Constructing S. eubayanus Maltose Transporter Strains

S. eubayanus IMK816 (SemalT1Δ) was constructed by transforming CBS 12357^T by electroporation (Gorter de Vries et al., 2017 (link)) with 200 ng of pUDP062 and 1 μg of 120 bp repair fragment obtained by mixing an equimolar amount of primers 11850 and 11851 (Table S5) (Mans et al., 2015 (link)) (Figure 1). As control, the same transformation was performed without including the repair DNA fragment. Transformants were selected on SM_AceG plates. Strain IMK817 (SemalT2Δ SemalT4Δ) and IMK818 (SemalT3Δ) were constructed in the same way. The SeMALT2/T4 deletion was constructed by co-transforming pUDP063 and a repair DNA fragment formed by primers 11328 and 11329, while the SeMALT3 deletion involved pUDP064 and a repair DNA formed by primers 11330 and 11331 (Table S5). Deletion of SeMALT1, SeMALT2/T4, and SeMALT3 was verified by diagnostic PCR, using primers pairs 11671/11672, 11673/11674, and 11675/11676 (Table S5), respectively (Figure 1C). Prior to storing at −80°C, transformants were successively streaked on SM_AceG and YPD plates. The genotype was verified after each plating round with the primers pairs mentioned above.
S. cerevisiae IMZ616 [mal1Δ mal2Δ mal3Δ mph2Δ mph3Δ suc2Δ ima1Δ ima2Δ ima3Δ ima4Δ ima5Δ pUDC156 (Spcas9 URA3 ARS4 CEN6)], which cannot grow on α-glucosides (Marques et al., 2018 (link)) was used as a host to test the functionality of individual S. eubayanus (putative) maltose transporter genes. S. cerevisiae IMX1253 was constructed by integrating the S. cerevisiae maltase gene ScMAL12 and the SeMALT1 transporter gene at the ScSGA1 locus of strain IMZ616 (Figure 2). The ScSGA1 gene encodes an intracellular sporulation-specific glucoamylase (Yamashita and Fukui, 1985 (link)) that is not expressed during vegetative growth (Knijnenburg et al., 2009 (link)). This integration site was shown suitable for expression of single or multiple genes as previously demonstrated in Mans et al. (2015 (link)), Kuijpers et al. (2016 (link)), Verhoeven et al. (2017 (link)), and Bracher et al. (2018 (link)) The fragment containing ScMAL12 was PCR amplified using Phusion High-Fidelity DNA polymerase (Thermo FisherScientific) from pUDE044 (Basso et al., 2011 (link)) with primers 9596 and 9355, which included a 5′ extension homologous to the upstream region of the S. cerevisiae SGA1 locus and an extension homologous to the co-transformed transporter fragment, respectively. The DNA fragment carrying the S. eubayanus SeMALT1 maltose symporter was PCR amplified from pUD479 using primers 9036 and 9039, which included a 5′ extension homologous to the co-transformed transporter fragment and an extension homologous to the downstream region of the S. cerevisiae SGA1 locus, respectively. To facilitate integration in strain IMZ616, the two PCR fragments were co-transformed with plasmid pUDR119, which expressed a gRNA targeting ScSGA1 (spacer sequence: 5′-ATTGACCACTGGAATTCTTC-3′) (van Rossum et al., 2016 (link)) (Figure 2A). The plasmid and repair fragments were transformed using the LiAc protocol (Gietz and Schiestl, 2007 (link)) and transformed cells were plated on SM_AceG. Correct integration was verified by diagnostic PCR with primers pairs 4226/5043 and 942/4224 (Figure 2, Table S5). Strains S. cerevisiae IMX1254, IMX1255, and IMX1365 were constructed following the same principle, but instead of using pUD479 to generate the transporter fragment, pUD480 pUD481 and pUD445 were used to PCR amplify SeMALT2/T4, SeMALT3, and ScAGT1 respectively. Correct integration was verified by diagnostic PCR with primers pairs 4226/5043 and 942/4224 (Figure 2, Table S5). All PCR-amplified gene sequences were Sanger sequenced (Baseclear, Leiden, The Netherlands).