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:

cmh022199@ecust.edu.cn.

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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