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Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

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Academic year: 2021

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(1)

Table S1. Primer for this study

Category Primer Sequence Annotation

Yeast assay

CC141 GATATCATGGACGCTTACTCC

Primers for cloning ScFAS1 with NdeI site CC142 GATATCTTAGGATTGTTCATACTTT

CC139 CGAGGCGATATCATTGAAAACTGGGCT

Primers for inverse PCR CC140 CGCGCGATATCCATAATGCTCTTAATTG

CC104 TTTCCATACAATTCAAGG

Primers for identifying fas1 mutant CC105 GGTAGGCTCGTCATCTGC

CC106 CTGCCTCTTTTTCTGGGA CC107 TTGCGGTTGCCATAAGAG

CC23 ATGGCGCTCCGCGTCA

Primers for cloning DH domains CC24 CTACTCCTCGGTGCTGGTGG

CC25 ATGTCCCGCCTCGGCGA CC26 CTACGAGGCGGCGGGCTC

TcPUFAs assay

KS13 CTTTCCCTGCGCCTTCGTCAAGGACGAGGTCA

Primers for mutated DH2 His active site into Ala (underline) KS14 CTTGACGAAGGCGCAGGGAAAGTACCAGTGGTC

KS15 CTTCGCGTGCGCCTTCTGGTTCGACTCGGTCA Primers for mutated DH2 His active site into Ala (underline) KS16 GAACCAGAAGGCGCACGCGAAGAACCAGTCGT

KS17 CCTATGACCTGGCACCCCAT

Primers for Inverse PCR KS18 CCTGCGCGAATTCGGATCCTGG

KS19 CATCCATGGCGATGGCGCTCCG

Primer for delete DH2 domain KS20 TTGGCGAAGAGCGAGGTGC

(2)

pGEM-T-ScFAS1

9162 bp

TA-cloning

pGEM-T-ScFAS1-M

7481 bp

pGEM-T-ScFAS1-M-DH1-DH2

10481 bp

B

lu

n

t

li

gation

pHVX2-ScFAS1-M-DH1-DH2

15009 bp

Sub-clone ScFAS1-M-DH1-DH2

fragment into pHVX2

(3)

R

es

tri

cti

o

n

di

g

es

ti

o

n

EcoR

V

/N

deI

Li

g

a

ti

o

n

R

es

tri

cti

o

n

di

g

es

ti

o

n

Eco

R

V

/N

deI

(4)

Inverse PCR

to delete DH1

Replace the original ORF-C

of the operon with mutated

ORF-C*

Replace DH1-DH2 with a

single DH2 in ORF-C

Figure

Table S1. Primer for this study
Fig. S1. Construction of the plasmids expressing engineered ScFAS1 swapped with individually DH domains of the PUFA synthase in yeast.
Fig. S2. Construction of the plasmids expressing a PUFA synthase with two individually mutated DH domains in E
Fig. S3. Construction of the plasmids expressing a PUFA synthase with a single DH domain in E

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