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Supplementary Information for
Engineered ethanol-driven biosynthetic system for improving production of
acetyl-CoA derived drugs in Crabtree-negative yeast
Yiqi Liua, Chenxiao Baia, Qi Liua, Qin Xua, Zhilan Qiana, Qiangqiang Penga, Jiahui Yua,
Mingqiang Xua, Xiangshan Zhoua, Yuanxing Zhanga,b, and Menghao Caia,*
aState Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, ChinabShanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road,
Shanghai 200237, China
*Corresponding author. Tel./fax: +86-21-64253306. E-mail address:
This supplementary file includes:
Supplementary Methods
Suppl. Figs. S1 to S12
Suppl. Tables S1 to S6
References for SI reference citations
Supplementary Methods
Construction of ADH3, ALD1 and ACS1 Deletion and Complemented Strains. The 5’
and 3’ flanking regions (~1000 bp) of ADH3, ALD1 and ACS1 gene were firstly cloned
from P. pastoris GS115 genome by primer pairs of pUC18-ADH-up-F/ADH-up-R,
ADH-down-F/pUC18-ADH-down-R for ADH3; primer pairs of pUC18-ALD-up-F/ALD-
up-R, ALD-down-F/pUC18-ALD-down-R for ALD1; primer pairs of pUC18-ACS-up-
F/ACS-up-R, ACS-down-F/pUC18-ACS-down-R for ACS1, respectively. A DNA
fragment was then cloned from the plasmid pPIC3.5K (Invitrogen) by primer pair of
HIS4-F/HIS4-R, which was then fused with the obtained 5’ and 3’ flanking regions of
ADH3, ALD1 and ACS1 to obtain the targeted knockout sequences with selective marker
HIS4, respectively. Then a DNA fragment was cloned from the plasmid pUC18 by
primer pair of pUC18-F/pUC18-R, and it was then fused with each targeted knockout
fragment by seamless cloning (ClonExpressTM II one-step cloning kit, Vazyme Biotech
Co., Ltd., China) to produce knockout plasmids of pUC_Δadh3, pUC_Δald1 and
pUC_Δacs1. Each plasmid was extracted and used as PCR template to obtain knockout
fragment for each gene, respectively. Primer pairs of testADH3-up-F/testADH3-down-R,
testALD-up-F/testALD-down-R or testACS1-up-F/testACS1-down-R was used for
cloning of knockout fragment for each specific gene, respectively. The obtained fragment
was then transformed into competent P. pastoris GS115 by electroporation and positive
transformants were screened (Pichia protocols)1. Accordingly, target gene was knocked
out by double crossover and gene knockout strains of Pp/Δadh3, Pp/Δald1, Pp/Δacs1
were obtained finally. For gene complementation, each gene was expressed by the
promoter of PGAP in a pGAPZα A (Invitrogen) plasmid with selective marker Sh ble
against zeocin, generating plasmids of pZ_PGAP-ADH3, pZ_PGAP-ALD1, pZ_PGAP-ACS1.
They were then linearized by BlnI and inserted into the homologous GAP site in P.
pastoris genome by single crossover separately, and positive transformants of
Pp/Δadh3_Re, Pp/Δald1_Re, Pp/Δacs1_Re, were screened by zeocin (Pichia protocols)1.
Construction of Strains Expressing eGFP by Ethanol Inducible Promoters. Ethanol
inducible promoters, i.e., PADH3, PALD1, PACS1, PICL1, were cloned from P. pastoris genome
using primer pairs of pADH3-F/ZBTT~pADH3-R, pALD-F/ZBTT~pALD-R,
ZBBgl~pACS1-F/ ZBTT~pACS1-R, ZB~pICL1-F/ZB~pICL1-R, respectively. Then a
linearized DNA fragment removing PAOX1 was harvested from the pPICZ B plasmid
(Invitrogen) by double digestion with BglІІ/EcoRІ. It was then fused with each ethanol
inducible promoter by seamless cloning to generate plasmids of pZ_PADH3, pZ_PALD1,
pZ_PACS1 and pZ_PICL1, respectively. Afterwards, various fragments of egfp were
amplified by upstream primer of pADH3~GFP-F, pALD~GFP-F, pACS1~GFP-F or
pICL1~GFP-F, paired with downstream primer of TT~GFP-R separately. The products
were correspondingly inserted into vectors generated by digesting plasmids of pZ_PADH3,
pZ_PALD1, pZ_PACS1, pZ_PICL1 with XhoІ/SalІ, respectively. Accordingly, expression
plasmids of pZ_PADH3-eGFP, pZ_PALD1-eGFP, pZ_PACS1-eGFP, pZ_PICL1-eGFP with
selective marker Sh ble (zeocin resistance) were constructed, respectively. Linearized
plasmids, i.e., pZ_PADH3-GFP (by EcoRІ), pZ_PALD-GFP (by SacІ), pZ_PACS1-GFP (by
BlnІ), pZ_PICL1-GFP (by SpeІ), were transformed into the competent P. pastoris GS115
by electroporation (Pichia protocols)1 and eGFP expression yeast strains were obtained.
Single copy expression strains were then identified and designated as Pp/PADH3-eGFP,
Pp/PALD-eGFP, Pp/PACS1-eGFP, Pp/PICL1-eGFP, respectively.
A methanol induced eGFP expression strain and a constitutive eGFP expression
strain was constructed as control. An egfp fragment amplified by primer pair of ZB~GFP-
F/ZBTT~GFP-R was cloned into a vector of EcoRІ/SalІ digested pPICZ B (Invitrogen)
by seamless cloning, resulting an expression plasmid, pZ_PAOX1-eGFP. Similarly, an egfp
fragment amplified by primer pair of GAP~GFP-F/GAPTT~GFP-R was cloned into a
vector of KpnІ/BspT104І digested (α-factor removed) pGAPZα A (Invitrogen) by
seamless cloning, resulting an expression plasmid pZ_PGAP-GFP. Afterwards, the
pZ_PAOX1-eGFP was linearized by PmeІ and the pZ_PGAP-eGFP was linearized by BlnІ.
The linearized plasmids were then transformed into competent P. pastoris GS115 by
electroporation, respectively, and positive transformants were screened by zeocin (Pichia
protocols)1. Single copy expression strains were then identified and named as Pp/PAOX1-
eGFP and Pp/PGAP-eGFP, respectively.
Construction of Strains Expressing eGFP by ESAD and CSAD. Two fragments were
amplified from a plasmid of pP-GFP2 with primer pairs of lacO-cAOX1F/pPcAGR and
pPcAGF/lacO-pPICR. They were then fused to an intact promoter by seamless cloning,
resulting a plasmid of pPlacO1cAG that containing a hybrid lacO-cPAOX1 (Supplementary
Table 2). Then a primer pair Bgl~LacOcAOX-F/TT~cAOX-R was used to further
amplify the hybrid promoter sequence, which was then cloned into a vector of
BglІI/EcoRІ digested pPICZ B (Invitrogen) by seamless cloning, resulting a plasmid of
pZ_lacO-cPAOX1. An egfp fragment was amplified with primer pair of
ZB~GFP-F/ZBTT~GFP-R and then cloned into the EcoRІ/SalІ digested pZ_lacO-cPAOX1,
resulting pZ_lacO-cPAOX1-eGFP. Afterwards, an expression cassette of lacO-cPAOX1-gfp-
TT was amplified by primer pair 35K~LacOcAOX-F/35K~TT-R, which was then cloned
into a PAOX1 removed pPIC3.5K and resulted a plasmid of pK-lacO-cPAOX1-eGFP. Then
this plasmid was linearized at HIS4 by SalІ, and integrated into P. pastoris genome to
obtain a single copy expression strain of Pp/lacO-cPAOX1-eGFP. Then, a lacI fragment
was amplified from E. coli genome by primer pair of pICL~lacI-F/mit1~lacI-R and a
MITIAD fragment was cloned from P. pastoris genome by primer pair of
lacI~mit1-F/TT~LacIMit1-R. They were then fused together by overlap PCR and
involved a GGGGS linker, which then produced the chimeric activator encoded gene. It
was then cloned into a vector of XhoI/SalІ digested pZ_PICL1 by seamless cloning to
produce a plasmid of pZ_PICL1-LacI-Mit1AD. The lacI-MIT1AD fragment was amplified
from this plasmid by primer pair of pGAP~LacImit1-F/TT~LacIMit1-R and cloned into a
BspT104І/KpnІ digested pGAPZα A (α-factor removed) by seamless cloning, resulting an
expression plasmid of pZ_PGAP-LacI-Mit1AD. Afterwards, competent cells of Pp/lacO-
cPAOX1-eGFP were prepared following Pichia protocols1. The pZ_PICL1-LacI-Mit1AD and
pZ_PGAP-LacI-Mit1AD were linearized by SpeІ and BlnІ, respectively, and transformed
into competent cells of Pp/lacO-cPAOX1-eGFP by single crossover. Positive transformants
were screened by zeocin and strains with single expression cassette copy of egfp and lacI-
MIT1AD were identified and named as Pp/ESAD-eGFP (ethanol induced expression) and
Pp/CSAD-eGFP (constitutive expression), respectively. In addition, single expression
strains involving LacI-Gal4AD or LacI-VP16 instead of LacI-Mit1AD in Pp/ESAD-
eGFP, were constructed using the same methods.
Construction of 6-Methylsalicylic Acid Producing Strains with Different Systems.
The biosynthesis of 6-MSA needs heterologous co-expression of 6-MSA synthase gene
atX and phosphopantetheinyl transferase gene npgA. Therefore, we firstly amplified both
genes from the previously constructed plasmids of pPICZ B-AtX (pZ_AtX) and
pPIC3.5K-NpgA (pK_NpgA)3, respectively, with primer pairs of pGAP~AtX-F/TT~AtX-
R and pGAP~NpgA-F/TT~NpgA-R. They were then cloned into a vector of
KpnІ/BspT104І digested (α-factor removed) pGAPZα A (Invitrogen) by seamless cloning
separately, resulting plasmids of pGAPZ_AtX and pGAPZ_NpgA. Then a PGAP-npgA-TT
fragment with a single SpeІ site was amplified from the pGAPZ_NpgA plasmid with
primer pair of 35K~SpepGAP-F/35KTT~NpgA-R, which was then cloned into a
BamHІ/BlnІ digested vector of pPIC3.5K by seamless cloning and a plasmid of pK_PGAP-
NpgA was obtained. Afterwards, a PGAP-atX-TT fragment was amplified from the
pGAPZ_AtX plasmid and then cloned into the SpeІ linearized pK_PGAP-NpgA by
seamless cloning, resulting a plasmid of pK_PGAP-AtX+NpgA. This plasmid was then
linearized by BspEІ and transformed into competent P. pastoris GS115 by
electroporation. Positive transformants were screened against zeocin (Pichia protocols)1
and the strains carrying single copy expression cassette of atX and npgA were designated
as Pp/PGAP-XN.
Three fragments, i.e., BamHІ/BlnІ digested pPIC3.5K, atX-TT amplified from the
pZ_AtX3 plasmid with primer pair of pAOX~AtX-F/KanaHis~TT-R, PAOX1-npgA
amplified from the pK_NpgA3 plasmid with primer pair of TT~pAOX-F/35KTT~NpgA-
R, were fused to generate a plasmid of pK_PAOX1-XN. This plasmid was linearized by
BspEІ and transformed into competent P. pastoris GS115 by electroporation. Positive
transformants were screened by HIS4 (Pichia protocols)1, and the strains carrying single
copy expression cassette of atX and npgA were designated as Pp/PAOX1-XN.
Afterwards, an atX fragment was amplified from pZ_AtX3 by primer pair of
cAOXXho~AtX-F/TTSal~AtX-R. Also, an npgA fragment was amplified from
pPIC3.5K-NpgA by primer pair of cAOXXho~NpgA-F/TTSal~NpgA-R. Each fragment
was cloned into a vector of XhoI/SalІ digested pZ_lacO-cPAOX1 by seamless cloning,
resulting plasmids of pZ_lacO-cPAOX1-AtX and pZ_lacO-cPAOX1-NpgA. A lacO-cPAOX1-
npgA-TT fragment was cloned from the pZ_lacO-cPAOX1-NpgA plasmid with primer pair
of 35K~placO-F/KanaHis~TT-R. A linearized, PAOX1 and TT removed pPIC3.5K vector
was obtained by PCR using pPIC3.5K as template and TT~KanaHis-F/Amp~KanaHis-R,
KanaHis~Amp-F/lacOcAOX~Amp-R as primer pairs. They were then fused by seamless
cloning to produce a plasmid of pK_lacO-cPAOX1-NpgA. Afterwards, a lacO-cPAOX1-atX-
TT fragment was cloned from the pZ_lacO-cPAOX1-AtX with primer pair of 35K~placO-
F/lacOcAOX~TT-R. It was then cloned into the SpeІ linearized pK_lacO-cPAOX1-NpgA
by seamless cloning, resulting a plasmid of pK_lacO-cPAOX1-AtX+NpgA. This plasmid
was digested by BspEІ and transformed into competent P. pastoris GS115 by
electroporation. Positive transformants were screened based on HIS4 marker (Pichia
protocols)1, and the strains with single copy expression cassette of atX and npgA
designated as Pp/lacO-cPAOX1-XN. Afterwards, the plasmids of pZ_PICL1-LacI-Mit1AD
and pZ_PGAP-LacI-Mit1AD were linearized by SpeІ and BlnІ, respectively, and separately
transformed into competent cells of Pp/lacO-cPAOX1-XN. Positive transformants was
screened by zeocin (Pichia protocols)1, and the strains with single copy expression
cassette of atX, npgA and lacI-MIT1AD were designated as Pp/ESAD-XN (ethanol
induced expression) and Pp/CSAD-XN (constitutive expression), respectively.
Construction of Dihydromonacolin L Producing Strains with the ESAD System.
Gene fragments of lovB, lovC, lovG and npgA were firstly cloned from the previously
constructed plasmids of pPICZ-LovB (by primer pair of lacOcAOX~LovB-F/TT~lovB-
R), pPICZ-LovC (by primer pair of lacOcAOX~LovC-F/TT~lovC-R), pPICZ-LovG (by
primer pair of lacOcAOX~lovG-F/TT~lovG-R) and pPICZ-NpgA (by primer pair of
lacOcAOX~npgA-F/TT~npgA-R), respectively4. Each gene fragment was cloned into a
vector of XhoІ/SalІ digested pZ_lacO-cPAOX1, generating plasmids of pZ_lacO-cPAOX1-
LovB, pZ_lacO-cPAOX1-LovC, pZ_lacO-cPAOX1-LovG and pZ_lacO-cPAOX1-NpgA.
Afterwards, a lacO-cPAOX1-lovC-TT fragment was cloned from the pZ_lacO-cPAOX1-LovC
plasmid with primer pair of TT~lacOcAOX-F1/BamH~TT-R. It was then cloned into a
vector of BamHІ digested pZ_lacO-cPAOX1-LovB by seamless cloning to generate a
plasmid of pZ_lacO-cPAOX1-BC. Similarly, a lacO-cPAOX1-lovG-TT fragment was cloned
from the pZ_lacO-cPAOX1-LovG plasmid with the same primer pair and then cloned into
the BamHІ digested pZ_lacO-cPAOX1-BC to generate a plasmid of pZ_lacO-cPAOX1-BCG.
Then using the same method, a plasmid of pZ_lacO-cPAOX1-BCGN was further obtained.
Finally, a PICL1-lacI-MIT1AD-TT fragment was amplified from the pZ_PICL1-LacI-
Mit1AD plasmid with primer pair of TT~pICL1-F/BamH~TT-R and cloned into the
linearized pZ_lacO-cPAOX1-BCGN, resulting a plasmid of pZ_PICL1-LM_lacO-cPAOX1-
BCGN. This plasmid was then linearized by SpeІ and transformed into competent P.
pastoris. Positive transformants were screened by zeocin (Pichia protocols)1, and the
strains with single copy expression cassette of lovB, lovC, lovG, npgA and lacI-MIT1AD
were designated as Pp/ESAD-BCGN.
Metabolic Engineering on Ethanol Metabolic Pathway in Dihydromonacolin L
Producing Strains. Ethanol catabolic related genes of ADH2, ALD6 and ACS1 were
firstly cloned from S. cerevisiae genome with primer pairs of
pGAP~ScALD6-F/TT~ScALD6-R, pGAP~ScADH2-F/TT~ScADH2-R and
pGAP~ScACS1-F/pGAP-ScACS1-R, respectively. A site mutated ACS1* was then
obtained by PCR with primer pair of pGAP-ScACS1-F/ACS1mut-R. These genes were
cloned into a vector of KpnІ/BspT104І digested (α-factor removed) pGAPZα A
(Invitrogen) by seamless cloning separately, resulting plasmids of pZ_PGAP-ScADH2,
pZ_PGAP-ScALD6 and pZ_PGAP-ScACS1*.
Two fragments were obtained from the pPIC3.5K vector by primer pairs of
35KdelHis-1-F/35KdelHis1-R, 35KdelHis-2-F/35KdelHis-2-R, by which the HIS4
selective marker was removed. Then the expression cassette of ADH2 was amplified
from the pZ_PGAP-ScADH2 plasmid with primer pair of
pAOXSpe~pGAP-F/35KdelHisTT-R. These three fragments were then fused to form a
plasmid of pKdH_PGAP-ScADH2 by seamless cloning. The plasmids of pKdH_PGAP-
ScALD6 and pKdH-PGAP-ScACS1* were then constructed by the same method. These
plasmids were then linearized by SpeІ and transformed into competent cells of Pp/ESAD-
BCGN, and positive transformants were screened by G418 (Pichia protocols)1. The
strains with single copy expression cassette were designated as Pp/ESAD-BCGN_PGAP-H,
Pp/ESAD-BCGN_PGAP-D, Pp/ESAD-BCGN_PGAP-S, respectively.
To combinatorially overexpressed the Adh2, Ald6 and Acs1*, plasmids for co-
expression of various genes were required. Firstly, an expression vector of pGAP*Z was
constructed from the pGAPZα A by PCR with primer pairs of
Z~pGAPmutBln-F/pGAPmutBln-R, pGAPmutBln-F/TT~pGAPmutBln-R followed by
an overlap PCR experiment. By this way, the BlnІ site was lost in PGAP in the pGAP*Z.
Then plasmids of pZ_PGAP*-ScADH2, pZ_PGAP*-ScALD6 and pZ_PGAP*-ScACS1* were
constructed using the same construction strategy as described above. Expression cassette
of each gene was cloned from the corresponding plasmid by primer pair of
pAOXSpe~pGAP-F/pGAP~TT-R. The obtained fragments were then combinatorially
cloned into the SpeІ linearized pKdH_PGAP-ScADH2 by seamless cloning, resulting
expression plasmids of pKdH_PGAP-ScADH2+PGAP*-ScALD6 and pKdH_PGAP-ScADH2+
PGAP*-ScACS1*. Based on this, expression cassette of ACS1* was cloned and fused into
the SpeІ linearized pKdH_PGAP-ScADH2+PGAP*-ScALD6, resulting an expression
plasmid of pKdH_PGAP-ScADH2+PGAP*-ScALD6+ScACS1*. These plasmids were
linearized by BlnІ and transformed into competent cells of Pp/ESAD-BCGN separately,
and positive transformants was screened by G418 (Pichia protocols)1. The strains with
single copy of each expression cassette were designated as Pp/ESAD-BCGN_PGAP-HD,
Pp/ESAD-BCGN_PGAP-HS, Pp/ESAD-BCGN_PGAP-HSD, respectively.
Overexpression of acetyl-CoA carboxylase gene was also performed. A S1132A site
mutated ACC1* was cloned from P. pastoris genome with primer pair of pGAP~ACC1-
F/ACC1mut-R and ACC1mut-F/TT~ACC1-R by overlap PCR. The ACC1* fragment
was then cloned into a vector of KpnІ/BspT104І digested (α-factor removed) pGAPZα A
by seamless cloning, resulting a plasmid of pZ_PGAP-ACC1*. Afterwards, a PGAP-acc1*-
TT fragment was then amplified from the pZ_PGAP-ACC1* plasmid with primer pair of
pAOXSpe~pGAP-F/35K~TT-R. It was then cloned into a vector of BamHІ/BlnІ digested
pPIC3.5K by seamless cloning, resulting a plasmid of pK_PGAP-ACC1*. This plasmid
was digested by BlnІ and transformed into competent cells of Pp/ESAD-BCGN_PGAP-HS,
and positive transformants with was screened by HIS4 (Pichia protocols)1 The strains
with single copy of each expression cassette were designated as Pp/ESAD-BCGN_PGAP-
HSC.
Construction of Strains for Coculture Strategy for Monacolin J Production. Gene
fragments of slovA and cpr were amplified from our previously constructed plasmids of
pPICZ-sLovA and pPICZ-CPR4, respectively, with primer pairs of lacOcAOX~sLovA-
F/TT~sLovA-R and lacOcAOX~CPR-F/TT~CPR-R. They were then cloned into a vector
of XhoІ and SalІ digested pZ_lacO-cPAOX1 separately, resulting plasmids of pZ_lacO-
cPAOX1-sLovA and pZ_lacO-cPAOX1-CPR. Afterwards, a lacO-cPAOX1-slovA-TT fragment
was amplified from the pZ_lacO-cPAOX1-sLovA with primer pair of
Amp~lacOcAOX-F/KanaHis~TTSpe-R. Two other fragments were amplified from the
pPIC3.5K vector with primer pairs of TT~KanaHis-F/Amp~KanaHis-R and
KanaHis~Amp-F/lacOcAOX~Amp-R, respectively. These three fragments were fused
and formed a plasmid of pK-lacO-cPAOX1-sLovA by seamless cloning. Similarly, a lacO-
cPAOX1-cpr-TT fragment was amplified from the pZ_lacO-cPAOX1-CPR plasmid with
primer pair of TT~lacOcAOX-F2/KanaHis~TTSpe-R. It was cloned into a vector of SpeІ
linearized pK-lacO-cPAOX1-sLovA, resulting a plasmid of pK_lacO-cPAOX1-sAR. Then,
fragments of PICL1-lacIMIT1-TT and PGAP-lacIMIT1-TT were amplified from the plasmids
of pZ_PICL1-LacI-Mit1 (with primer pair of TT~pICL1-F/BamH~TT-R) and pZ_PGAP-
LacI-Mit1 (with primer pair of TT~pGAP-F/BamH~TT-R), respectively. They were
cloned into a vector of SpeІ linearized pK_lacO-cPAOX1-sAR, resulting plasmids of
pK_PICL1-LM_lacO-cPAOX1-sAR and pK_PGAP-LM_lacO-cPAOX1-sAR, respectively. Then
the obtained plasmids were linearized by SalІ and transformed into competent P. pastoris
GS115 separately. Positive transformants were screened by HIS4 (Pichia protocols)1. The
strains with single copy of each expression cassette were designated as Pp/ESAD-sAR
and Pp/CSAD-sAR, respectively.
To knock down expression of FAS1 on methanol, we involved a promoter PHXT1 and
its expression depends on existence of glucose5. A single copy eGFP expression strain,
Pp/PHXT1-eGFP, was firstly constructed (similar steps to the Pp/PHXT1-eGFP) to test its
expression activity on ethanol. For construction of FAS1 knock-down strain, a HXT1
promoter sequence was cloned from P. pastoris genome with primer pairs of
ZEcoR~pHXT1-F/PpFAS1~pHXT1-R; and an upstream FAS1 flanking region was
amplified from P. pastoris genome with primer pairs of
pHXT1~PpFAS1-F/HisSal~PpFAS1-R. Both fragments were cloned into the EcoRI/SalI
digested pPICZ B by seamless cloning to generate a plasmid named pZ_PHXT1-FAS1up.
Then an expression cassette of PHXT1-FAS1up was amplified from the pZ_PHXT1-FAS1up
with primer pairs of pAGBamH~pHXT1-F/pAGBgl~TT-R, which was subsequently
cloned into a BamHI/BglII digested pAG32 by seamless cloning and resulting an
expression plasmid of pAG-PHXT1-FAS1up. It was then linearized by XbaI and
transformed into the Pp/ESAD-BCGN_PGAP-HSC strain and inserted into its native gene
loci by single crossover and hygromycin was used to screen positive transformants. By
this method, the native FAS1 expression cassette was destroyed and expression of FAS1
can only be controlled by the PHXT1. The resulted strain was named as Pp/ESAD-BCGN-sAR_PGAP-HSC_PHXT1-F.
Subcellular Localization Analysis. The eGFP was used as a reporter for subcellular
localization. The samples expressing eGFP were visualized by inverted microscope
DMI3000B (Leica) using a 100× oil immersion objective. Images were processed using
the Leica application suite, version 2.8.1. Cells were cultured in YPD medium to OD600 of
6.0. It was then centrifuged (5000 g) to harvest cells, followed by two rounds of washing
in sterile ddH2O and centrifugation. Then cells were resuspended and inoculated in YPD
medium or YNE medium (YNB+0.5% [v/v] ethanol) to a final density (OD600=1.0) for
culture. After OD600 reached 3.0~4.0, 500 µL broth was centrifuged and washed, and then
added by 200 µL DAPI staining solution (C1006, Beyotime Biotech. Co. Ltd. China) and
incubated at 30°C for 30 min. Afterwards, cells were collected by centrifugation (12000
g, 2 min), and washed and centrifuged twice. Finally, cells were resuspended by sterile
ddH2O and observed by the fluorescence microscope.
Measurement of Intracellular Fatty Acids. Culture steps refer to Fig. S1. Culture
samples were prepared before ethanol feedings. Total intracellular fatty acid content was
determined by gas chromatography-mass spectrometry (GC-MS), adapted from a
previously report6. For fatty acids analysis, samples were collected at 12, 36, 60 and 84 h,
respectively. Residual ethanol level was detected by a Biosensor equipment (SBA-40E,
Shandong Academy of Sciences, China). Suitable volume of P. pastoris broth was
collected and centrifuged immediately after sampling from shake flask. The obtained cell
precipitate was then stored at -20°C and used for fatty acids analysis after fermentation.
Cells samples were dried at 60°C to constant weight and put into 15-mL centrifuge tube,
followed by adding 2 mL solution 1 (4.48 g KOH dissolved in 200 mL methanol) and
putting in 70°C water bath for 30 min. After cooling to room temperature, 2 mL solution
2 (3.2 mL H2SO4 dissolved in 200 mL methanol) was added and mixed, followed by
addition of 1 mL BF3-CH3OH solution (stored at -20°C, Sigma) and incubation in 70°C
water bath for 30 min. Then 2 mL n-hexane was added, vortexed, stood for 10 min and
added double distilled H2O to 10 mL solution. The obtained sample was then centrifuged
at 3000 g for 5 min. Afterwards, 1 mL oil layer was harvested, added with 0.1 g Na2SO4
to dehydration for over 3 h. The sample was then centrifuged at 3000 g for 5 min to
harvest the supernatant. The supernatant was then mixed with the same volume of
internal standard solution (0.01 g methylnonadecanoate dissolved in 25 mL n-hexane)
and used for GC-MS analysis. Standards of fatty acid methyl ester (FAME), FAME MIX
GLC-80 and FAME MIX GLC-10 (Sigma) that containing FAME of (C13:0), (C14:0),
(C15:0), (C16:0), (C17:0), (C18:1), (C18:2), (C18:3) and (C18:0), were used. They were
mixed with a methyl palmitoleate standard (C16:1), followed by adding the same volume
of internal standard solution, and used for preparation of standard curves for
determination of fatty acids in fermentation samples. Samples were analyzed by a GC-
MS System (HIMADZU QP2010 SE) equipped with a fused-silica capillary column HP-
5MS (Thickness 0.25 μm, I.D. 0.32 mm, Length 30 m, Agilent Technologies, USA). The
injection volume was 1 μL at a split ratio of 10:1. The oven temperature was set as 160
°C for 2 min initially and increased by 5°C/min to 230°C and held for 2 min. The injector
port was set at 250°C and the FIDdetector was set at 280°C. Helium was used as the
carrier gas at a constant flow rate of 1.0 mL/min. The temperatures of quadrupole and ion
source temperature were 150°C and 230°C, respectively. A SIM-Scan mode was used
and molecule weight of 20~400 was scanned. Each FAME peak was identified by
comparing its retention time and ion fragmentation information to those of reference
standards. Quantification of individual FAME was accomplished by incorporating the
known amount of internal standard.
Calculating Methods for Specific Productivity, Biomass yield and Product Yield on
Ethanol. These methods were referred to literatures7-9 and described as the following
equations.
(Equation 1)
(Equation 2)
(Equation 3)
(Equation 4)
Where µ is for specific growth rate, qP is for specific productivity, YX/S is for biomass
yield on ethanol, YP/S is for product yield on ethanol, t is for culture time (t1 is followed
by t2); X is for biomass concentration (time point for X1 is followed by that for X2); P is
for compound titre (time point for P1 is followed by that for P2); S is for the total
consumed ethanol from fermentation start to a certain culture time point (time point for
S1 is followed by that for S2).
Supp. Fig. S1. A recapitulative scheme for culture steps in various experiments.
Suppl. Fig. S2. Spotting growth of wild type P. pastoris on acetate. Cells were cultured on YPD
plates (pH=4.5) for 2 days with gradient concentration of acetate. Various cell densities
(OD600=0.1, 0.01, 0.001, 0.0001) were used for spottings.
Results description: Acetate easily impaired cell growth of P. pastoris. Acetate higher than 30
mM severely repressed cell growth. Cell even cannot grow on agar plate with 40 mM acetate,
which was even lower than the limited level of 100 mM toleranted by S. cerevisiae10.
Suppl. Fig. S3. Residual ethanol levels in culture broth of various strains. These results support
the ethanol catabolic capacities of various strains in Fig. 1e&f. Culture steps refer to Fig. S1. The
independent-sample t-test was used to determine statistical significance in various experiments.
Statistical significance of residual ethanol in culture of various strains relative to the wild type
strain at each time point was shown. ## P<0.01, # P<0.05 at 24 h; ** P<0.01 at 48 h; ++ P<0.01, +
P<0.05 at 72 h; n.s., Not significance.
Suppl. Fig. S4. Nuclear localization function analysis of LacI and Output of synthetic
transcriptional signal amplification devices with various transactivation domains. A LacI protein
from lac operon of E. coli functioned as a nuclear localization signal in P. pastoris (A). The
constitutive promoter of PGAP was used to express eGFP, LacI-eGFP and SV40-LacI-eGFP. The
SV40 nuclear localization signal (NLS), an active and widely used NLS in P. pastoris11, was
fused at N-terminus of LacI-eGFP to be a positive control. DAPI was used to stain the cell
nucleus. Fluorescence microscopy of the constructed strains were carried out under the inverted
microscope DMI3000B (Leica) with a 100× oil immersion objective. The eGFP fluorescence
intensity activated by different transactivation domains were shown in (B). Methanol-activated
and ethanol-repressed promoter, PAOX1, was used as a control. The transactivation domains were
constructed to C-terminus of a LacI protein from lac operon of E. coli by a linker of GGGGS. A
lacO sequence from lac operon of E. coli was flanked to 5’ end of cPAOX1 by PCR. Sequence of
Gal4AD and codon optimized VP16 were shown in Supplementary Table 2. The error bars
represent the standard deviation of three biological replicates (each with two or three technical
replicates), assayed in duplicate. Culture steps refer to Fig. S1. Every 24 h, 0.5% (v/v) ethanol or
methanol, 2% (w/v) glucose or 1% (w/v) glycerol was added. D, glucose; G, glycerol; E, ethanol.
One mL sample was pipetted out and centrifuged (5000 g, 4°C) every 8 h, supernatant was
discarded, and cells were harvested and stored immediately at -80°C for the following
fluorescence analysis using an enzyme-labeled instrument (Synergy 2, BioTek Instruments) at an
excitation wavelength of 485 nm and an emission wavelength of 525 nm. Statistical significance
of eGFP fluorescence by various expression system relative to that by the LacI-Mit1AD on
ethanol is at P<0.01 at each time point/substrate. Thus, we uniformly marked it at the LacI-
Mit1AD site as ## (16 h); ** (24 h), respectively.
Results description: LacI functioned well as a nuclear localization signal in P. pastoris (A).
Although the LacI-Gal4AD and LacI-VP16 successfully activated eGFP expression especially
under ethanol induction condition, their expression intensity were much lower than that by the
LacI-Mit1AD.
Suppl. Fig. S5. Transcription analysis of egfp for function evaluation of the engineered
transcriptional signal amplification device (TSAD). The mRNA levels were normalized to the
levels of mRNA of housekeeping gene ACT1 in each sample. The relative expression level
indicated on the y-axis (2-CT) for each gene at different carbon sources was normalized for its
expression by PAOX1 on methanol. Samples were collected from 4 h culture. The error bars
represent the standard deviation of three biological replicates (each with two technical replicates)
assayed in duplicate. Error bars smaller than the plot symbols not displayed. The independent-
sample t-test was used to determine statistical significance of various groups relative to the ESAD
(E) group. ** P<0.01. M, methanol; D, glucose; E, ethanol. Total RNA was extracted using
RiboPure™-Yeast Kit (Ambion), according to manufacturer’s protocol and this was treated with
DNase I to exclude the genomic DNA contaminant. Reverse transcription was performed
following ReverTra Ace transcription kit (Toyobo).
Results description: These transcription results accorded with the eGFP expression results by
various systems. Our engineered TSAD functioned well as that, the ethanol induced ESAD
generated the highest egfp transcriptional level that was even higher than that by the natural
strongest PAOX in P. pastoris. Besides, the constitutive CSAD also produced high transcriptional
levels of egfp on either ethanol or glucose. The low transcriptional level of egfp by the ESAD on
glucose indicated that it showed a good regulation mode of glucose-repressed and ethanol-
induced.
Suppl. Fig. S6. The eGFP fluorescence intensity of the synthetic ethanol induced expression
system (ESAD) on gradient levels of different substrates. Glucose/Glycerol (A); Ethanol/Acetate
(B). Fluorescence analysis was conducted referring to Fig. S3. The error bars represent the
standard deviation of three biological replicates assayed in duplicate. Culture steps refer to Fig.
S1.
Results description: Glucose level higher than 1% (w/v) repressed eGFP expression. Ethanol
level of 0.5% or 1% (v/v) well induced eGFP expression. However, the repression effect of
glycerol was not good at various levels. Expression of eGFP on acetate was weak.
Suppl. Fig. S7. Comparison of leaked production of 6-MSA by expression systems based on
ESAD and PAOX1. Constitutive PGAP and CSAD were used as control. The error bars represent the
standard deviation of three biological replicates assayed in duplicate. Error bars smaller than the
plot symbols not displayed. Culture steps refer to Fig. S1. Cells were inoculated in YND medium
(YNB+2% (w/v) glucose) to a final density (OD600=1.0) for culture. During culture, samples were
analyzed every 24 h. Glucose of 2% (w/v) was fed every 24 h after each sample pipetted out.
Results description: Under this condition, 6-MSA by the PGAP and CSAD was constitutively
produced but 6-MSA by the PAOX1 was almost completely blocked by glucose. The ESAD was
also highly repressed by glucose as compared to the PGAP and CSAD, meaning that this
engineered ESAD produced a low leaked production of the target compound. As 6-MSA caused
some damage to cells12, a higher 6-MSA titre led to a lower cell density for these strains.
Suppl. Fig. S8. Cell growth of dihydromonacolin L (DML) producing strains with various
expression systems. This figure corresponds with Fig. 4A&B in text. M, methanol; E, ethanol.
PAOX1 represents the Pp/PAOX1-BCGN strain; ESAD represents the Pp/ESAD-BCGN strain. The
error bars represent the standard deviation of three biological replicates assayed in duplicate or
triplicate. Error bars smaller than the plot symbols not displayed. Culture steps refer to Fig. S1.
Ethanol of 0.5% (v/v) was fed every 24 h.
Results description: The Pp/ESAD-BCGN produced DML with a higher level than Pp/PAOX1-
BCGN. Also, it grew better than the Pp/PAOX1-BCGN.
Suppl. Fig. S9. Extracellular acetate concentrations (A) and cell growth (B) of recombinant
strains overexpressing genes for metabolic pathways of ethanol to acetyl-CoA and acetyl-CoA to
malonyl-CoA. These genes were combinatorially overexpressed in the Pp/ESAD-BCGN strain.
The Pp/ESAD-BCGN were used as control. The wild type strain of GS115 was also tested as
compared to the Pp/ESAD-BCGN, which was used to evaluate the effects of overexpression of
the heterologous biosynthetic genes on acetate metabolism. This figure corresponds with Fig. 3d
in text. The error bars represent the standard deviation of at least three biological replicates (each
with two or three technical replicates) assayed in duplicate or triplicate. Error bars smaller than
the plot symbols not displayed. Culture steps refer to Fig. S1. Cells were inoculated in YNE
medium (YNB+0.5% (v/v) ethanol) to a final density (OD600=1.0) for culture. During culture,
samples were analyzed every 24 h. Ethanol of 0.5% (v/v) was fed every 24 h after each sample
pipetted out.
Results description: P. pastoris strains of overexpression of S. cerevisiae acetaldehyde
dehydrogenase Ald6, Adh3+Ald6, Adh3+Ald6+Acs1*, i.e., Pp/ESAD-BCGN_PGAP-D, Pp/ESAD-
BCGN_PGAP-HD, Pp/ESAD-BCGN_PGAP-HSD, produced higher levels of acetate comparing with
other strains. The accumulated acetate also caused damage to cell growth of the three strains.
1:0.1
1:0.2
1:0.5 1:1
1:1
.5 1:2 1:0.1
1:0.2
1:0.5 1:1
1:1
.5 1:20
30
60
90
120
150
180Ti
tre
of p
rodu
cts
(mg/
L)DMLMLMJ
ESAD-sAR CSAD-sAR
Downstream strain
Suppl. Fig. S10. Coculture of the upstream strain Pp/ESAD-BCGN_PGAP-HSC with different
downstream strain of Pp/ESAD-sAR or Pp/CSAD-sAR. Culture steps refer to Fig. S1. The error
bars represent the standard deviation of three biological replicates (each with two or three
technical replicates) assayed in duplicate.
Results description: It caused severe accumulation of intermediates when using the Pp/ESAD-
sAR as the downstream strain and accumulation of intermediates aggravated with the increase of
inoculation ratio of the upstream strain. While it only produced low levels of intermediates when
employing the Pp/CSAD-sAR as the downstream strain. The concentrations of intermediates
decreased with the increase of inoculation ratio of the downstream strain. The optimal
combination is Pp/ESAD-BCGN_PGAP-HSC:Pp/CSAD-sAR=1:0.2.
Suppl. Fig. S11. Expression behaviors of the promoter PHXT1 (a) on glucose and ethanol. The glucose-repressed and ethanol-induced promoter PICL1 (b) was used as a control. Culture steps
refer to Fig. S1. The error bars represent the standard deviation of at least three biological
replicates assayed in duplicate. Statistical significance of eGFP fluorescence by PHXT1 from
various runs relative to the PHXT1-2%D was shown. #P<0.01, ## P<0.01 at 16 h; *P<0.01, **P<0.01
at 24 h; n.s., not significance. Statistical significance of eGFP fluorescence by PICL1 from various
runs relative to the PICL1-0.5%E was also shown. ## P<0.01 at 16 h; *P<0.05, **P<0.01 at 24 h;
n.s., not significance. 1% or 2%D indicates 1% or 2% (w/v) glucose; 0.5% or 1% E indicates
0.5% or 1% (v/v) ethanol.
Results description: The results showed that PHXT1 was induced by glucose and
repressed by ethanol. Thus, it is suitable to be used for knock-down of the FAS1 when cells were
shifted to ethanol culture phase (Refer to Fig. 4 in text).
Suppl. Fig. S12. Comparison of total intracellular fatty acids between Pp/ESAD-BCGN_PGAP-HSC and Pp/ESAD-BCGN_PGAP-HSC_PHXT1-F cultured on ethanol. Cell density (a), residual
ethanol (b) and intracellular fatty acids (c) were determined. FAS1, Pp/ESAD-BCGN_PGAP-HSC; FAS1-knock down, Pp/ESAD-BCGN_PGAP-HSC_PHXT1-F. The error bars represent
the standard deviation of two or three biological replicates assayed in duplicate. Culture steps
refer to Fig. S1. Fatty acids analysis was described in Supplementary Methods. Ethanol of 1%
(v/v) was fed every 24 h.
Results description: Content of total intracellular fatty acids in Pp/ESAD-BCGN_PGAP-HSC was higher than that in the FAS1 knock-down strain of Pp/ESAD-BCGN_PGAP-HSC_PHXT1-F (C), despite that growth and ethanol utilization of both
strains were similar. Cell density reached a higher level than the control (Fig. S8) because of the
enhanced ethanol-to-acetyl-CoA pathway and the increased ethanol feedings (1%, v/v).
Suppl. Fig. S13. Biomass yield and MJ yield of strains of Pp/ESAD-BCGN-sAR_PGAP-HSC and Pp/ESAD-BCGN-sAR_PGAP-HSC_PHXT1-F from the substrate of ethanol. This
figure corresponds with Fig. 4b&c in text. The results were from further analysis of the data
obtained from bioreactor fermentation shown in Fig. 4b&c. Culture steps refer to Fig. S1. DCW,
dry cell weight; MJ, monacolin J.
Results description: The strain of Pp/ESAD-BCGN-sAR_PGAP-HSC_PHXT1-F showed higher MJ yield and lower biomass yield from ethanol after 80 h, comparing with the Pp/ESAD-BCGN-sAR_PGAP-HSC.
Suppl. Table S1. List of oligonucleotides used in this study*.
Primer name Sequence (5’ to 3’)Primers for constructionpUC18-ADH-up-F GGAAACAGCTATGACCATGATTACCGATTGCCCCTCTACAGG
ADH-up-R TAAGCTTGCACAAACGAACGTTTCGTAAAGTAAATAAGATAAAAGCTAGT
ADH-down-F AACCAATTAACCAATTCTGAGCCGAATAGTTTGTATACGTCTT
pUC18-ADH-down-R TGCCAAGCTTGCATGCCTGCAGAGAAATGGACGGTGTTTTGGA
pUC18-ALD-up-F GGAAACAGCTATGACCATGATTACAGACCAGCAGTTTAACTACGC
ALD-up-R AGCTTGCACAAACGAACGCTTTTCTTTGGGCAAGGAAAAATCAAG
ALD-down-F ACCAATTAACCAATTCTGAACTGAGTATTTATGACCTTATATATTATTA
pUC18-ALD-down-R TGCCAAGCTTGCATGCCTGCAGAAATCAATCGTCAGTTCAATCAAG
pUC18-ACS-up-F GGAAACAGCTATGACCATGATTACAGCAAAATCATCTGGCTCAG
ACS-up-R TAAGCTTGCACAAACGAACGAATTGATCAACAACTAAGTCGTATCC
ACS-down-F AACCAATTAACCAATTCTGAGCATCTGATTAGGACTTACACTTC
pUC18-ACS-down-R TGCCAAGCTTGCATGCCTGCAGTCTGATTCCAAAACCTTTTGATCAT
pUC18-F CTGCAGGCATGCAAGCTT
pUC18-R TAATCATGGTCATAGCTGTTTCC
testADH3-up-F CGATTGCCCCTCTACAGG
testADH3-down-R AGAAATGGACGGTGTTTTGGA
testALD-up-F AGACCAGCAGTTTAACTACGC
testALD-down-R AAATCAATCGTCAGTTCAATCAAG
testACS1-up-F AGCAAAATCATCTGGCTCAG
testACS1-down-R TCTGATTCCAAAACCTTTTGATCAT
HIS4-F CGTTCGTTTGTGCAAGCT
HIS4-R TCAGAATTGGTTAATTGGTTGTAACAC
pADH3-F TTTGGTCATGAGATCCGCAGCGTTTTCTGACG
ZBTT~pADH3-R CAATGATGATGATGATGATGTTTCGTAAAGTAAATAAGATAAAAG
pALD-F TTTGGTCATGAGATCAGACCAGCAGTTTAACTAC
ZBTT~pALD-R GCTACAAACTCAATGATGATGATGATGATGCTTTTCTTTGGGCAAGGAAA
ZBBgl~pACS1-F TTGGTCATGAGATCAGATCTAAAACCACCAGCTAGTACAG
ZBTT~pACS1-R GCTGGGCCACGTGAATTCAATTGATCAACAACTAAGTCGT
ZB~pICL1-F TTTGGTCATGAGATCAGATCTTCATCTAACACTTTGTATAGCACATCG
ZB~pICL1-R GCTGGCGGCCGCCGCGGCTGCAGTCTTGATATACTTGATACTGTGTTCTT
pADH3~GFP-F TTTTATCTTATTTACTTTACGAAAACCATGGGTTCTAAAGGTGAA
pALD~GFP-F TTCCTTGCCCAAAGAAAAGACCATGGGTTCTAAAGGTGAA
pACS1~GFP-F AGTTGTTGATCAATTGAATTCACCATGGGTTCTAAAGGTG
pICL1~GFP-F CAAACTCAATGATGATGATGATGATGCTATTTGTACAATTCATCCATACC
TT~GFP-R CTCAATGATGATGATGATGATGCTATTTGTACAATTCATCCATACCAT
ZB~GFP-F AACAACTAATTATTCGAAACGAGACCATGGGTTCTAAAGGTGAAG
ZBTT~GFP-R AACTCAATGATGATGATGATGATGCTATTTGTACAATTCATCCATACCAT
GAP~GFP-F TTTCAATCAATTGAACAACTATACCATGGGTTCTAAAGGTGAAGA
GAPTT~GFP-R GCGGCCGCCGCGGCTCGCTATTTGTACAATTCATCCATACCATGG
lacO-cAOX1F GAATTGTGAGCGGATAACAATTTCACACAGGGCCCCTAACCCCTACTTGACAGCA
pPcAGR CTGATGTTACTGAAGGATCAGATCACGCAT
pPcAGF TGATCCTTCAGTAACATCAGAGATTTTGAG
lacO-pPICR TTGTTATCCGCTCACAATTCCACACACTCGAGGAGCTCGTTCCCGATCTGCGTCTA
35KDpAOX-F CGCTCACAATTCCACACAAGATCTCGAATAATAACTGTTATTTTT
35K~TT-R ATCGATAAGCTTGCACAAACGAACTTCTCACTTAATCTTCTGTACTCTGA
35KDpAOX-R TCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCGAT
Bgl~LacOcAOX-F GGATTTTGGTCATGAGATCAGATCTTGTGTGGAATTGTGAGCG
TT~cAOX-R AGCTGGCGGCCGCCGCGGCTCGAGTTCGAATAATTAGTTGTTTTTTGATCT
35K~LacOcAOX-F AAAAATAACAGTTATTATTCGAGATCTTGTGTGGAATTGTGAGCG
pICL~lacI-F ACACAGTATCAAGTATATCAAGAATGGGTGTTAAGCCAGT
mit1~lacI-R TTAACAGAGCCGCCGCCACCTTGTCCAGACTCCAATCTAGAGACT
lacI~mit1-F GGACAAGGTGGCGGCGGCTCTGTTAACAACTCCATGAAGGATTTC
TT~LacIMit1-R ACTCAATGATGATGATGATGATGTTATTCTTCAACATTCCAGTAGTCA
pGAP~LacImit1-F TCAATCAATTGAACAACTATATGGGTGTTAAGCCAGTTAC
ZBAOXup-F GTCTGACGCTCAGTGGAAC
pGAP~AtX-F ATCAATTGAACAACTATTTCGAAACGAGGACCATGGGTATGGAGGTACATGGAGA
TT~AtX-R GCTAAAACTCAATGATGATGATGATGATGTAGAAAGCTGGCGGCC
pGAP~NpgA-F TCAATTGAACAACTATTTCGAAACGAGGACCATGGGTATGGTGCAAGACACATCA
TT~NpgA-R AAAACTCAATGATGATGATGATGATGGGATAGGCAATTACACACC
35K~SpepGAP-F AACAACTAATTATTCGAAGACTAGTCTTTTTGTAGAAATGTCTTGGTGTC
35KTT~NpgA-R AATTAATTCGCGGCCGCCCTAGGTCAATGATGATGATGATGATGGGATAG
pGAP~TT-R ACACCAAGACATTTCTACAAAAATCTCACTTAATCTTCTGTACTCTGAAG
TT~pAOX-F TCGTCTTTGGATGTTAGATCTTCTCACTTAATCTTCTGTAC
pAOX~TT-R GTACAGAAGATTAAGTGAGAAGATCTAACATCCAAAGACGA
cAOXXho~AtX-F AAAACAACTAATTATTCGAAATGGAGGTACATGGAGATGA
TTSal~AtX-R AACTCAATGATGATGATGATGATGTAGAAAGCTGGCGGCC
cAOXXho~NpgA-F ATCAAAAAACAACTAATTATTCGAAATGGTGCAAGACACATCAAG
TTSal~NpgA-R AAACTCAATGATGATGATGATGATGGGATAGGCAATTACACACCC
pAOX~AtX-F CAAAAAACAACTAATTATTCGAAATGGAGGTACATGGAGATGAAG
pAOX~NpgA-F AACTAATTATTCGAAGGATCCTACGTAACCATGGTGCAAGACACATCAAG
35K~placO-F TAACAGTTATTATTCGGAGCTCTGTGTGGAATTGTGAGCG
KanaHis~TT-R GCTTGCACAAACGAACTACTAGTTCTCACTTAATCTTCTGTACTCT
TT~KanaHis-F CAGAAGATTAAGTGAGAACTAGTAGTTCGTTTGTGCAAGC
KanaHis~Amp-F GGAGATTTCATGGTAAATTTCTCTGA
Amp~KanaHis-R TCAGAGAAATTTACCATGAAATCTCC
lacOcAOX~Amp-R CCACACAGAGCTCCGAATAATAACTGTTATTTTTCAGTGT
lacOcAOX~TT-R TCCGCTCACAATTCCACACAAGATCTTCTCACTTAATCTTCTGTAC
lacOcAOX~LovB-F AAACAACTAATTATTCGAAATGGCTCAATCTATGTATCCT
TT~lovB-R AACTCAATGATGATGATGATGATGTGCCAGCTTCAGGGC
TT~lovC-R AACTCAATGATGATGATGATGATGCGGCCCCTCGAGC
lacOcAOX~LovC-F AACAACTAATTATTCGAAATGGGCGACCAGCC
lacOcAOX~lovG-F AACAACTAATTATTCGAAATGCGTTACCAAGCATCT
TT~lovG-R ACTCAATGATGATGATGATGATGCTCCAATGTCTGGGCC
lacOcAOX~npgA-F AACAACTAATTATTCGAAATGGTGCAAGACACATCAA
TT~npgA-R ACTCAATGATGATGATGATGATGGGATAGGCAATTACACACCC
TT~lacOcAOX-F1 TTAAGTGAGACCTTCGTTTGTGCAGATCTTGTGTGGAATTGTGA
BamH~TT-R AAGCTATGGTGTGTGGGGGATCCGCACAAACGAAGGTCTC
TT~pICL1-F AGTGAGACCTTCGTTTGTGCAGATCTTCATCTAACACTTTGTATAG
ACS1mut-R GCGGCCGCCGCGGCTCGAGGCAACTTGACCGAATCAATTGGATGTC
pGAP~ScALD6-F TTTCAATCAATTGAACAACTATATGACTAAGCTACACTTTGACAC
TT~ScALD6-R ATTAATTCGCGGCCGCCCTAGGTTACAACTTAATTCTGACAGCTTTTACT
pGAP~ScADH2-F CTATTTCAATCAATTGAACAACTATATGTCTATTCCAGAAACTCAAAAAG
TT~ScADH2-R GGCGAATTAATTCGCGGCCGCCCTAGGTTAATGATGATGATGATGATGTTTAGAAGTGTCAACAACGTA
pGAP~ScACS1-F TCAATCAATTGAACAACTATATGTCGCCCTCTGCCG
pGAP-ScACS1-R GCGGCCGCCGCGGCTCGAGGCAACTTGACCGAATCAATTAGATGTC
pAOXSpe~pGAP-F AACAACTAATTATTCGAAGACTAGTCTTTTTGTAGAAATGTCTTGGTGTC
35KdelHisTT-R GACACCAAGACATTTCTACAAAAAGACTAGTCTTCGAATAATTAGTTGTT
35KdelHis-2-R CTTCAAGTTTATTTAGAGATTTTAACTTACATTTAGATTCGATAGATCCA
35KdelHis-1-F AAGTTAAAATCTCTAAATAAACTTGAAGTCGGACAGTGAG
35KdelHis1-R CGAGGCAGAGATCATGAGATAAATTTCA
35KdelHis-2-F TGAAATTTATCTCATGATCTCTGCCTCG
pGAP-ACC1-F AAACAACTAATTATTCGAAATGAGTAGTGTTAACCACTCT
TTHis6~ACC1-R ACTCAATGATGATGATGATGATGTGACTTGATCTTAGATAAAATTGATTC
Z~pGAPmutBln-F CATGCATGAGATCAGATCTTTTTTGTAGAAATGTCTTGGT
TT~pGAPmutBln-R GAGACGGCCGGCTGGGCCACGTGAATTCTTCGAAATAGTTGTTCAATTGATTGAAATAGG
pGAPmutBln-F GTTACCGTCGCTAGGAAATTTTAC
pGAPmutBlnNei-R GTAAAATTTCCTAGCGACGGTAAC
pGAP~TT-R ACCAAGACATTTCTACAAAAATCTCACTTAATCTTCTGTACTCTGA
pGAP~ACC1-F CAATCAATTGAACAACTATATGAGTAGTGTTAACCACTCT
ACC1mut-F ATAGAGCAGTTGCTGTCTCC
ACC1mut-R GGAGACAGCAACTGCTCTAT
TT~ACC1-R CAATGATGATGATGATGATGTGACTTGATCTTAGATAAAATTGATTCCT
35K~ACC1-R CAATGATGATGATGATGATGTGACTTGATCTTAGATAAAATTGATTCCT
lacOcAOX~sLovA-F AACAACTAATTATTCGAAATGACTGTTGACGCTTTG
TT~sLovA-R ACTCAATGATGATGATGATGATGCAAAGAACCTGGCAATCTAAT
lacOcAOX~CPR-F AACAACTAATTATTCGAAATGGCTCAACTCGACAC
TT~CPR-R ACTCAATGATGATGATGATGATGTGACCACACGTCCTCCT
Amp~lacOcAOX-F TAACAGTTATTATTCGGAGCTCTGTGTGGAATTGTGAGCG
KanaHis~TTSpe-R GCTTGCACAAACGAACTACTAGTTCTCACTTAATCTTCTGTACTCT
TT~lacOcAOX-F2 GTACAGAAGATTAAGTGAGAAGATCTTGTGTGGAATTGTG
ZEcoR~pHXT1-F AACAACTAATTATTCGAAACGAGGGTACCCAATTGATTAAGTTCAGTGAAATTTCAAACC
PpFAS1~pHXT1-R CCGGATGTAGCACTCATATTATATTATGGGGAATAATGAAGAGAAGGGGApHXT1~PpFAS1-F CCTTCTCTTCATTATTCCCCATAATATAATATGAGTGCTACATCCGGAGTTGTHisSal~PpFAS1-R CAAACTCAATGATGATGATGATGATGGTTCAGATTCAAACCGTAAAGTGATTGAGpAGBamH~pHXT1-F AAGCTTCGTACGCTGCAGGTCGACGGGTACCCAATTGATTAAGTTCAGTG
pAGBgl~TT-R CCCGGCGGGGACAAGGCAAGCTAAACTCTCACTTAATCTTCTGTACTCTGAAGAG
* The designed restriction enzyme site or mutated site is underlined.
Suppl. Table S2. List of major plasmids constructed in this study.
Plasmid name Description Resistance Addgene IDpUC_Δadh3 Δahd3 Amp 126707pUC_Δald1 Δald Amp 126708pUC_Δacs1 Δacs1 Amp 126709pZ_PGAP-ADH3 PGAP-ADH3 Zeocin 126710
pZ_PGAP-ALD1 PGAP-ALD Zeocin 126711
pZ_PGAP-ACS1 PGAP-ACS1 Zeocin 126712
pZ_PADH3-eGFP PADH3-egfp Zeocin 126713
pZ_PALD-eGFP PALD-egfp Zeocin 126714
pZ_PACS1-eGFP PACS1-egfp Zeocin 126715
pZ_PICL1-eGFP PICL1-egfp Zeocin 126716
pZ_PGAP-eGFP PGAP-egfp Zeocin 126717
pZ_PAOX1-eGFP PAOX1-egfp Zeocin 126718
pZ_PHXT1-eGFP PHXT1-egfp Zeocin 126719
pZ_lacO-cPAOX1-eGFP lacO-cPAOX1-egfp Zeocin 126720
pZ_PICL1-LacI-Mit1AD PICL1-lacI-MIT1AD Zeocin 126721
pZ_PGAP-LacI-Mit1AD PGAP-lacI-MIT1AD Zeocin 126722
pZ_PICL1-LacI-VP16 PICL1-lacI-vp16 Zeocin 126723
pZ_PICL1-LacI-Gal4AD PICL1-lacI-GAL4AD Zeocin 126724
pK_PGAP-NpgA+AtX PGAP-npgA, atX Amp; Kan 126725
pK_PAOX1-NpgA+AtX PAOX1-npgA, atX Amp; Kan 126726
pK_lacO-cPAOX1-AtX+NpgA lacO-cPAOX1-npgA, atX Amp; Kan 126727
pZ_lacO-cPAOX1-LovB lacO-cPAOX1-lovB Zeocin 126728
pZ_lacO-cPAOX1-LovC lacO-cPAOX1-lovC Zeocin 126729
pZ_lacO-cPAOX1-LovG lacO-cPAOX1-lovG Zeocin 126730
pZ_lacO-cPAOX1-NpgA lacO-cPAOX1-npgA Zeocin 126731
pZ_lacO-cPAOX1-BCGN lacO-cPAOX1-lovB,lovC,lovG,npgA Zeocin 126732
pZ_PICL1-LM_lacO-cPAOX1-BCGN PICL1-lacI-MIT1AD; lacO-cPAOX1-lovB,lovC,lovG,npgA Zeocin 126733
pKdH_PGAP-ScADH2 PGAP-ScADH2 Amp; Kan 126734
pKdH_PGAP-ScALD6 PGAP-ScALD6 Amp; Kan 126735
pKdH_PGAP-ScACS1* PGAP-ScACS1* Amp; Kan 126736
pKdH_PGAP-ScADH2+PGAP*-ScALD6 PGAP-ScADH2+ScALD6 Amp; Kan 126737
pKdH_PGAP-ScADH2+PGAP*-ScACS1* PGAP-ScADH2+ScACS1* Amp; Kan 126738
pKdH_PGAP-ScADH2+PGAP*-ScALD6+ScACS1* PGAP-ScADH2+ScALD6+ScACS1* Amp; Kan 126739
pK_PGAP-ACC1* PGAP-PpACC1* Amp; Kan 126740
pZ_lacO-cPAOX1-sLovA lacO-cPAOX1-slovA Zeocin 126741
pZ_lacO-cPAOX1-CPR lacO-cPAOX1-cpr Zeocin 126742
pK_lacO-cPAOX1-sAR lacO-cPAOX1-slovA+cpr Amp; Kan 126743
pK_PICL1-LM_lacO-cPAOX1-sAR PICL1-lacI-MIT1AD; lacO-cPAOX1-slovA+cpr Amp; Kan 126744
pK_PGAP-LM_lacO-cPAOX1-sAR PGAP-lacI-MIT1AD; lacO-cPAOX1-slovA+cpr Amp; Kan 126745
pZ_PHXT1-FAS1up PHXT1-FAS1up Zeocin 126746
pAG_PHXT1-FAS1up PHXT1-FAS1up Amp; Hyg 126747
Suppl. Table S3. List of major strains constructed in this study.
Strains Description Characteristics
Pichia pastoris GS115 Wild type his4
Saccharomyces cerevisiae BY4741 Wild type MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0
Pp/Δadh3 Δahd3 HIS4
Pp/Δald1 Δald1 HIS4
Pp/Δacs1 Δacs1 HIS4
Pp/Δadh3_Re Pp/Δadh3 carrying plasmid: pZ_PGAP-ADH3 HIS4; Sh ble
Pp/Δald1_Re Pp/Δald1 carrying plasmid: pZ_PGAP-ALD1 HIS4; Sh ble
Pp/Δacs1_Re Pp/Δacs1 carrying plasmid: pZ_PGAP-ACS1 HIS4; Sh ble
Pp/PADH3-eGFP GS115 carrying plasmid: pZ_PADH3-eGFP Sh ble
Pp/PALD1-eGFP GS115 carrying plasmid: pZ_PALD1-eGFP Sh ble
Pp/PACS1-eGFP GS115 carrying plasmid: pZ_PACS1-eGFP Sh ble
Pp/PICL1-eGFP GS115 carrying plasmid: pZ_PICL1-eGFP Sh ble
Pp/PGAP-eGFP GS115 carrying plasmid: pZ_PGAP-eGFP Sh ble
Pp/PAOX1-eGFP GS115 carrying plasmid: pZ_PAOX1-eGFP Sh ble
Pp/PHXT1-eGFP GS115 carrying plasmid: pZ_PHXT1-eGFP Sh ble
Pp/lacO-cPAOX1-eGFP GS115 carrying plasmid: pZ_lacO-cPAOX1-eGFP HIS4
Pp/ESAD-eGFP Pp/lacO-cPAOX1-eGFP carrying plasmid: pZ_PICL1-LacI-Mit1AD HIS4; Sh ble
Pp/CSAD-eGFP Pp/lacO-cPAOX1-eGFP carrying plasmid: pZ_PGAP-LacI-Mit1AD HIS4; Sh ble
Pp/ESAD(GAL4AD)-eGFP Pp/lacO-cPAOX1-eGFP carrying plasmid: pZ_PICL1-LacI-Gal4AD HIS4; Sh ble
Pp/ESAD(VP16)-eGFP Pp/lacO-cPAOX1-eGFP carrying plasmid: pZ_PICL1-LacI-VP16 HIS4; Sh ble
Pp/PGAP-NX GS115 carrying plasmid: pK_PGAP-NpgA+AtX Sh ble
Pp/PAOX1-NX GS115 carrying plasmid: pK_PAOX1-NpgA+AtX Sh ble
Pp/lacO-cPAOX1-XN GS115 carrying plasmid: pK_lacO-cPAOX1-AtX+NpgA HIS4; G418R
Pp/CSAD-NX Pp/lacO-cPAOX1-XN carrying plasmid: pZ_PGAP-LacI-Mit1AD HIS4; Sh ble; G418R
Pp/ESAD-NX Pp/lacO-cPAOX1-XN carrying plasmid: pZ_PICL1-LacI-Mit1AD HIS4; Sh ble; G418R
Pp/PAOX1-BCGN GS115 carrying plasmid: pZ_BCGN Sh ble
Pp/ESAD-BCGN GS115 carrying plasmid: pZ_PICL1-LM_lacO-cPAOX1-BCGN Sh ble
Pp/ESAD-BCGN_PGAP-H Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScADH2 Sh ble; G418R
Pp/ESAD-BCGN_PGAP-D Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScALD6 Sh ble; G418R
Pp/ESAD-BCGN_PGAP-S* Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScACS1* Sh ble; G418R
Pp/ESAD-BCGN_PGAP-HD Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScADH2+PGAP*-ScALD6 Sh ble; G418R
Pp/ESAD-BCGN_PGAP-HS* Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScADH2+PGAP*-ScACS1* Sh ble; G418R
Pp/ESAD-BCGN_PGAP-HDS* Pp/ESAD-BCGN carrying plasmid: pKdH_PGAP-ScADH2+PGAP*-ScALD6+ScACS1* Sh ble; G418R
Pp/ESAD-BCGN_PGAP-HCS* Pp/ESAD-BCGN_PGAP-HS* carrying plasmid: pK-PGAP-ACC1* HIS4; Sh ble G418R
Pp/ESAD-sAR GS115 carrying plasmid: pZ_PICL1-LM_lacO-cPAOX1-sAR Sh ble
Pp/CSAD-sAR GS115 carrying plasmid: pZ_PGAP-LM_lacO-cPAOX1-sAR Sh ble
Suppl. Table S4. Identified three homologs of S. cerevisiae Adh2, Ald6 and Acs1 in P. pastoris by BLAST. NCBI reference No., Genbank accession No., gene sequence, and amino acid alignment results were shown.
Alcohol dehydrogenase (Adh)
S. cerevisiae ADH2
NCBI:
NM_001182812.1
ATGTCTATTCCAGAAACTCAAAAAGCCATTATCTTCTACGAATCCAACGGCAAGTTGGAGCATAAGGATATCCCAGTTCCAAAGCCAAAGCCCAACGAATTGTTAATCAACGTCAAGTACTCTGGTGT
CTGCCACACCGATTTGCACGCTTGGCATGGTGACTGGCCATTGCCAACTAAGTTACCATTAGTTGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTTAAGGGCTGGAAGA
TCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTATGGCCTGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTACACCCACGACGGTTCTTTCCAA
GAATACGCTACCGCTGACGCTGTTCAAGCCGCTCACATTCCTCAAGGTACTGACTTGGCTGAAGTCGCGCCAATCTTGTGTGCTGGTATCACCGTATACAAGGCTTTGAAGTCTGCCAACTTGAGAG
CAGGCCACTGGGCGGCCATTTCTGGTGCTGCTGGTGGTCTAGGTTCTTTGGCTGTTCAATATGCTAAGGCGATGGGTTACAGAGTCTTAGGTATTGATGGTGGTCCAGGAAAGGAAGAATTGTTTA
CCTCGCTCGGTGGTGAAGTATTCATCGACTTCACCAAAGAGAAGGACATTGTTAGCGCAGTCGTTAAGGCTACCAACGGCGGTGCCCACGGTATCATCAATGTTTCCGTTTCCGAAGCCGCTATCGA
AGCTTCTACCAGATACTGTAGGGCGAACGGTACTGTTGTCTTGGTTGGTTTGCCAGCCGGTGCAAAGTGCTCCTCTGATGTCTTCAACCACGTTGTCAAGTCTATCTCCATTGTCGGCTCTTACGTGG
GGAACAGAGCTGATACCAGAGAAGCCTTAGATTTCTTTGCCAGAGGTCTAGTCAAGTCTCCAATAAAGGTAGTTGGCTTATCCAGTTTACCAGAAATTTACGAAAAGATGGAGAAGGGCCAAATTG
CTGGTAGATACGTTGTTGACACTTCTAAATAA
P. pastoris
ADH3
NCBI:
XM_002491337.1
ATGTCTCCAACTATCCCAACTACACAAAAGGCTGTTATCTTCGAGACCAACGGCGGTCCCCTAGAGTACAAGGACATTCCAGTCCCAAAGCCAAAGTCAAACGAACTTTTGATCAACGTTAAGTACT
CCGGTGTCTGTCACACTGATTTGCACGCCTGGAAGGGTGACTGGCCATTGGACAACAAGCTTCCTTTGGTTGGTGGTCACGAAGGTGCTGGTGTCGTTGTCGCTTACGGTGAGAACGTCACTGGA
TGGGAGATCGGTGACTACGCTGGTATCAAATGGTTGAACGGTTCTTGTTTGAACTGTGAGTACTGTATCCAAGGTGCTGAATCCAGTTGTGCCAAGGCTGACCTGTCTGGTTTCACCCACGACGGAT
CTTTCCAGCAGTATGCTACTGCTGATGCCACCCAAGCCGCCAGAATTCCAAAGGAGGCTGACTTGGCTGAAGTTGCCCCAATTCTGTGTGCTGGTATCACCGTTTACAAGGCTCTTAAGACCGCTGA
CTTGCGTATTGGCCAATGGGTTGCCATTTCTGGTGCTGGTGGAGGACTGGGTTCTCTTGCCGTTCAATACGCCAAGGCTCTGGGTTTGAGAGTTTTGGGTATTGATGGTGGTGCCGACAAGGGTGA
ATTTGTCAAGTCCTTGGGTGCTGAGGTCTTCGTCGACTTCACTAAGACTAAGGACGTCGTTGCTGAAGTCCAAAAGCTCACCAACGGTGGTCCACACGGTGTTATTAACGTCTCCGTTTCCCCACAT
GCTATCAACCAATCTGTCCAATACGTTAGAACTTTGGGTAAGGTTGTTTTGGTTGGTCTGCCATCTGGTGCCGTTGTCAACTCTGACGTTTTCTGGCACGTTCTGAAGTCCATCGAGATCAAGGGATC
TTACGTTGGAAACAGAGAGGACAGTGCCGAGGCCATCGACTTGTTCACCAGAGGTTTGGTCAAGGCTCCTATCAAGATTATCGGTCTGTCTGAACTTGCTAAGGTCTACGAACAGATGGAGGCTG
GTGCCATCATCGGTAGATACGTTGTGGACACTTCCAAATAA
S. cerevisiae Adh2 vs. Score = 463 bits (1191), Expect = 2e-163, Method: Compositional matrix adjust. Identities = 256/347 (74%), Positives = 285/347 (82%), Gaps = 0/347 (0%)
P. pastoris Adh3
Acetaldehyde dehydrogenase (Ald)
S. cerevisiae ALD6
NCBI:
NM_001183875.1
ATGACTAAGCTACACTTTGACACTGCTGAACCAGTCAAGATCACACTTCCAAATGGTTTGACATACGAGCAACCAACCGGTCTATTCATTAACAACAAGTTTATGAAAGCTCAAGACGGTAAGACCT
ATCCCGTCGAAGATCCTTCCACTGAAAACACCGTTTGTGAGGTCTCTTCTGCCACCACTGAAGATGTTGAATATGCTATCGAATGTGCCGACCGTGCTTTCCACGACACTGAATGGGCTACCCAAGA
CCCAAGAGAAAGAGGCCGTCTACTAAGTAAGTTGGCTGACGAATTGGAAAGCCAAATTGACTTGGTTTCTTCCATTGAAGCTTTGGACAATGGTAAAACTTTGGCCTTAGCCCGTGGGGATGTTAC
CATTGCAATCAACTGTCTAAGAGATGCTGCTGCCTATGCCGACAAAGTCAACGGTAGAACAATCAACACCGGTGACGGCTACATGAACTTCACCACCTTAGAGCCAATCGGTGTCTGTGGTCAAATT
ATTCCATGGAACTTTCCAATAATGATGTTGGCTTGGAAGATCGCCCCAGCATTGGCCATGGGTAACGTCTGTATCTTGAAACCCGCTGCTGTCACACCTTTAAATGCCCTATACTTTGCTTCTTTATGTA
AGAAGGTTGGTATTCCAGCTGGTGTCGTCAACATCGTTCCAGGTCCTGGTAGAACTGTTGGTGCTGCTTTGACCAACGACCCAAGAATCAGAAAGCTGGCTTTTACCGGTTCTACAGAAGTCGGTA
AGAGTGTTGCTGTCGACTCTTCTGAATCTAACTTGAAGAAAATCACTTTGGAACTAGGTGGTAAGTCCGCCCATTTGGTCTTTGACGATGCTAACATTAAGAAGACTTTACCAAATCTAGTAAACGGT
ATTTTCAAGAACGCTGGTCAAATTTGTTCCTCTGGTTCTAGAATTTACGTTCAAGAAGGTATTTACGACGAACTATTGGCTGCTTTCAAGGCTTACTTGGAAACCGAAATCAAAGTTGGTAATCCATT
TGACAAGGCTAACTTCCAAGGTGCTATCACTAACCGTCAACAATTCGACACAATTATGAACTACATCGATATCGGTAAGAAAGAAGGCGCCAAGATCTTAACTGGTGGCGAAAAAGTTGGTGACAA
GGGTTACTTCATCAGACCAACCGTTTTCTACGATGTTAATGAAGACATGAGAATTGTTAAGGAAGAAATTTTTGGACCAGTTGTCACTGTCGCAAAGTTCAAGACTTTAGAAGAAGGTGTCGAAAT
GGCTAACAGCTCTGAATTCGGTCTAGGTTCTGGTATCGAAACAGAATCTTTGAGCACAGGTTTGAAGGTGGCCAAGATGTTGAAGGCCGGTACCGTCTGGATCAACACATACAACGATTTTGACTC
CAGAGTTCCATTCGGTGGTGTTAAGCAATCTGGTTACGGTAGAGAAATGGGTGAAGAAGTCTACCATGCATACACTGAAGTAAAAGCTGTCAGAATTAAGTTGTAA
P. pastoris ALD1
NCBI:
XM_002491373.1
ATGCTTAGAACTTCTCCAGCTACTAAGAAAGCTCTCAAGTCGCAGATTAACGCCTTCAACGTTGCTGCCTTGAGATTCTACTCCTCATTGCCTTTGCAGGTTCCAATTACCTTGCCAAACGGTAAGAC
CTACAATCAGCCAACAGGTTTGTTTATCAACAATGAGTTCGTTCCTTCTAAGCAAGGTAAGACCTTTGCTGTTTTAAACCCTTCCACTGAGGAGGAGATTACTCACGTCTACGAGTCCAGAGAGGAC
GACGTTGAGTTAGCCGTTGCAGCCGCTCAAAAGGCTTTCGACTCAACCTGGTCCACCCAGGACCCTGCTGAGAGAGGTAAGGTCTTGAACAAGTTGGCTGACCTGATCGAGGAGCACTCTGAGA
CCCTTGCCGCCATCGAGTCCTTGGACAACGGTAAGGCCATTTCCTCCGCTAGAGGTGATGTTGGTCTGGTTGTCGCCTACTTGAAGTCCTGTGCCGGTTGGGCCGACAAGGTTTTCGGTAGAGTTG
TTGAAACCGGAAGCTCCCACTTCAACTACGTTAGAAGAGAGCCATTGGGTGTTTGTGGTCAGATTATCCCATGGAACTTTCCTCTTCTGATGTGGTCCTGGAAAGTTGGTCCAGCTTTGGCCACTGG
TAACACTGTTGTCCTGAAGACAGCCGAGTCTACTCCTCTGTCCGCCCTGTACGTTTCCCAATTGGTCAAGGAGGCCGGTATCCCAGCTGGTGTCCACAACATTGTGTCCGGTTTCGGTAAGATTACTG
GTGAAGCTATTGCTACTCATCCTAAGATCAAGAAGGTTGCCTTCACTGGTTCTACCGCCACTGGTCGTCACATCATGAAGGCTGCTGCCGAATCCAACTTGAAGAAGGTTACTTTGGAGTTGGGTGG
TAAATCTCCTAACATCGTGTTCAACGATGCTAACATTAAGCAAGCTGTCGCCAACATCATCCTCGGTATTTACTACAACTCTGGAGAAGTTTGTTGTGCTGGTTCCAGAGTTTATGTTCAATCCGGTATT
TACGACGAGCTTTTGGCCGAATTCAAGACTGCTGCTGAGAATGTCAAGGTTGGTAACCCATTCGACGAGGACACCTTCCAAGGTGCTCAAACCTCTCAGCAACAATTGGAGAAGATTTTGGGTTTC
GTTGAGCGTGGTAAGAAGGACGGTGCTACTTTGATTACTGGTGGTGGCAGATTAGGTGACAAGGGTTACTTCGTCCAGCCAACTATCTTCGGTGATGTTACACCAGAGATGGAGATTGTCAAGGA
AGAGATCTTTGGTCCTGTTGTCACTATCAGCAAGTTTGACACCATTGATGAGGTTGTCGACCTTGCTAACGACTCTCAATACGGTCTTGCTGCTGGTATCCACTCTGACGATATCAACAAGGTCATTGA
CGTTGCTGCTAGAATCAAGTCCGGTACCGTGTGGGTCAACACCTACAACGATTTCCACCAAATGGTTCCATTCGGTGGATTTGGCCAATCCGGTATTGGTCGTGAGATGGGTGTTGAAGCTTTGGAA
AACTACACCCAATACAAGGCTATCCGTGTCAAGATCAACCACAAGAACGAGTAA
S. cerevisiae Ald6 vs.
P. pastoris Ald1
Score = 578 bits (1489), Expect = 0.0, Method: Compositional matrix adjust. Identities = 269/499 (54%), Positives = 373/499 (75%), Gaps = 4/499 (1%)
Acetyl-CoA synthetase (Acs); A mutant of S. cerevisiae Acs1* (L707P, the underlined red CTA was mutated to CCA) was used for overexpression
S. cerevisiae ACS1
GenBank: AY723758.1
ATGTCGCCCTCTGCCGTACAATCATCAAAACTAGAAGAACAGTCAAGTGAAATTGACAAGTTGAAAGCAAAAATGTCCCAGTCTGCCGCCACTGCGCAGCAGAAGAAGGAACATGAGTATGAACA
TTTGACTTCGGTCAAGATCGTGCCACAACGGCCCATCTCAGATAGACTGCAGCCCGCAATTGCTACCCACTATTCTCCACACTTGGACGGGTTGCAGGACTATCAGCGCTTGCACAAGGAGTCTATT
GAAGACCCTGCTAAGTTCTTCGGTTCTAAAGCTACCCAATTTTTAAACTGGTCTAAGCCATTCGATAAGGTGTTCATCCCAGACCCTAAAACGGGCAGGCCCTCCTTCCAGAACAATGCATGGTTCCT
CAACGGCCAATTAAACGCCTGTTACAACTGTGTTGACAGACATGCCTTGAAGACTCCTAACAAGAAAGCCATTATTTTCGAAGGTGACGAGCCTGGCCAAGGCTATTCCATTACCTACAAGGAACTA
CTTGAAGAAGTTTGTCAAGTGGCACAAGTGCTGACTTACTCTATGGGCGTTCGCAAGGGCGATACTGTTGCCGTGTACATGCCTATGGTCCCAGAAGCAATCATAACCTTGTTGGCCATTTCCCGTAT
CGGTGCCATTCACTCCGTAGTCTTTGCCGGGTTTTCTTCCAACTCCTTGAGAGATCGTATCAACGATGGGGACTCTAAAGTTGTCATCACTACAGATGAATCCAACAGAGGTGGTAAAGTCATTGAG
ACTAAAAGAATTGTTGATGACGCGCTAAGAGAGACCCCAGGCGTGAGACACGTCTTGGTTTATAGAAAGACCAACAATCCATCTGTTGCTTTCCATGCCCCCAGAGATTTGGATTGGGCAACAGAA
AAGAAGAAATACAAGACCTACTATCCATGCACACCCGTTGATTCTGAGGATCCATTATTCTTGTTGTATACGTCTGGTTCTACTGGTGCCCCCAAGGGTGTTCAACATTCTACCGCAGGTTACTTGCTG
GGAGCTTTGTTGACCATGCGCTACACTTTTGACACTCACCAAGAAGACGTTTTCTTCACAGCTGGAGACATTGGCTGGATTACAGGCCACACTTATGTGGTTTATGGTCCCTTACTATATGGTTGTGC
CACTTTGGTCTTTGAAGGGACTCCTGCGTACCCAAATTACTCCCGTTATTGGGATATTATTGATGAACACAAAGTCACCCAATTTTATGTTGCGCCAACTGCTTTGCGTTTGTTGAAAAGAGCTGGTG
ATTCCTACATCGAAAATCATTCCTTAAAATCTTTGCGTTGCTTGGGTTCGGTCGGTGAGCCAATTGCTGCTGAAGTTTGGGAGTGGTACTCTGAAAAAATAGGTAAAAATGAAATCCCCATTGTAGAC
ACCTACTGGCAAACAGAATCTGGTTCGCATCTGGTCACCCCGCTGGCTGGTGGTGTTACACCAATGAAACCGGGTTCTGCCTCATTCCCCTTCTTCGGTATTGATGCAGTTGTTCTTGACCCTAACAC
TGGTGAAGAACTTAACACCAGCCACGCAGAGGGTGTCCTTGCCGTCAAAGCTGCATGGCCATCATTTGCAAGAACTATTTGGAAAAATCATGATAGGTATCTAGACACTTATTTGAACCCTTACCCT
GGCTACTATTTCACTGGTGATGGTGCTGCAAAGGATAAGGATGGTTATATCTGGATTTTGGGTCGTGTAGACGATGTGGTGAACGTCTCTGGTCACCGTCTGTCTACCGCTGAAATTGAGGCTGCTAT
TATCGAAGATCCAATTGTGGCCGAGTGTGCTGTTGTCGGATTCAACGATGACTTGACTGGTCAAGCAGTTGCTGCATTTGTGGTGTTGAAAAACAAATCTAGTTGGTCCACCGCAACAGATGATGAA
TTACAAGATATCAAGAAGCATTTGGTCTTTACTGTTAGAAAAGACATCGGGCCATTTGCCGCACCAAAATTGATCATTTTAGTGGATGACTTGCCCAAGACAAGATCCGGCAAAATTATGAGACGTAT
TTTAAGAAAAATCCTAGCAGGAGAAAGTGACCAACTAGGCGACGTTTCTACATTGTCAAACCCTGGCATTGTTAGACATCTAATTGATTCGGTCAAGTTGTAA
P. pastoris ACS1
NCBI:
XM_002491656.1
ATGCCATTAGATAACGAACACTTACTTCATGAAAATTCCATTGACCCACCAAAGGGATTCTTTGAAAGACACCCTGGAACTCCTAATATACCAGGCGGTTGGGAAGAATACTTGAAGCTGTACAATCA
GTCCATCGAGAACCCCTCAAAGTTTTTTGGAGAAAAAGCAAAGGAATTCTTGTCATGGGCTACTCCTTTCACTGACGCTCGTTACCCACCTGGTAATGGATTTCAGAATGGTGACTCCGCCGCTTGG
TTTCTGAATGGTGAGTTGAACGCGTCGTACAACTGTGTTGATAGACATGCTTTAAAGAATCCAGACAAACCTGCCATTATTTATGAGGCTGATGAACCTAATCAAGGCCGTACGGTTACCTATGGAGA
GTTGCTGAAGGATGTTTGTCGAATTGCCCAAGTATTGACTGACCTGGGTGTGAAAAAGGGTGACACTGTTGCTGTTTACCTGCCTATGGTTCCAGAAGCTATCACCACTTTATTGGCTATCGTTAGAA
TCGGTGCTATCCACTCTGTTGTCTTCGCAGGTTTTTCAGCTGGTTCTCTACGTGATCGTATATTGGATGCTGATTCTAGAATTGTTATCACTTCTGATGAATCTCTGAGAGGTGGGAAGATCATCGAGA
CTAAGAAGATTGTTGACGAGGCTCTGAAGTCTTGCCCAGATGTTCGTAATGTGCTGGTCTTCAAAAGAACAGGTACACCACATCTTCCATGGGTTGAGGGTCGTGATCTTTGGTGGCACGAGGAAA
TCATTAAGCATGTTCCGTACTCTCCCCCAGTGAATGTTAGATCTGAAGATACTTCATTTTTGCTTTACACTTCTGGCTCTACCGGAAAGCCTAAAGGTATCCAGCATTCAACTGCTGGCTACTTACTGGG
AGCTCTTTTGACCACCAAGTATGTCTTTGATGTTCAGGGTGATGATATTTTATTCACTGCTGGTGATGTGGGCTGGATCACAGGGCATTCTTATGTAGTTTACGGTCCACTTTTAAACGGGGCTACGAC
AGTTGTTTTTGAGGGCACCCCAGCTTACCCAGACTATTCACGTTATTGGGATATCGTTGACAAACACAAAGTTACTCAGTTTTATGTAGCACCAACTGCTCTTAGGTTGCTGAAGAGAGCTGGTAGC
AAGTATGTCCAGAATCATGATTTGTCTTCAATCAGGGTTTTGGGTTCCGTTGGTGAACCTATAGCCGCTGAAGTTTGGGAATGGTACAACGAGTATGTTGGAAGAGGAAAAGCTCATATTTGTGATA
CGTATTGGCAAACAGAGACTGGTTCTCACATTATTGCTCCAATAGCTGGTGTGTCAAAGACCAAACCAGGTTCAGCATCTTTCCCCTTCTTCGGTATTGATCCGGTTATTCTAGATGCTACTACTGGAG
AGGAACTCAAAGGTAATAATGTTGAAGGTGTTTTGGCTATCAGAAATCCATGGCCATCTATGGCTAGAACAGTCTGGAAGGACTACAACCGTTTCCTGGATACATATCTCAGGCCATATGAAGGTTAT
TACTTCACTGGTGATGGAGCTGCCAGAGATCAGGAAGGATTTTATTGGGTTCTGGGTAGAGTTGATGATGTTGTTAATGTGTCAGGTCACAGATTGTCTACTGCCGAGATTGAAAGCGCTCTAATCG
AACACAATTTGGTAGGAGAGTCTGCTGTCGTCGGATTCCCTGACGAGCTGACTGGTTCTGCTGTGGCCGCGTTTGTGTCTTTGAAGAAGGACGTCGACAATCCAGCGGAAGTGAAAAAGGAGTTA
ATCCTTACTGTCAGAAAAGAGATTGGACCATTCGCTGCACCTAAACTCATCATCTTGGTAAGTGATCTTCCAAAGACCAGATCAGGTAAGATAATGAGACGTATTCTCAGAAAGGTTTTGGCTGGAG
AGGAAGACTCTCTGGGCGACATTTCAACTCTTTCAAACCCTTCGATTGTGGAAGAGATAATCTCTACCGTTAAAAGGGATGCCCGCAAATGA
S. cerevisiae Acs1 vs.
P. pastoris Acs1
Score = 890 bits (2299), Expect = 0.0, Method: Compositional matrix adjust. Identities = 430/641 (67%), Positives = 518/641 (81%), Gaps = 10/641 (2%)
Suppl. Table S5. Coding sequences of transactivation domains used for chimeric transcription factors and hybrid promoter lacO-cPAOX1*.
Activation domain
DNA sequence
LacI-Mit1AD ATGGGTGTTAAGCCAGTTACTTTGTATGACGTTGCTGAATACGCTGGAGTTTCCTACCAAACTGTCTCTAGAGTTGTTAATCAAGCTTCTCATGTCTCCGCTAAGACTAGAGAGAAGGTTGAGGCTGCTATGGCTGAATTGAACTATATTCCAAATAGAGTTGCTCAGCAGTTGGCTGGAAAGCAATCTTTGTTGATTGGAGTCGCTACTTCTTCTTTGGCTTTGCATGCTCCATCTCAGATTGTTGCTGCTATTAAGTCCAGAGCTGACCAGTTGGGAGCTTCTGTTGTTGTTTCTATGGTTGAGAGATCTGGAGTTGAGGCTTGCAAGGCTGCTGTTCATAACTTGTTGGCTCAGAGAGTTTCTGGATTGATTATTAATTACCCATTGGACGATCAAGACGCTATTGCCGTTGAGGCCGCTTGTACCAACGTCCCAGCTTTGTTCTTGGACGTTTCCGATCAAACTCCAATTAATTCTATTATTTTTTCTCACGAGGATGGAACTAGATTGGGAGTTGAACACTTGGTTGCTTTGGGACATCAACAGATTGCTTTGTTGGCTGGACCATTGTCTTCCGTTTCTGCTAGATTGAGATTGGCCGGATGGCACAAGTACTTGACCAGAAACCAGATTCAACCAATTGCTGAGAGAGAGGGAGATTGGTCTGCTATGTCTGGATTCCAGCAGACTATGCAGATGTTGAACGAAGGAATTGTCCCAACCGCTATGTTGGTCGCTAATGACCAAATGGCTTTGGGAGCTATGAGAGCTATTACTGAATCTGGATTGAGAGTCGGAGCTGACATTTCTGTTGTTGGATATGATGACACTGAGGATTCTTCTTGCTACATTCCACCATTGACTACTATTAAGCAAGACTTCAGATTGTTGGGACAGACTTCTGTTGATAGATTGTTGCAGTTGTCCCAAGGACAAGCTGTTAAAGGAAACCAATTGTTGCCAGTTTCTTTGGTTAAGAGAAAGACTACTTTGGCTCCAAACACTCAGACTGCTTCCCCAAGAGCTTTGGCTGACTCTTTGATGCAATTGGCTAGACAAGTCTCTAGATTGGAGTCTGGACAAGGTGGCGGCGGCTCTGTTAACAACTCCATGAAGGATTTCTTAGGCAAGAAAACGGTGGATGGAGCTGATAGTCTCAATTTGGCCGTGAATCTGCAACAACAGCAGAGTTCAAACACAATTGCCAATCAATCGCTTTCCTCAATTGGATTGGAAAGTTTTGGTTACGGCTCTGGTATCAAAAACGAGTTTAACTTCCAAGACTTGATAGGTTCAAACTCTGGCAGTTCAGATCCGACATTTTCAGTAGACGCTGACGAGGCCCAAAAACTCGACATTTCCAACAAGAACAGTCGTAAGAGACAGAAACTAGGTTTGCTGCCGGTCAGCAATGCAACTTCCCATTTGAACGGTTTCAATGGAATGTCCAATGGAAAGTCACACTCTTTCTCTTCACCGTCTGGGACTAATGACGATGAACTAAGTGGCTTGATGTTCAACTCACCAAGCTTCAACCCCCTCACAGTTAACGATTCTACCAACAACAGCAACCACAATATAGGTTTGTCTCCGATGTCATGCTTATTTTCTACAGTTCAAGAAGCATCTCAAAAAAAGCATGGAAATTCCAGTAGACACTTTTCATACCCATCTGGGCCGGAGGACCTTTGGTTCAATGAGTTCCAAAAACAGGCCCTCACAGCCAATGGAGAAAATGCTGTCCAACAGGGAGATGATGCTTCTAAGAACAACACAGCCATTCCTAAGGACCAGTCTTCGAACTCATCGATTTTCAGTTCACGTTCTAGTGCAGCTTCTAGCAACTCAGGAGACGATATTGGAAGGATGGGCCCATTCTCCAAAGGACCAGAGATTGAGTTCAACTACGATTCTTTTTTGGAATCGTTGAAGGCAGAGTCACCCTCTTCTTCAAAGTACAATCTGCCGGAAACTTTGAAAGAGTACATGACCCTTAGTTCGTCTCATCTGAATAGTCAACACTCCGACACTTTGGCAAATGGCACTAACGGTAACTATTCTAGCACCGTTTCCAACAACTTGAGCTTAAGTTTGAACTCCTTCTCTTTCTCTGACAAGTTCTCATTGAGTCCACCAACAATCACTGACGCCGAAAAGTTTTCATTGATGAGAAACTTCATTGACAACATCTCGCCATGGTTTGACACTTTTGACAATACCAAACAGTTTGGAACAAAAATTCCAGTTCTGGCCAAAAAATGTTCTTCATTGTACTATGCCATTCTGGCTATATCTTCTCGTCAAAGAGAAAGGATAAAGAAAGAGCACAATGAAAAAACATTGCAATGCTACCAATACTCACTACAACAGCTCATCCCTACTGTTCAAAGCTCAAATAATATTGAGTACATTATCACATGTATTCTCCTGAGTGTGTTCCACATCATGTCTAGTGAACCTTCAACCCAGAGGGACATCATTGTGTCATTGGCAAAATACATTCAAGCATGCAACATAAACGGATTTACATCTAATGACAAACTGGAAAAGAGTATTTTCTGGAACTATGTCAATTTGGATTTGGCTACTTGTGCAATCGGTGAAGAGTCAATGGTCATTCCTTTTAGCTACTGGGTTAAAGAGACAACTGACTACAAGACCATTCAAGATGTGAAGCCATTTTTCACCAAGAAGACTAGCACGACAACTGACGATGACTTGGACGATATGTATGCCATCTACATGCTGTACATTAGTGGTAGAATCATTAACCTGTTGAACTGCAGAGATGCGAAGCTCAATTTTGAGCCCAAGTGGGAGTTTTTGTGGAATGAACTCAATGAATGGGAATTGAACAAACCCTTGACCTTTCAAAGTATTGTTCAGTTCAAGGCCAATGACGAATCGCAGGGCGGATCAACTTTTCCAACTGTTCTATTCTCCAACTCTCGAAGCTGTTACAGTAACCAGCTGTATCATATGAGCTACATCATCTTAGTGCAGAATAAACCACGATTATACAAAATCCCCTTTACTACAGTTTCTGCTTCAATGTCATCTCCATCGGACAACAAAGCTGGGATGTCTGCTTCCAGCACACCTGCTTCAGACCACCACGCTTCTGGTGATCATTTGTCTCCAAGAAGTGTAGAGCCCTCTCTTTCGACAACGTTGAGCCCTCCGCCTAATGCAAACGGTGCAGGTAACAAGTTCCGCTCTACGCTCTGGCATGCCAAGCAGATCTGTGGGATTTCTATCAACAACAACCACAACAGCAATCTAGCAGCCAAAGTGAACTCATTGCAACCATTGTGGCACGCTGGAAAGCTAATTAGTTCCAAGTCTGAACATACACAGTTGCTGAAACTGTTGAACAACCTTGAGTGTGCAACAGGCTGGCCTATGAACTGGAAGGGCAAGGAGTTAATTGACTACTGGAATGTTGAAGAATAA
VP1613
(Optimized
GCTCCACCAACCGACGTTTCTTTGGGTGACGAGTTGCACTTGGACGGTGAAGATGTTGCCATGGCTCATGCTGACGCTTTGGACGACTTCGACTTGGACATGTTGGGTGACGGTGATTCTCCA
forP. pastoris)
GGTCCAGGTTTCACTCCACACGATTCTGCTCCATACGGTGCTTTGGACATGGCCGACTTCGAGTTTGAGCAGATGTTCACCGACGCTTTGGGTATTGACGAGTACGGTGGTTAA
Gal4AD13,14 GCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGTAA
lacO-cPAOX1 TGTGTGGAATTGTGAGCGGATAACAATTTCACACACTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAA
* For VP16, CAI was increased from 0.58 to 0.88; GC content was decreased from 62.74% to 53.10%; CFD was reduced from 13% to zero. LacI showed nuclear localization function in P. pastoris (Supplementary Fig. 3a). For the LacI-Mit1AD, the coding sequence for LacI was marked as blue and the linker of GGGGS was shown in red. Fusion strategy of LacI-Gal4AD and LacI-VP16 refers to LacI-Mit1AD. The lacO was underlined in blue in the sequence of lacO-cPAOX1.
Suppl. Table S6. Gene sequence for S1132A (in red) site mutated Acc1* in P. pastoris.
Acetyl-CoA carboxylase 1* (Acc1*)P. pastorisACC1*
ATGAGTAGTGTTAACCACTCTCTCCGTCATTCAAAGCTACCGCCGCATTTCCTTGGTCTCAACTCGGTTGAAGTCGCTGCTCCCTCCAAGGTCAGAGACTTTGTCAGGGACCATGGTGGCCACTCGGTCATCACGAGAGTGCTGATCGCAAACAACGGTATAGCTGCCGTGAAAGAAATTCGTTCCGTCAGGAAATGGGCGTATGAAACGTTTGGTAACGATAGAGCCATTCAATTTATTGTTATGGCTACCCCAGAGGATCTTGAAGCTAATGCTGAATATATTCGAATGGCTGACCAGTATGTCATGGTCCCAGGAGGAACTGCAAACAACAACTATGCGAACGTCGACCTCATTGTAGAAATAGCAGAATCTACTGATGCTCATGCTGTTTGGGCTGGTTGGGGTTTTGCCTCCGAAAATCCCCATTTGCCTGAGCAACTGGCCGCTTCTCCTAAGAAGATTATCTTCATTGGCCCTCCGGGCTCTGCCATGCGATCTCTTGGTGACAAGATTTCCTCTACTATTGTCGCACAACATGCTAAAGTCCCATGTATTCCTTGGTCAGGAACTGGTGTCGATCAGGTTATAATCGACCCCGTAAGCAATTTGGTTTCCGTTGATGAAGAAACGTACGCCAAAGGATGCTGTTCCGATCCACAGGACGGTTTGGCAAAAGCCAAGGCTATTGGTTTCCCTGTGATGATTAAAGCTTCCGAAGGTGGTGGTGGTAAAGGAATTAGAAAAGTTGACAGGGAGGAAGATTTTCTTTCTCTTTATGATCAAGCTGCTAATGAAATTCCAGGTTCCCCAATTTTTATCATGAAGCTTGCTGGAGATGCCAGGCATTTGGAAGTTCAATTACTTGCTGATCAATATGGAACCAACATCTCCCTTTTTGGAAGAGATTGTTCCGTTCAAAGAAGACACCAAAAGATCATAGAAGAGGCACCAGTTACCATTGCCAAACAAGACACTTTCAGGCAAATGGAACAAGCCGCTGTCAGACTGGGTCAATTGGTTGGATACGTTTCTGCCGGTACCGTTGAGTATCTATATTCACACGCTGAGGACAAGTTCTACTTCTTGGAACTGAACCCTCGTCTTCAAGTTGAGCATCCAACCACAGAAATGGCCACAGGTGTCAATCTTCCAGTTGCCCAGTTGCTAATTGCAATGGGTATTCCTTTGAATAGAATCAGAGATATCAGGGTACTTTACGGACTTGAACCAAATGGCGCTACAGAAATTGACTTTGAATTCAAAACTGAAGAAAGCTTGAAGAGTCAAAGAAAACCCATTCCAAAGGGTCACACTATTGCATGTCGTATCACATCTGAAGATCCTGGTGAAGGTTTTAAGCCTTCTGGTGGTGCTCTATATGAGCTAAATTTCAGATCTTCTTCTAGCGTTTGGGGTTACTTCAGTGTAGGAAACAAATCCTCAATTCATTCTTTCAGTGACTCTCAATTTGGTCATATATTCTCGTTTGGCGAAAACCGTCAAATCGCCAGAAAAAATATGGTCGTCGCCTTGAAAGAGCTTTCTATTCGTGGTGACTTTAGAACTACAATTGAGTACTTAATAAAACTGTTGGAAACAGCTGATTTCGAGAACAACACCATCACTACTGGTTGGTTGGACGAACTGATCTCGAAGAAGCTGACTGCTGAAAGACCTGATGAAACCACAGCAATTTTATGTGGTGCTGAAAAAGGTCAAATCCCAGGCAAAGAACTTCTTCGTACTATTTTCCCAATTGAATTTATTTATGAAGGAAAGAAGTACAAGTTTACTGTGGTTCAGGCTGCATTTGACAAATACAACGTCTTTGTCAACGGATGTATGATTACTGTAAGTGTAACCCATTTGAAGGATGGCAGTTTATTGGTAGCACTTGATGGTAAATCCCATTCTGTCTATTACTTGCAGGAAGAAGTCGGAAATACTAGGTTGTCGGTGGATGGTAAATCTTGCATTTTAGAAGTTGAGCATGAGCCAACTGAACTTCGTACTCCATCTCCAGGTAAACTTATCAAATATCTTGTGGAACACGGTGATCACGTCAAAATTGGACAACCTTACGCTGAAGTTGAAGTAATGAAGATGTGTATGCCTTTGGTCAGTCAGGAGAATGGAACTATCAGGTTATTGAAGCAGCCAGGATCTTCGGTTGCCGCTGGAGACATCCTTGCTATTCTTGCATTGGATGATCCCAGCAAGGTGAAGCATGCTTTGCCATTCGATGGTACAATCCCTGATATGAAACAGCCATTTATCCATAGCAACAAACCAGTTTATAAGTTCATTTCTCTTCTCTCCGTGCTGAAAAACATTTTAGCAGGGTATGATAATCAAGTTGTGATGAACGATACTCTGCAGAGTCTATTGGATGTGTTGAAGAACCCTGAACTTCCTTATTCGGAATGGAATCATTCGATATCTGCACTTCATTCAAGGTTACCAATTCATTTGGACGAACAATTGACCAGTTTGATTGAGAGATCGCATCAACGTGGTGCAGACTTTCCAGCTAAGCACTTGCTCAAGCTTTTGGACAAGGAGCAGGCTGTTAATCCTGATCCACTTTTCTCCCAGGTCATTGCGCCTCTTACTGCTGTTGCCAAAAGCTACGAACATGGACTTGAAGTTCATGAACACAATGTATTCGCCGATTTGATCACCCAATACTACGACATAGAGAGCTTGTTTGCCGATAAAAGGGAGGAAGATGTTATTTTACAGCTACGTGATGAGAACAAATCGTCCCTTGACAAGGTCATCGATGTCGTCTTGTCACATTCCAGAGTTGGAGCTAAGAACCATTTAATCAGAGCTATTCTGGAAATTTATCAAACTATCTGCCAAAATGATCTCCAAGCTGCAACCATTTTGAAGAAACCTTTGAAAAAGATTGTTGAGCTAGATTCTAGATTTACAGCAAAGGTTTCGTTAAAAGCTAGAGAGATTTTGATTCAATGTTCCCTTCCCTCTATCAAAGAACGTTCAGACCAGCTCGAGCATATCCTTCGATCTTCAGTTGTACAAACTCAGTACGGAGAGAGCTTCAATGGAAACTACAAACTGCCTAACTTGGACGTTATACAAGACGTAATTGATTCCAAGTACATTGTATTCGATGTTTTGACACAATTTGTTGTTAGCCCAAACAAGTATATATTTGCAGCAGCAGCCGAGGTGTATCTGCGAAGAGCTTACAGGGCTTACTCGGTGAGAGAAGTTAAACATCATTTCGTAGGTGATTCTGCTCTCCCAATTGTGGAATGGAAGTTCCAATTGCCGCTGTTATCAACAGCTGCTTACAATTCCGTGCCTGAAGCTATGAGAAACTCCTCCAGTAACCGATCCTCTATTTCAATGGATAGAGCAGTTGCTGTCTCCGATTTGACCTTCATGATCAACAAGAATGATTCTCAACCTTTGAGAACAGGTATCATAATTCCCACAAACCACTTAGATGACATTGAGGAGTCCTTGTCATCTGCCATTGATGTCTTCCCTAAACGTCCACGTAACAATGGACCAGCTCCTGACAGAACTAATGTGGCTCCTGAGCAACCTACTAACGTATGCAATGTTTTCATTGCCAATGTTTCTGGCTACAACAGTGAGGCTGAGATCGTTGACAAGATTAGCAGCGTTCTTTCTGAGTTGAAAGACGACCTCAGGGCTAGTGGCGTTCGAAGAGTTACCTTTGTCTTGGGAGACAAGGTTGGAACTTATCCAAAATACTATACCTTCAAATTTCCAGACTATTTTGAAGACGAGACAATCCGTCACATAGAGCCTGCTCTTGCGTTCCAGCTGGAACTAAGAAGATTGTCCAATTTCAATATTAAACCTGTTCCAACTGAGAATAGAAATATTCATGTGTATGAGGCAGTTGCCAAAAATACTTCATGCATTGACAGGAGATTTTTTACTAGGGGTATCATCAGAACAAGCAGAATCAGAGAGGATGTGACTATCTCTGAATACCTTATCAGCGAAGCTAATCGTCTTATGAGTGACATTTTGGACGCTCTTGAGATTATTGATACCTCCAACACTGATTTGAACCATATATTCATCAATTTCTCTGCTGTTTTCAATGTCACGCCAGATGACGTTGA
AGCAGCGTTCGGTGGTTTCTTAGAAAGGTTTGGACGTAGGCTGTGGAGACTACGTGTTTCTGCTGCTGAAATCCGTATTATGTGCACGGACCCTGAGACTGGTATCCCATTCCCACTTCGTGCTTTAATTAACAACGTTTCAGGATACGTTGTGAAATCTGAAATGTATCAAGAGGTGAAAAATGATCATGGGGAATGGGTTTTCAAAAGTCTTGGTCCTACACCAGGTTCAATGCACCTTAGACCAATTTCAACACCATACCCAACCAAAGAATGGCTTCAACCAAAACGTTACAAAGCTCATCTTATGGGTACTACTTACGTGTATGATTTCCCTGAATTATTCCGTCAAGCTACGCTCTCCCAATGGAAAAAATACTCTCCTACTGCGAGAGTTCCTTCTGATGTGTTTGTGGCCAATGAATTGATCGTCGATGATTCAGGTGAACTAACTGAAGTAAGCAGAGAACCCGGCGCCAACGTTGTGGGTATGGTGGCCTTCAAGGTAACCGCAAAAACTCCTGAGTATCCACGCGGTCGCCATTTCATCATAATTGCTAATGATATCACCTTCAAGATCGGATCCTTTGGCCCTCAAGAAGATGAATATTTCAACAAGGCCACACAACTTGCAAGAAAATTGGGCATTCCTCGAATTTATCTGTCAGCCAACTCGGGTGCTAGAATTGGAGTTGCTGAAGAACTTCTTCCATTATTCAAAGTAGCCTGGAAGGAAGAAGGTAAACCAAGCAAGGGATTTGAATACTTATACCTCACATCGGAAGATCTTACTCTATTGGAAAAGTCCGGAAAGTCTAACAGCGTTACCACTCAAAGAATAGTTGAAGAAGGCGAAGAACGCCACGTTATAACTGCCATCATTGGAGCTAGTGATGGACTGGGTGTTGAATGTCTAAGAGGTTCCGGTTTGATCGCTGGTGCTACATCTCGGGCGTACAAGGACATCTTCACTATCACATTGGTCACCTGTAGATCTGTTGGTATTGGTGCTTACTTGGTCAGATTGGGTCAACGAGCCATTCAAATTGAAGGACAACCAATAATTTTGACTGGTGCCCCTGCTATTAATAAGTTGTTGGGTAGGGAAGTGTACTCTTCCAACCTGCAACTTGGTGGTACCCAGATTATGTACAAGAACGGTGTTTCACACTTAACCGCCAATGATGATCTCGCAGGTGTCGAAAAGATTATGGATTGGTTAGCTTATGTGCCTGCTAAGAGAAACATGCCTGTTCCTATTTTAGAATCACTTCATGACAAATGGGACAGAGATGTGGACTATAAGCCTACAAGAAATGAGCCGTACGACGTCAGATGGATGATCAGTGGACGTGAAACTCCTGATGGTGAGTTCGAATCTGGATTGTTTGACTCTGGGTCCTTCACTGAAACTTTGAGTGGATGGGCTAAAGGTGTAGTCGTCGGAAGAGCCCGTTTAGGTGGTATTCCTATGGGAGTCATTGGTGTTGAAACTAGAGTCACAGAAAACCTGATTCCAGCTGATCCCGCCAATCCAGACTCAACCGAAATGATGATTCAAGAAGCTGGTCAAGTCTGGTACCCTAACAGTGCCTTCAAGACTGCACAAGCTATCAACGATTTCAACAATGGTGAACAGCTACCCTTGATGATTTTGGCCAACTGGAGAGGTTTCTCTGGTGGTCAAAGAGACATGTACAATGAAGTTTTGAAATACGGTTCTTTCATTGTGGATGCTTTAGTCGACTTCAAGCAGCCTATCTTCACTTACATTCCTCCCACTGCTGAGTTGAGAGGTGGATCTTGGGTTGTTGTAGACCCTACCATCAATGAAGACATGATGGAAATGTATGCAGACGTCGAATCAAGAGCAGGTGTTTTGGAACCAGAAGGTATGGTAGGTATCAAATACCGTAAGGACAAACTCCTTGCTACTATGGAACGATTGGATGCCAAATATGCTGAGCTTAAATCCAAGGTTAGCGATACTAGTCTTTCAGAAAAGGATGTTTCCGAGATCAAGAAACAAATTGAGCAGAGAGAGAAGCAATTGTTGCCAATTTATGCACAAATCTCTATTCAATTTGCTGATCTTCATGACAGATCTGGTCGTATGTTGGCCAAGGGTGTCATTAAAAAGGAACTGGAATGGGTTAATTCTCGTCGTTTCTTCTTCTGGAGAGTCCGTCGTCGTTTGAACGAGGAATACCTCATTAAGCGTATTACCGAATTCCTATCTGCTTCTGCTACCAGATTGGACAAGATCTCGAGGATCAATTCTTGGTTGCCAACATCGATTGATTTGGAAGATGACCAGAAGGTTGCCATTTGGTTGGAAGAAAACCGTAAAGCTCTTGACGCCAATATCAAGGAGCTCAGGGCTGAGCATGTTAGAAGAACTCTGGCTACTCTTGTCAGAACTGATATGGATACTACTTCCAAGAGTTTGGCTGAATTGATCAACCTTCTTCCTGAAACCGAAAAGGAATCAATTTTATCTAAGATCAAGTCATGA
Mutation site in Acc1 of S. cerevisiae15 and P. pastoris
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