TableS1 Primers used in this study
Primers / Oligonucleotide sequences(5’-3’)ori-amyA-F / CCCTAGGGCCGCACCGTTTAACG
ori-amyA-R / CGCGGCCGCTCAATGATGATGATGATGATGAGGCCATGCCACCA
opt-amyA-F / CCTAGGGCTGCTCCATTCAACGG
opt-amyA-R / GCGGCCGCTTAATGATGATGATGATGATGTGGCCAAGCAACCAA
AOX1-HAC1-F / GAATTCATGCCCGTAGATTCTTC
AOX1-HAC1-R / CGGCCGCTTATTCCTGGAAGAATACAAAGTCATTTAAATCAAATGCATTAGCGGTAAATGGTGCTGCTGGATGATGCAAC
GAP-HAC1-F / CAATTGAACAACTATATGCCCGTAGATTCTTC
GAP-HAC1-R / ATGAGTTTTTGTTCTTATTCCTGGAAGAATACAAA
T-PAOX1-F / AGATCTAACATCCAAAGACGAA
T-PAOX1-R / ACGTTTCGAATAATTAGTTGTT
T-HAC1-F / AATACGGGCATCTAAGAAGAGTA
T-HAC1-R / ATTATTCCTGGAAGAATACAA
T-GAP-F / AATGGCTATCACTGTCGGTATT
T-GAP-R / ATTAAGCCTTAGCAACGTGTTG
RT-Paox-F / GAAGCTGCCCTGTCTTAAACCTT
RT-Paox-R / CAAAAGCTTGTCAATTGGAACCA
RT-actin-F / CTCCAATGAACCCAAAGTCCAA
RT-actin-R / CTCCAATGAACCCAAAGTCCAA
RT-opt-amyA-R / CACAATACAACATTCCATCCT
RT-opt-amyA-R / CCTTCTCTAGTCCATCCAAT
RT-HAC1-F / CTGAATATGACGACGAAGAA
RT-HAC1-R / CTCCTGCTTGATAGATGTG
5’-AOX1 / GACTGGTTCCAATTGACAAGC
α-factor primer / TACTATTGCCAGCATTGCTGC
3’-AOX1 / GCAAATGGCATTCTGACATCC
TableS2 Plasmids used in this study
Plasmids / Description / SourcepPICZB / Expression vector for intracellular expression / Invitrogen
pPIC9K / Expression vector for extracellular expression / Invitrogen
pGAPZB / Expression vector for intracellular expression / Invitrogen
pGAPH / Expression vector for intracellular expression / This study
pPIC9K-ori-amyA / Vector for extracellular expression Gs4j-amyA / This study
pPIC9K-opt-amyA / Vector for extracellular expression Gs4j-amyA / This study
pPCIZB-HAC1 / Vector for intracellular expression HAC1p / This study
pGAPH-HAC1 / Vector for intracellular expression HAC1p, Hygromycin B / This study
pGAPH-(HAC1)2 / Vector for intracellular expression HAC1p, Hygromycin B, with 2 expression cassette HAC1 / This study
pGAPH-(HAC1)4 / Vector for intracellular expression HAC1p, Hygromycin B, with 4 expression cassette HAC1 / This study
pMD19-T / Commercial plasmid for standard curve construction / Takara
pMD19-T-GAP / Plasmid for GAPDH standard curve construction / This study
pMD19-T-HAC1 / Plasmid for HAC1 standard curve construction / This study
pMD19-T-opt-amyA / Plasmid for opt-amyA standard curve construction / This study
pMD19-T-ori-amyA / Plasmid for PAOX1 standard curve construction / This study
Table S3 Usage of codons for ori-amyA and opt-amyA
Amino acid / Codon / Host fraction / ori-amyA / opt-amyAGly / GGG / 0.1 / 11 / 0
GGA / 0.32 / 14 / 47
GGT / 0.44 / 3 / 0
GGC / 0.14 / 19 / 0
Glu / GAG / 0.43 / 1 / 0
GAA / 0.57 / 18 / 19
Asp / GAT / 0.58 / 21 / 41
GAC / 0.42 / 20 / 0
Ala / GCG / 0.06 / 3 / 0
GCA / 0.23 / 5 / 0
GCT / 0.45 / 8 / 32
GCC / 0.26 / 16 / 0
Arg / AGG / 0.16 / 2 / 0
AGA / 0.48 / 1 / 21
CGG / 0.05 / 6 / 0
CGA / 0.1 / 2 / 0
CGT / 0.16 / 2 / 0
CGC / 0.05 / 8 / 0
Lys / AAG / 0.53 / 5 / 28
AAA / 0.47 / 25 / 2
Ser / AGT / 0.15 / 0 / 0
AGC / 0.09 / 0 / 0
TCG / 0.09 / 5 / 0
TCA / 0.19 / 2 / 0
TCT / 0.29 / 3 / 1
TCC / 0.2 / 6 / 27
Stop / TAG / 0.53 / 1 / 1
Asn / AAT / 0.49 / 7 / 3
AAC / 0.51 / 15 / 19
Met / ATG / 1 / 10 / 10
Ile / ATA / 0.19 / 0 / 0
ATT / 0.5 / 8 / 21
ATC / 0.3 / 13 / 0
Thr / ACG / 0.11 / 10 / 0
ACA / 0.24 / 8 / 0
ACT / 0.4 / 6 / 44
ACC / 0.25 / 20 / 0
Cys / TGT / 0.65 / 0 / 1
TGC / 0.35 / 1 / 0
Trp / TCG / 1 / 23 / 23
Leu / TTG / 0.33 / 9 / 33
TTA / 0.16 / 5 / 0
CTG / 0.16 / 0 / 0
CTA / 0.11 / 0 / 0
CTT / 0.16 / 0 / 0
CTC / 0.08 / 0 / 0
Phe / TTT / 0.54 / 14 / 1
TTG / 0.46 / 10 / 23
Gln / CAG / 0.39 / 4 / 0
CAA / 0.91 / 13 / 17
His / CAT / 0.57 / 6 / 11
CAC / 0.43 / 5 / 0
Pro / CCC / 0.15 / 13 / 0
CCG / 0.09 / 4 / 0
CCA / 0.41 / 4 / 25
CCT / 0.35 / 4 / 0
Fig.S1Sequence alignment of ori-amyA and opt-amyA.
Fig.S2Standard curves for real time-qPCR. Each curve was the result of three biological repeated experiments and the amplification efficiency was calculated using the E=101/-slope-1. And the standard curves were used for the calculation for gene dosage integrated into the genome. a: standard curve for opt-amyA; b: standard curve for GAP; c: standard curve for HAC1; d: standard curve for ori-amyA.
Fig.S3Aox enzyme activity in GS-9K and GS-opt-A12. All the samples were harvested after 10 h of methanol induction in shake flasks. The intracellular protein was obtained by grinder with zircon beads. Data are presented as the means ± standard deviation (SD) of triplicate determination.
Fig. S4Prediction of glycosylastion sites in the deduced animo acid sequence. Four potential glycosylation sites were detected according to the glycosylation site prediction website (