Supplemental Figure S1
1 TATCCTCTTAAGCGGGTATTTTGATGGTAAATCTACAAGCCCTCGGCTTTAATTATTACCATGGTCATAAAAAGTTACTT
81 ACAATGCATACATTTTTCTTTTAGGAAGAAATAAAATATAAAAGACTTATCGTTCAAATCGGGTCATAAAGGGAACTAAA
161 GGTATACTAAGGTAAGACGGGGTTCTCATCTGCACGTAATTTTTTATTATTTTTAGTGAGGGAGAACTTTACCTCTAAGA
241 TTGTTTATATAGGTATACACATTTTTGTAAAACTGATTATTCGTTTTTTTTTTTGAAACAAGTTATTAGTACAAAAATCA
321 AGCTCTCACTACTTACCAGTAGTTATTTTAGTAGAATATTATTAACTCTACCGAATAAGTACGATATGAGAAATTGCTGA
401 TTTACTTGTGTGTGTTCCTATTATTATCCAGGAGCGCCTCTAATCTATGCTTATAATTTTACGTTGAAAAAAAAGGAGTC
481 TGTATAATTATTTCAAATTCATCCTTTTCTCACGGATATTGGGTGCTCTTTTCACAAAATTCGTCGCGCAATTATTTACC
561 TCATAGGGTACACAGACACGCTCGACTTCCTGTCTTCCAGTCTGGAGTCGGCTTCCAATTTCGTCACACAACAAGATTTC
641 TAGCAGCGGCCTTCAGGCTTGAAACAAAGAATCGAAGGTTCTGGAATGGTGGGAATCAGTTTACTACCACATGCTATGAT
721 GTCTACCGCACTCTCCAGAGCAAAAGTTTCGTTCAATCACACTCTTACACTCTCTCTCACACAATTGTTCGAATCGTGTG
801 ACAACAGCAACTTGTTCTCACACAGTCTCTTTTCTTCTAACCAAGGGGTTGTTTGGGCTAGTAAAGCTTTGTGGAAACTT
881 AGATTTACATATATATAAAGTTGAACGAATTAGTGAATGCTAAAGAAATATATAATTGACCTTTTTCTCTAGATAGTTTT
961 TTGAACTTGTTAGATGTATTTTAATTCTTTCTCTGTTTTTTTTATTTCTTCTTTTCACAGATCATCAAGGAAGTAATTAT
1041 CTACTTTTTGCAACAAATATAAAACAATGAGATTCCCATCTATTTTCACTGCTGTTTTGTTCGCTGCTTCTTCTGCTTTG
1121 GCTGGTATTATTAATACTTTGCAAAAATACTACTGTAGAGTTAGAGGTGGTAGATGTGCTGTTTTGTCTTGTTTGCCAAA
1201 AGAAGAACAAATTGGTAAATGTTCTACTAGAGGTAGAAAATGTTGTAGAAGAAAAAAATGAGGCGCGCCACTTCTAAATA
1281 AGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACT
1361 CTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGTATGAG
1441 GTCGCTCTTATTGACCACACCTCTACCGGCAGATCCGCTAGGGATAACAGGGTAATATAGTCGACCTCGAGTCATGTAAT
1521 TAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTC
1601 TAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTA
1681 CAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCG
1761 GCCGGTACCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTAAGGGTTCTCGACGTTTTCGACACTGGATGGC
1841 GGCGTTAGTATCGAATCGACAGCAGTATAGCGACCAGCATTCACATACGATTGACGCATGATATTACTTTCTGCGCACTT
1921 AACTTCGCATCTGGGCAGATGATGTCGAGGCGAAAAAAAATATAAATCACGCTAACATTTGATTAAAATAGAACAACTAC
2001 AATATAAAAAAACTATACAAATGACAAGTTCTTGAAAACAAGAATCTTTTTATTGTCAGTACTGATTAGAAAAACTCATC
2081 GAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAG
2161 GAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATA
2241CAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAA
2321 TGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAAACGAGCGTAATGGCTGGCCTGCATCAAAATCACTCGCATCAA
2401 CCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGA
2481 ATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTG
2561 GAATGCTGTTTTGCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAA
2641 GAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTC
2721 AGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCA
2801 TTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAAACGTGAGTCTTTTCCTTACCCATGGTTG
2881 TTTATGTTCGGATGTGATGTGAGAACTGTATCCTAGCAAGATTTTAAAAGGAAGTATATGAAAGAAGAACCTCAGTGGCA
2961 AATCCTAACCTTTTATATTTCTCTACAGGGGCGCGGCGTGGGGACAATTCAACGCGTCTGTGAGGGGAGCGTTTCCCTGC
3041 TCGCAGGTCTGCAGCGAGGAGCCGTAATTTTTGCTTCGCGCCGTGCGGCCATCAAAATGTATGGATGCAAATGATTATAC
3121 ATGGGGATGTATGGGCTAAATGTACGGGCGACAGTCACATCATGCCCCTGAGCTGCGCACGTCAAGACTGTCAAGGAGGG
3201 TATTCTGGGCCTCCATGTCGCTGGCCGGGTGACCCGGCGGGGACGAGGCAAGCTAAACAGATCTCTAGACCTAATAACTT
3281 CGTATAGCATACATTATACGAAGTTATATTAAGGGTTGTCTTAATTAAGGGTAACGATTACTAAAAATATCCAAAAGAAA
3361 AACAATTGGATCTTTTTTGACTTCTCTACATTACAATTTCAGTAAAGCAGCTTTCAACTTAATTTAATTTTGAACAATGC
3441 CGCCAGCTAGTACTAGTACTACCAATGATATGATAACCGAAGAACCTACTTCTCCACACCAAATCCCAAGGCTTACAAGG
3521 AGACTTACGGGGTTTCTTCCCCAAGAAATCAAGTCAATTGACACGATGATTCCTTTAAAGTCAAGAGCGTTATGGAATAA
3601 GCATCAAGTCAAAAAATTTAACAAGGCAGAAGATTTTCAAGATAGATTCATTGACCATGTGGAAACTACATTAGCACGTT
3681 CCCTATATAATTGTGATGACATGGCTGCTTATGAAGCTGCTTCGATGAGTATTCGTGACAATTTGGTCATTGACTGGAAC
Figure S1. DNA sequence of CM-51-INT. Nts 1-520: S. cerevisiae chromosome XVI (859735-860254), Nts 521-904: S. bayanus PGK
promoter (GenBank Accession Number AACA01000006.1; 48563- 48946), Nts 1067-1126: Synthetic construct mating factor alpha
preproprotein (mfapp) (GenBank Accession Number JN193564.1), Nts 1127-1262: Codon optimised ORF human beta defensin-3.
(databank sequence; GENE ID: 55894 DEFB103B | defensin, beta 103B [Homo sapiens]), Nts 1263-1499: S. cerevisiae ADH1
3'untranslated and terminator sequences Chromosome XV (159538-159562), Nts 1500-1771: S. cerevisiae CYC1 3' untranslated and
terminator sequences Chromosome X (526684-526939) Nts 1772-3437: Lox-KanMx-Lox cassette, Nts 3437-: S. cerevisiae
chromosome XVI. The location of primers used for amplification of sections of the gene construct are listed in Table 1.
Supplemental Material
Integration of HBD3 into Lager yeast strain CMBS-33.
The DNA sequences for insertion were amplified as three overlapping fragments (1, 2 and 3; Fig 1B). Fragment
1, corresponding to the S. bayanus (PGK) gene promoter (GenBank Accession Number AACA01000006.1;
48563- 48946) was amplified from the lager yeast strain CMBS-33 genomic DNA using primers A and B (Table
1). The 5’ region of primer A contains DNA sequences from YPR159C-A (Table 1, represented by an open
rectangle in Fig 1B) while the 5’ end of primer B has reverse complementarity (30 nucleotides) downstream to
the start codon (ATG) of the signal peptide in the HBD3 construct in pGREGHBD3-505. Fragment 2 was
amplified with Primers C and D and contains sequences spanning from the signal peptide sequence (Primer C)
and the 5’ end of the KanMx gene of the HBD3 construct in pGREGHBD3-505 (Primer D). Similarly, primers E
and F were used to amplify Fragment 3 encompassing the remainder of the KanMx gene. Primer E has
extensive overlap with Fragment 2 while the 5’ 30 nucleotides of primer F has reverse complementarity to
sequences in the intergenic region YPR159C-YPR160W of chromosome XVI in the lager yeast genome (Usher
and Bond, 2009), upstream of the start codon YPR160W (Table 1). The three fragments were amplified
separately and purified. Equimolar amounts of the three fragments were used in a transformation reaction
with the yeast lager strain CMBS-33 with the following exceptions; the PEG-lithium acetate incubation was at
o
30 C for 16 hours and the recovery period was extended to 24 hours. The cells were plated on YEPM-agar
plates containing 200 µg/mL G418 and incubated up to 72 hours. The three fragments were assembled and
inserted into the chromosome by in vivo homologous recombination as described previously (Jansen et al.,
2005), To identify integrants at the YPR159A site, colony PCRs were carried out using primers anchored in the
YPR159C-A region of the genomic DNA and various segments of the HBD3 gene construct. PCR products were
sequenced on both strands.