pYEDIS+GOI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Five micrograms of the purified amplicon was then electroporated into P. pastoris GS115 (his4 −), and the transformants were selected on minimal medium without histidine.	Positive transformants were identified by colony PCR and characterized by partial sequencing (Fig. S5). The resulting strain was named P. pastoris YEDIS.

Protocol tips
Five micrograms of the purified amplicon was then electroporated into P. pastoris GS115 (his4 −), and the transformants were selected on minimal medium without histidine.
Downstream tips
Positive transformants were identified by colony PCR and characterized by partial sequencing (Fig. S5). The resulting strain was named P. pastoris YEDIS.

Publication protocol

The P. pastoris necessary as a recipient for the expression of the pYEDIS+GOI plasmids obtained from S. cerevisiae (Fig. 1C) was obtained by integration, into the AOX1 promoter of P. pastoris GS115, of a linear DNA fragment generated as follows. The pYEDIS vector was digested with NotI and re‐ligated to remove the GAL1 promoter. The re‐ligated vector was then used as template for the amplification, with primers AOX‐FW and MATα‐RV, of a 1221 bp DNA fragment carrying the AOX1 promoter fused with the coding sequence for the S. cerevisiae α‐factor signal peptide, which ends in a stop codon introduced with the MATα‐RV primer (Fig. S5). On the other hand, a second 2731 bp fragment containing the P. pastoris HIS4 gene was amplified from the genome of P. pastoris X33, using primers HIS4‐FW and HIS4‐RV, which introduced ends overlapping with the previous fragment. The two PCR products were fused by their two ends with the Gibson Assembly Cloning Kit, generating a circular 3904 bp DNA molecule (Fig. S5). This circular DNA fragment was then used as template in a third PCR, with primers PAOXmid‐FW and PAOXmid‐RV, amplifying the whole circular molecule from the middle of the AOX1 promoter (Fig. S5). Five micrograms of the purified amplicon was then electroporated into P. pastoris GS115 (his4 −), and the transformants were selected on minimal medium without histidine. Positive transformants were identified by colony PCR and characterized by partial sequencing (Fig. S5). The resulting strain was named P. pastoris YEDIS.

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