Table 1. Strains, plasmids, and oligonucleotide primers used in this study.
| Strains, plasmid or primer | Description | Source or reference |
|---|---|---|
| Strains | ||
| E. coli DH5α | F− ɸ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 (rk-, mk+) supE44 thi-1 gyrA relA1 | Life Technologies |
| Y. lipolytica | Strain SKU507; isolated from soil sample | [15] |
| P. pastoris KM71H | Expression host. aox1:ARG4,arg4 | Life Technologies |
| Plasmids | ||
| pGEM-T-Easy | ori pMB1, Ampr | Promega |
| pPICZαA | P. pastoris 3.6-kb protein expression and secretion vector carrying a methanol-inducible promoter (PAOX1), 5′ AOX1 region, MFα1s; Zeor | Invitrogen |
| pPICZαA-Sp-YlLac | 5′AOX1 region; Zeor; with YlLac for expression in P. pastoris | This study |
| pPICZαA-Wsp-YlLac | 5′AOX1 region, MFα1s; Zeor; with YlLac for expression in P. pastoris | This study |
| Primers (5′-3′) | ||
| CuI | CAYTGGCAYGGNTTYTTYCA | [16] |
| CuIV | TGRAARTCDATRTGRCARTG | |
| SPR1 | AGGTGGCTATGG TACCAGAACGTTCCAGC | This study |
| SPR2 | GCAGCAGTGAAGTCGTATAGGAACGAGTTG | This study |
| SPR3 | GGACACTGGTTGACGAAAGCAGGTCCGTCC | This study |
| SPF1 | CGTCCTTGGCTGCAGACGGACAATCCGG | This study |
| SPF2 | TGTTCACACACATATCTATCTCCCGGCCGC | This study |
| SPF3 | CGGCACGGCTGGCGACAATGTCACCATCCG | This study |
| YlLac-f1 | ATGAACTTTGTGACCGCACTCCCACTG | This study |
| YlLac-r1 | TTGCAGATCCGGGCCTAAACGTCCACG | This study |
| Sp-YlLac-f1 | ctcgagATGAACTTTGTGACCGCACTCCCAC a | This study |
| Sp-YlLac-r1 | tctagaTTGCAGATCCGGGCCTAAACGTCCAC b | This study |
| Wsp-YlLac-f1 | ggtaccGCCATTGGCCCGGTGACC c | This study |
| Wsp-YlLac-r1 | tctagaTTGCAGATCCGGGCCTAAACGTCCAC b | This study |
| AD1 | NTGCANTNTGCNGTT | [17] |
| AD2 | NGTCAGNNNGANANGAA | |
| AD3 | NGTGNGANANCANCANAG | |
| AD4 | TGNGNGANANCANAG | |
| AD5 | AGNGNAGNANCANAGC | |
aThe XhoI site is in small letters.
bThe XbaI site is in small letters.
cThe KpnI site is in small letters.
W = A / T, S = G / C, N = A/T / G / C