > 95 °C for more than 10 min. Next, 15 µL of the resultant lysate was separated on a 15% polyacrylamide gel using SDS-PAGE (e-PAGEL [Cat. No. E-R15L, ATTO, Tokyo, Japan]) or 5–20% gradient polyacrylamide gel (m-PAGEL [Cat. No. M-520L, ATTO]) using SDS-PAGE and stained with CBB Stain One (Nacalai Tesque).

Small-scale nanobody production

For the production of ALX-0081 using iSynP or P_KpAOX1, cells were grown overnight at 30 °C and 170 rpm in BMDY (for iSynP) or BMGY (for P_KpAOX1) media and collected by centrifugation. After washing, 5% of the cell pellet was resuspended in 2 mL of BMDY medium supplemented with DAPG (10 µM) for DAPG induction and BMMY medium for methanol induction, and incubated for 60 h. For the selective production of two nanobodies with single yeast, 10% of the cell pellet prepared with the overnight yeast cell culture (30 °C, 170 rpm) in BMDY medium was resuspended in 2 mL of BMDY media supplemented with DAPG (10 µM) and Dox (30 µg/mL) in different combinations and incubated for 49 h to induce nanobody secretion. After incubation, the cell pellet was harvested via centrifugation, and 50 µL of the supernatants were then subjected to SDS-PAGE analysis to evaluate nanobody production per volume.

Directed evolution of PhlTA in S. cerevisiae

The directed evolution of PhlTA was performed as described previously¹³. Briefly, the wild-type phlTA gene along with short untranslated sequences (i.e., 104-bp upstream and 85-bp downstream sequence of phlTA) was amplified via error-prone PCR using Takara Taq^TM DNA polymerase (Takara Bio, Shiga, Japan) and MnCl₂ (50 µM, Nacalai Tesque) using the MT415 and MT416 primers (see Supplementary Data 4). The resulting fragment and the digested expression vector were used to transform yeast strain ScMT487, wherein these two DNA fragments were assembled via GAP-repair cloning. The resulting transformants (equivalent to 4 × 10⁴ colonies) were recovered from the SD + Met media and subjected to three successive incubations: first in the presence of 4.8 mg/mL Zeocin, then in the presence of 5 or 0.5 µM DAPG, and finally with both 5 or 0.5 µM DAPG and 4.8 mg/mL Zeocin. Between each incubation step, the yeast cell populations were mixed with glycerol and stored at − 80 °C until subsequent analysis. Selected cells were then propagated on agar plates, and individual clones were incubated both in the presence and absence of DAPG, followed by flow cytometry analysis.

Fed-batch fermentation for secreted protein production

A 5-L fed-batch fermentation was performed following a previously described protocol⁴⁸, with some modifications. In particular, BMDY media was used throughout the process, including seed culture preparation. Briefly, a preculture was incubated with 100 mL BMDY media in a 1-L Erlenmeyer flask at 30 °C under shaking at 180 rpm and was then transferred to 1.5-L BMDY media in a 5-L fermenter (BMS-P; ABLE & Biott Co., Ltd., Tokyo, Japan) (initial OD₆₆₀~ 1.0). This was followed by fermentation under the following conditions: dissolved oxygen (DO), temperature, and pH set at 30%, 30 °C, and 6.0, respectively. Automatically controlled stirring at 200–800 rpm using a Proportional Integral-Differential Controller (DPC-4; ABLE & Biott Co., Ltd.) and compressed 98% O₂ gas (ITO-08-1(2); IBS Co. Ltd., Osaka, Japan) were used to maintain a constant DO level. Glucose solution (450 g/L) supplemented with 12.0 mL/L PTM1 solution was fed to the medium at different feeding rates ranging from 0.2 to 1.2 g/min (Supplementary Fig. 17). OD₆₆₀ was monitored at each time point, and the protein levels in the supernatant were determined by SDS-PAGE. Bovine serum albumin solutions were used as calibration standards at various concentrations (120, 60, 30, 15, and 7.5 mg/L).

Preparation of RBD^om via yeast fermentation

The recombinant yeast colony was inoculated into 25 mL BMDY media containing 50 g/L hipolypeptone in nine large test tubes (Cat. No. 245920, EYELA, Tokyo, Japan) and incubated overnight (30 °C, 170 rpm). The supernatant was then replaced with the same volume of fresh medium supplemented with 10 µM DAPG and incubated for another 60 h (18 °C, 170 rpm). Subsequently, 225 mL supernatant was collected by centrifugation (2380 × g, 5 min), and the His-tag protein was column-purified using two sets of Capturem His-tag Purification Maxiprep Kits (Takara Bio), followed by desalination in PBS buffer (Cat. No. 7575-31, Nacalai Tesque) using Prepacked Disposable PD-10 columns (Code No. 17085101, Cytiva, Marlborough, MA, USA). The recovered protein concentration was then determined using a Qubit^TM Protein Assay Kit (Code No. Q33212, Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific) before being stored at 4 °C until use. Before peptide mapping and intact mass analysis, the stored sample was subjected to gel filtration chromatography using Superdex 200 increase (Cytiva) and concentrated 10-fold using Amicon Ultra-15 Ultracel-10K (Cat. No. UFC901096, Merk Millipore, MA, USA). This process was performed twice and then used for analysis.

Peptide mapping and intact mass analysis of RBD^om by liquid chromatography/mass spectrometry (LC/MS)

Peptide mapping and intact mass analysis via LC/MS were performed using a Vanquish UHPLC System (Thermo Fisher Scientific) and an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). For peptide mapping, the sample was prepared as previously reported^50,51, with slight modifications. Briefly, after reduction and carboxymethylation, the RBD^om protein (10 µg) was deglycosylated overnight at 37 °C using 10 units of PNGaseF (Roche, Mannheim, Germany). Subsequently, the carboxymethylated protein was digested using 2 µg of chymotrypsin (sequencing grade, 1 mg/mL; Promega, Madison, WI, USA) at 37 °C overnight. This was followed by desalting using an Oasis HLB μElution plate (Waters, Manchester, UK). A sample of the digested peptide solution was analyzed via LC/MS using data-dependent higher-energy collisional dissociation. The resulting peptide fragments were then identified using BioPharma Finder 3.1 (Thermo Fisher Scientific). Precursor mass tolerance was set to ± 5 ppm. Carboxymethylation (+ 58.005 Da) was set as a static modification of cysteine residues. Oxidation (+ 15.995 Da) of methionine, tryptophan, and tyrosine, and deamidation (+ 0.984 Da) of asparagine and glutamine were set as variable modifications. For intact mass analysis, RBD^om protein (10 µg) was deglycosylated for 3 days at 37 °C using 1 µL of PNGaseF (Waters, Milford, MA, USA), and analyzed via LC/MS on a polyphenyl column (Waters, BioResolve RP mAb, 2.7 µm, 2.1 × 150 mm) with a column temperature of 30 °C. Mobile phases A and B were prepared by adding 0.1% formic acid (Kanto Chemical, Tokyo, Japan) to distilled water and acetonitrile, respectively. Proteins were then eluted using a linear gradient of mobile phase B from 5% to 90% over 15 min at a flow rate of 200 μL/min. The parameters for mass spectrometry were as follows: electrospray voltage, 3.5 kV in positive ion mode; ion transfer temperature, 300 °C; full mass scan range, m/z 500–4500; full mass scan Orbitrap resolution, 15,000; and in-source collision voltage, 30 V. Finally, proteins were identified using BioPharma Finder 3.1 (Thermo Fisher Scientific). The search parameters were as follows: a merge tolerance of 10 ppm, a deconvolution mass tolerance of 10 ppm, and a peak detection quality measure of 95%. Mass spectrometric data have been deposited at ProteomeXchange/jPOST⁵².

Chicken immunization of RBD protein using a SARS-CoV-2 omicron variant

Chicken immunization was performed as described previously with some modifications⁵³. Briefly, three female chickens of 34-day-old (Boris Brown, Mie Hiyoko Co. Ltd., Mie, Japan) were used for immunization. The antigen was mixed with Freund’s Complete Adjuvant (Cat. No. 014-09541, Fujifilm Wako Pure Chemical) or Freund’s Incomplete Adjuvant (Cat. No. 011-09551, Fujifilm Wako Pure Chemical) for first and subsequent immunizations, respectively, together with D-PBS (Cat. No. 045-29795, Fujifilm Wako Pure Chemical). A total of five immunizations were performed, in which 1 mL of mixed antigen per dose was administered intraperitoneally (from the first to the fourth immunizations) and intravenously (for the fifth immunization). Three days after the final immunization, the immunized chickens were euthanized, and the spleens were collected. Total RNA was extracted from the spleens, and cDNA was obtained via reverse transcription using the PolyATtract® mRNA Isolation Systems (Cat. No. Z5310, Promega) and the PrimeScript II 1^st Strand cDNA Synthesis Kit (Cat. No. 6210 A, Takara Bio).

Phage-based biopanning

Using the above cDNA mixtures, a scFv phage library was constructed using a previously described method⁵³ and used for a panning assay⁵⁴. Briefly, the scFv DNA fragments were cloned into a phagemid vector, and the resulting library plasmid was electroporated into E. coli XL1-Blue Electroporation Competent Cells (Cat. No. 200228, Stratagene, La Jolla, CA, USA). The recovered E. coli transformants were then incubated with VCSM13 helper phage (Cat. No. 200251, Stratagene) at 37 °C with vigorous shaking, and the medium was replaced with one containing isopropyl β-D-thiogalactopyranoside, and the culture was incubated at 30 °C overnight. After incubation, the supernatant was filtrated to obtain a phage display library. For their selection, 50 μL (final conc. 5 µg/mL) of RBD^om (Cat. No. 40592-V08H121, Sino Biological Inc., Beijing, China) was coated on a Maxisorp Nunc-Immuno^TM plate (Cat. No. 442404, Thermo Fisher Scientific). At each round, absorption panning to deplete scFv variants that were bound to the domain was performed by using wild-type RBD produced with the recombinant K. phaffii strain PpYI1924 (Supplementary Data 2). After the 11^th repetition of the panning procedure, scFv variants that showed strong binding to RBD^om but not to wild-type RBD were isolated and sequenced to identify complementarity-determining region (CDR) sequences. Full-length scFv sequences were sequenced via sanger-sequencing using primer 5´-GGAAACAGCTATGACCATG-3´ at Eurofins Genomics Inc. (Tokyo, Japan), and each CDR sequence was identified according to the Kabat numbering scheme. Based on these sequencing data, the DNA sequences coding the heavy and light chains were cloned into a transient expression vector for transformation into Expi293F cells (Cat. No. A14528, Thermo Fisher Scientific). The resulting secreted IgY antibody was then purified using Ni-Sepharose Excel (Cat. No. 17-3712-01, Thermo Fisher Scientific) and desalted on a PD-10 column (Cytiva) using PBS buffer.

ELISA

ELISA was performed as described previously⁵⁴. Briefly, plate wells were coated with 50 µL (1 µg/mL) of SARS-CoV-2 Spike trimer (Cat. No. SPN-C52H9, Acro Biosystems, Tokyo, Japan) or SARS-CoV-2 Spike trimer Omicron/B.1.1.529 (Cat. No. SPN-C52Hz, Acro Biosystems), followed by blocking for 1 h at 37 °C with 25% BlockAce in PBS. Next, the different concentrations of monoclonal IgY antibody were added to each well of the plate, followed by incubation at 37 °C for 1 h. The bound antibodies were then washed with PBS-T buffer and incubated with 500 ng/mL HRP-anti-chicken IgG (Cat. No.5220-0373, SeraCare Life Sciences, Milford, MA, USA) for 1 h at 37 °C. After washing with PBS-T, SureBlue^TM (Cat. No. 5120-0074, SeraCare Life Sciences) was added and incubated for 30 min. Finally, the TMB stop solution (Cat. No. 5150-0019, SeraCare Life Sciences) was added to stop the reaction. Absorbance was measured at 450 and 650 nm using a microplate reader (Cytation 5 imaging reader from BioTek, Winooski, VT, USA).

Western blotting

An equivalent of 10 ng SARS-CoV-2 Spike trimer (Cat. No. SPN-C52H9, Acro Biosystems) and SARS-CoV-2 Spike trimer Omicron/B.1.1.529 (Cat. No. SPN-C52Hz, Acro Biosystems) were analyzed via SDS-PAGE using a NuPAGE 4–12% Bis-Tris gel (Cat. No. NP0329BOX, Thermo Fisher Scientific) and MOPS buffer (Cat. No. NP0001, Thermo Fisher Scientific). The proteins were then transferred to iBlot2 PVDF Mini Stacks (Cat. No. IB240002, Thermo Fisher Scientific) using an iBlot2 Gel Transfer Device (Cat. No. IB21001, Thermo Fisher Scientific). After blocking with Blocking One solution (Cat. No. 03953-95, Nacalai Tesque) for 30 min at room temperature, the membrane was incubated with #Omi-7 IgY (100 ng/mL) diluted in the same blocking buffer at 4 °C overnight. The membranes were then washed with TBS-T buffer (5 min, three times) before being incubated with a secondary antibody (HRP-anti-chicken-IgG, 50 ng/mL in blocking solution) at room temperature for 1 h, followed by additional washing in TBS-T. Finally, the membrane was developed using ECL^TM Prime Western Blotting Detection Regents (Cat. No. RPN2232, Cytiva), followed by analyzing the immunoreactive bands using a LuminoGraph (WSE-6100, ATTO). The His-tag proteins were detected using an HRP-conjugated His-tag antibody (Cat. No. HRP-66005, Proteintech, Rosemont, IL, USA) instead of primary and secondary antibodies.

Statistics and reproducibility

Statistical difference (p-value) was determined with a two-tailed Welch’s or paired t test. All yeast strains except for S. cerevisiae strains harboring centromeric plasmids were stored as glycerol stock and streaked onto the YPD plate before experiments. Experiments were performed with the resulting colonies in more than triplicate unless otherwise noted. No statistical method was used to predetermine sample size, and no data were excluded from the analyses otherwise noted.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54865-z