Kod one

The standard transformation procedure of R. toruloides was performed as described previously [46 (link)]. In short, overnight cultures of R. toruloides strains and A. tumefaciens strains were diluted to OD₆₀₀ of 0.6. Subsequently, 100 μL R. toruloides culture and equivoluminal A. tumefaciens culture were mixed and coated on induction medium (IM) plate. After 2–3 days of incubation in a 28 °C incubator, the colony on IM plate was collected with 1 mL sterile water, centrifuged and re-suspended in 200 μL sterile water. 100 μL cells was then plated on selective YPD media and incubated for 4–5 days until the transformants appeared. To obtain the correct transformants, transformants grown on selective YPD media were randomly selected for genomic DNA extraction, which served as a template for PCR amplification using 2xEs Taq MasterMix (CWBIO, China) or KOD One (TOYOBO, Japan), and the PCR products were verified by agarose gel electrophoresis and sanger sequencing (Tsingke, China).

Using a replication-incompetent VSIV containing the green fluorescent protein (GFP) instead of the receptor-binding VSIV glycoprotein (G) gene (VSVΔG*-G), VSIVs pseudotyped with S proteins of SARS CoVs (VSVΔG*-SCoV and VSVΔG*-SCoV-2) were generated as described previously (41 (link), 42 (link)). Briefly, 24 h after transfection of HEK293T cells with pCAGGS expressing the S protein of SARS-CoV (Tor2 strain; GenBank accession number NC_004718.3) or SARS-CoV-2 (strain WHU01; GenBank accession number MN988668.1), the cells were incubated with VSVΔG*-G for 60 min at 37°C. After three washes with DMEM, the medium was replaced by DMEM with 10% FCS. After 24 h, the supernatants were harvested and stored at −80°C until use. Virus IUs in HEK293T cells were determined by counting the number of GFP-positive cells with an IN Cell Analyzer 2500HS (GE Healthcare). To produce VSIVs pseudotyped with the S proteins having the substitutions in RBD derived from the Alpha (N501Y), Beta (K417N, E484K, and N501Y), Gamma (K417T, E484K, and N501Y), and Delta (L452R and T478K) variants, the mutant S protein genes were constructed by site-directed mutagenesis with KOD One (Toyobo) based on the S protein gene of SARS-CoV-2 WHU01, which is an early isolate from Wuhan. Each mutation was confirmed by Sanger sequencing.

Genomic DNA extracted from small pieces of clonally propagated hygromycin-resistant rice calli or kanamycin-resistant tobacco calli using Agencourt chloropure (Beckman Coulter) according to the manufacturer's protocol was subjected to cleaved amplified polymorphic sequence (CAPS) analysis. PCR amplifications were performed with KOD FX neo or KOD ONE (TOYOBO) using the primer sets shown in Supplementary Table 1. PCR products were digested with restriction enzyme MfeI for OsALS and NtALS-B, XbaI for OsCly1, and HindIII for NtEPSPS-B and analyzed with MultiNA microchip electrophoresis system (Shimadzu).
PCR fragments derived from CAPS-positive calli or plants were cloned into pCR-BluntII-TOPO (Invitrogen) and subjected to sequence analysis using an ABI3130 sequencer (Applied Biosystems).

Plasmids were linearized with SwaI prior to the transformation of T.reesei, or the PCR product was transformed using a modified protoplast-PEG method^{64 (link)}, in which 20 mg/mL of Yatalase (Takara Bio) was used as the protoplasting enzyme instead of Novozyme 234 (Novozymes Bagsværd, Denmark). The transformed protoplasts were plated on minimal transformation medium [2.0% (w/v) glucose, 18.27% (w/v) sorbitol, 0.5% (w/v) (NH₄)₂SO₄, 0.2% (w/v) CaCl₂, 0.06% (w/v) MgSO₄, 0.21% (w/v) CsCl, and 0.1% (w/v) trace element solution in 100 mM KH₂PO₄ buffer (pH 5.5)] for the pyr4 marker. The trace element solution contained 500 mg FeSO₄·7H₂O, 200 mg CoCl₂, 160 mg MnSO₄·H₂O, and 140 mg ZnSO₄·7H₂O in 100 mL of distilled water. After two weeks of incubation at 30 °C, candidate transformants were streaked twice on selective plates (each minimal transformation medium without sorbitol) for several days at 30 °C for single-colony isolation. Single colonies were then transferred to PDA plates for one week at 30 °C to allow for the formation of conidia. According to the manufacturer’s protocol, one transformant was confirmed by colony PCR using KOD One (Toyobo). To transform the resulting transformants again using the PDA medium containing 0.2% (w/v) 5-fluoroorotic acid (5-FOA) monohydrate, a strain that acquired 5-FOA resistance again (pyr4 pop-out through homologous recombination) was selected.

To facilitate the future assembly of the synAC, sets of neighboring fragments consisting of five or six fragments were introduced into S. cerevisiae. These fragments were used to construct 32 initial fragments (~32 kb) using TAR assembly. To construct the initial fragment plasmids, functional vectors containing iterative assembly parts and homologous arms (500 bp homologous arms designed to be added to the ends of DNA fragments) were pre-constructed. The NEBuilder HiFi DNA Assembly Master Mix from NEB was employed to assemble the vectors and iterative parts into the pre-constructed functional vectors. Subsequently, the pre-constructed vectors were amplified using the KOD-one (a kind of DNA polymerase) PCR Master Mix from TOYOBO. Five or six linear fragments (100–200 ng of each fragment) and the pre-constructed functional vector (~100 ng) were co-transformed into BY4741^XT1-FCY and BY4742^XT2-URA yeast strains according to the design outlined in Supplementary information, Data S2.

Genomic DNA was extracted from hESCs or HCT116 cells using the DNeasy Blood and Tissue Kit (QIAGEN), and endpoint PCRs were performed using KOD One (TOYOBO). Sanger sequencing was performed with a cycle sequencing reaction using the BigDye, version 3.1, Cycle Sequencing Kit (Thermo Fisher Scientific) and analyzed with an ABI 3730XL DNA Analyzer (Thermo Fisher Scientific).
For knockdown experiments, siRNAs targeting OCT4 (Thermo Fisher Scientific, Silencer Select, s10872) and SOX2 (Thermo Fisher Scientific, Silencer Select, s13294) were used. Transfection of siRNAs was performed using TransIT X2 (Mirus Bio) following the manufacturer’s protocol. Seventy-two hours after transfection, cells were collected for RNA preparation.
RNA was extracted from hESCs or HCT116 cells using the RNeasy Kit (QIAGEN) and reverse transcribed using ProtoScript II Reverse Transcriptase (New England BioLabs). qRT-PCR was performed using KOD SYBR (TOYOBO) in a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Relative expression levels were calculated by the ΔΔCt method using ACTB as an internal control gene. Primer sequences are listed in Supplemental Table 3.