Strains

Characteristics

Sources

DWc9n

B. licheniformis DW2 with Cas9n integrated strain

Lab stock

Escherichia coli

DH5a

F– Φ80d/lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (rK–, mK+), phoA, supE44, λ–, thi-1, gyrA96, relA1

Takara

BL21(DE3)

F-, ompT, hsdSB(rB-mB-), gal (λcI857, ind1, Sam7, nin5, lacUV5-T7 gene1), dcm(DE3)

Takara

DH8

DWc9nΔpyk-xkdE::yugJ-ldh::tyrAfbr-xkdG::aroGfbr-PbacA-aroKΔpheAΔhisCΔdhaS ΔdhbCΔadhA

Lab stock

DH10

DWc9nΔpyk-xkdE::yugJ-ldh::tyrAfbr-xkdG::aroGfbr-PbacA-aroKΔpheAΔhisCΔdhaS ΔdhbCΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG

Lab stock

DH11

DWc9nΔpyk-xkdE::yugJ-ldh::tyrAfbr-yvnA::tyrAfbr-xkdG::aroGfbr-PbacA-aroKΔpheAΔhisCΔdhaSΔdhbCΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG

This study

DH12

DWc9nΔpyk-xkdE::yugJ-ldh::tyrAfbr-yvnA::tyrAfbr-xkdG::aroGfbr-PbacA-aroKΔpheAΔhisCΔdhaSΔdhbCΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG-PbacA-tkt

This study

DH13

DWc9nΔpyk-xkdE::yugJ-ldh::tyrAfbr-yvnA::tyrAfbr-xkdG::aroGfbr-PbacA-aroKΔpheAΔhisCΔdhaSΔdhbCΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG-PbacA-tkt-PbacA-zwf

This study

DHT11

DH8/pHY-PbacA-kivD-P43-hpaBC

Lab stock

DHT12

DH10/pHY-PbacA-kivDV461I-P43-hpaBC

This study

DHT13

DH10/pHY-PbacA-kivDV461I-PbacA-hpaBC

This study

DHT14

DH10/pHY-PbacA-kivDV461I-Pbay-hpaBC

This study

DHT15

DH10/pHY-P43-kivDV461I-P43-hpaBC

This study

DHT16

DH10/pHY-P43-kivDV461I-PbacA-hpaBC

This study

DHT17

DH10/pHY-P43-kivDV461I-Pbay-hpaBC

This study

DHT18

DH10/pHY-Pbay-kivDV461I-P43-hpaBC

This study

DHT19

DH10/pHY-Pbay-kivDV461I-PbacA-hpaBC

This study

DHT20

DH10/pHY-Pbay-kivDV461I-Pbay-hpaBC

This study

DHT21

DH11/pHY-P43-kivDV461I-Pbay-hpaBC

This study

DHT22

DH12/pHY-P43-kivDV461I-Pbay-hpaBC

This study

DHT23

DH13/pHY-P43-kivDV461I-Pbay-hpaBC

This study

DHT24

DH13/pHY-P43-kivDV461I-Pnark-hpaBC

This study

DWc9n/pHY-kivD

DWc9n harboring pHY-kivD, Tetr

This study

DWc9n/pHY-kivDS286G

DWc9n harboring pHY-kivDS286G, Tetr

This study

DWc9n/pHY-kivDE462G

DWc9n harboring pHY-PbacA-kivDE462G, Tetr

This study

DWc9n/pHY-kivDF542G

DWc9n harboring pHY-PbacA-kivDF542G, Tetr

This study

DWc9n/pHY-kivDV461G

DWc9n harboring pHY-PbacA-kivDV461G, Tetr

This study

DWc9n//pHY-kivDV461Y

DWc9n harboring pHY-PbacA-kivDV461Y, Tetr

This study

DWc9n/pHY-kivDV461T

DWc9n harboring pHY-PbacA-kivDV461T, Tetr

This study

DWc9n/pHY-kivDV461F

DWc9n harboring pHY-PbacA-kivDV461F, Tetr

This study

DWc9n/pHY-kivDV461H

DWc9n harboring pHY-PbacA-kivDV461H, Tetr

This study

DWc9n/pHY-kivDV461R

DWc9n harboring pHY-PbacA-kivDV461R, Tetr

This study

DWc9n/pHY-kivDV461A

DWc9n harboring pHY-PbacA-kivDV461A, Tetr

This study

DWc9n/pHY-kivDV461I

DWc9n harboring pHY-PbacA-kivDV461I, Tetr

This study

DWc9n/pHY-kivDV461M

DWc9n harboring pHY-PbacA-kivDV461M, Tetr

This study

DWc9n/pHY-kivDV461S

DWc9n harboring pHY-PbacA-kivDV461S, Tetr

This study

DWc9n/pHY-kivDV461N

DWc9n harboring pHY-PbacA-kivDV461N, Tetr

This study

DWc9n/pHY-kivDV461K

DWc9n harboring pHY-PbacA-kivDV461K, Tetr

This study

DWc9n/pHY-kivDV461D

DWc9n harboring pHY-PbacA-kivDV461D, Tetr

This study

DWc9n/pHY-kivDV461E

DWc9n harboring pHY-PbacA-kivDV461E, Tetr

This study

DWc9n/pHY-kivDV461W

DWc9n harboring pHY-PbacA-kivDV461W, Tetr

This study

DWc9n/pHY-kivDV461P

DWc9n harboring pHY-PbacA-kivDV461P, Tetr

This study

DWc9n/pHY-kivDV461Q

DWc9n harboring pHY-PbacA-kivDV461Q, Tetr

This study

DWc9n/pHY-kivDV461C

DWc9n harboring pHY-PbacA-kivDV461C, Tetr

This study

DWc9n/pHY-kivDV461L

DWc9n harboring pHY-PbacA-kivDV461L, Tetr

This study

DWc9n/pHY-300

DWc9n harboring pHY-300, Tetr

Lab stock

Plasmids

Characteristics

Source

pET-28a(+)

E. coli expression vector, Kanr

Takara

pET-28a(+)kivD

pET-28a(+) containing kivD

This study

BL21(DE3)/pET-28a(+)

BL21(DE3) harboring pET-28a(+), Kanr

Lab stock

BL21(DE3)/pET-28a(+)kivD

BL21(DE3) harboring pET-28a(+)kivD, Kanr

This study

BL21(DE3)/pET-28a(+)kivDV461I

BL21(DE3) harboring pET-28a(+)kivDVI, Kanr

This study

T2(2)-Ori

E. coli and B. subtilis shuttle vector, Kanr

Lab stock

pHY300PLK

E. coli-Bacillus shuttle vector; Ampr in E. coli, Tcr in both E. coli and B. licheniformis

Takara

pHY-kivD

pHY300 containing kivD gene with PbacA promoter

Lab stock

pHY-kivDS286G

pHY300 containing kivDS286G gene with PbacA promoter

This study

pHY-kivDE462G

pHY300 containing kivDE462G gene with PbacA promoter

This study

pHY-kivDF542G

pHY300 containing kivDF542G gene with PbacA promoter

This study

pHY-kivDV461G

pHY300 containing kivDV461G gene with PbacA promoter

This study

pHY-kivDV461Y

pHY300 containing kivDV461Y gene with PbacA promoter

This study

pHY-kivDV461T

pHY300 containing kivDV461T gene with PbacA promoter

This study

pHY-kivDV461F

pHY300 containing kivDV461F gene with PbacA promoter

This study

pHY-kivDV461H

pHY300 containing kivDV461H gene with PbacA promoter

This study

pHY-kivDV461R

pHY300 containing kivDV461R gene with PbacA promoter

This study

pHY-kivDV461A

pHY300 containing kivDV461A gene with PbacA promoter

This study

pHY-kivDV461I

pHY300 containing kivDV461I gene with PbacA promoter

This study

pHY-kivDV461M

pHY300 containing kivDV461M gene with PbacA promoter

This study

pHY-kivDV461S

pHY300 containing kivDV461S gene with PbacA promoter

This study

pHY-kivDV461N

pHY300 containing kivDV461N gene with PbacA promoter

This study

pHY-kivDV461K

pHY300 containing kivDV461K gene with PbacA promoter

This study

pHY-kivDV461D

pHY300 containing kivDV461D gene with PbacA promoter

This study

pHY-kivDV461E

pHY300 containing kivDV461E gene with PbacA promoter

This study

pHY-kivDV461W

pHY300 containing kivDV461W gene with PbacA promoter

This study

pHY-kivDV461P

pHY300 containing kivDV461P gene with PbacA promoter

This study

pHY-kivDV461Q

pHY300 containing kivDV461Q gene with PbacA promoter

This study

pHY-kivDV461C

pHY300 containing kivDV461C gene with PbacA promoter

This study

pHY-kivDV461L

pHY300 containing kivDV461L gene with PbacA promoter

This study

pHY-P43-hpaBC

pHY300 containing hpaBC with P43 promoter

Lab stock

pHY-PbacA-hpaBC

pHY300 containing hpaBC with PbacA promoter

Lab stock

pHY-PbacA-kivDV461I-P43-hpaBC

pHY300 containing kivDV461I with PbacA promoter and hpaBC genes with P43 promoter, respectively

This study

pHY-PbacA-kivDV461I-PbacA-hpaBC

pHY300 containing kivDV461I with PbacA promoter and hpaBC genes with PbacA promoter, respectively

This study

pHY-PbacA-kivDV461I-Pbay-hpaBC

pHY300 containing kivDV461I with PbacA promoter and hpaBC genes with Pbay promoter, respectively

This study

pHY-P43-kivDV461I-P43-hpaBC

pHY300 containing kivDV461I with P43 promoter and hpaBC genes with P43 promoter, respectively

This study

pHY-P43-kivDV461I-Pbay-hpaBC

pHY300 containing kivDV461I with P43 promoter and hpaBC genes with Pbay promoter, respectively

This study

pHY-Pbay-kivDV461I-P43-hpaBC

pHY300 containing kivDV461I with Pbay promoter and hpaBC genes with P43 promoter, respectively

This study

pHY-Pbay-kivDV461I-PbacA-hpaBC

pHY300 containing kivDV461I with Pbay promoter and hpaBC genes with PbacA promoter, respectively

This study

pHY-Pbay-kivDV461I-Pbay-hpaBC

pHY300 containing kivDV461I with Pbay promoter and hpaBC genes with Pbay promoter, respectively

This study

pHY-P43-kivDV461I-Pnark-hpaBC

pHY300 containing kivDV461I with Pbay promoter and hpaBC genes with Pnark promoter, respectively

This study

pHY-tyrAfbr

pHY300 containing tyrAfbr gene with PbacA promoter

Lab stock

T2(2)-yjiC-pheA

T2(2) containing yjiC up arm +pheA+ yjiC

down arm

Lab stock

T2(2)-yjiC-tyrAfbr

T2(2) containing yjiC up arm + tyrAfbr + yjiC

down arm

This study

T2(2)-PbacA-tkt

T2(2) containing Ptkt up arm+PbacA promoter+Ptkt down arm

This study

T2(2)-PbacAu12-zwf

T2(2) containing zwf up arm + PbacAu12 + zwf

down arm

This study

Primer name

Sequence of primer (5' to 3')

pHY-F

GTTTATTATCCATACCCTTAC

pHY-R

CAGATTTCGTGATGCTTGTC

amp-YF

CCCTGATCTCGACTTCGT

amp-YR

TTAAGGGGTCTGACGCTC

T2-F

ATGTGATAACTCGGCGTA

T2-R

GCAAGCAGCAGATTACGC

V461G-F

AATAATGATGGTTATACAGGTGAAAGAGAAATTCATGGA

V461Y-F

AATAATGATGGTTATACATACGAAAGAGAAATTCATGGA

V461T-F

AATAATGATGGTTATACAACAGAAAGAGAAATTCATGGA

V461F-F

AATAATGATGGTTATACATTTGAAAGAGAAATTCATGGA

V461H-F

AATAATGATGGTTATACACATGAAAGAGAAATTCATGGA

V461R-F

AATAATGATGGTTATACACGCGAAAGAGAAATTCATGGA

V461A-F

AATAATGATGGTTATACAGCGGAAAGAGAAATTCATGGA

V461I-F

AATAATGATGGTTATACAATTGAAAGAGAAATTCATGGA

V461M-F

AATAATGATGGTTATACAATGGAAAGAGAAATTCATGGA

V461S-F

AATAATGATGGTTATACATCTGAAAGAGAAATTCATGGA

V461N-F

AATAATGATGGTTATACAAACGAAAGAGAAATTCATGGA

V461K-F

AATAATGATGGTTATACAAAGGAAAGAGAAATTCATGGA

V461D-F

AATAATGATGGTTATACAGATGAAAGAGAAATTCATGGA

V461E-F

AATAATGATGGTTATACAGAGGAAAGAGAAATTCATGGA

V461W-F

AATAATGATGGTTATACATGGGAAAGAGAAATTCATGGA

V461P-F

AATAATGATGGTTATACACCCGAAAGAGAAATTCATGGA

V461Q-F

AATAATGATGGTTATACACAGGAAAGAGAAATTCATGGA

V461C-F

AATAATGATGGTTATACATGCGAAAGAGAAATTCATGGA

V461L-F

AATAATGATGGTTATACATTAGAAAGAGAAATTCATGGA

V461-R

TGTATAACCATCATTATTGATAATAAAGCAAATTGGATT

PbacA-kivD-R

ATCTCCTACTGTATACATATAAAAATTCTCCTTTTT

Pbay-kivD-R

ATCTCCTACTGTATACATACAAATCTCCCCCTTTGTTGTT

T5-yjiC-F

TTTTTAACCTCCCGTTATTTTTCG

T5-yjiC-R

CCATATGAAGGAACCCGGGAAATC

yjiC-tyrA-F

CGAAAAATAACGGGAGGTTAAAAAATGGTTGCTGAATTG

yjiC-tyrA-R

GATTTCCCGGGTTCCTTCATATGGAATGAAGGTATTGGGCTG

T2-T5(BamHI)-F

GGATCCCCCGGGCTGCAGGAATTC

T2-T5(SacI)-R

GAGCTCGTAGAAAAGATCAAAGGA

tkt-AF1

CTGCAGCCCGGGGGATCCAACTTGAGCTTTCCGAAGCG

tkt-AR1

TGAATCTCGCCGAAATCGCAGGACGCCTTGAAGATATTCCTGG

AT-F

CCAGGAATATCTTCAAGGCGTCCTGCGATTTCGGCGAGATTCA

AT-R

CAGATTTTAATTCAATCGTTTTCATATAAAAATTCTCCTTTTTG

tkt-BF1

CAAAAAGGAGAATTTTTATATGAAAACGATTGAATTAAAATCTG

tkt-BR1

GATCTTTTCTACGAGCTCTTCAAATGTCCCGCAAGCG

tkt-YF

TGTGACGCCTGAAAAGCT

tkt-YR

AAACGCTGCTTCACATTT

yjiC-YF

ACGAAGCAGCCCCTTTTC

yjiC-YR

CACTCCAATAATTGAGTA

zwf–A-F

GATCTTTTCTACGAGCTC TTCCTGCACGCGGTATCCA

zwf–A-R

TCTCGCCGAAATCGCAGGATAGCCTGGCGTGAAGCC

zwf-PbacAu12-F

GGCTTCACGCCAGGCTATCCTGCGATTTCGGCGAGA

zwf-PbacAu12-R

TATATATTCCTCCTTTCTAATATACTCACCATAATATTGGTCT

zwf–B-F

AGACCAATATTATGGTGAGTATATTAGAAAGGAGGAATATATAATGAAAAAAGATCAAATG

zwf–B-R

AACGAATTCCTGCAGCCCATTTCCTGATTCAAGGCT

zwf-YF

TGTGGCCTCCCCGCAGTC

zwf-YR

ATATCTCGCTCTGTCTTC

PnarK-hpaBC-F

TTTTTTATAACAGGAATTCCCAGCTTTTCCTTTACG

PnarK-hpaBC-R

GAAATCTTCTGGTTTCATTGCGTTTTTGCTCCTTTC

T5-300-hpaBC-R

AATTCCTGTTATAAAAAAAGGATCA

T5-hpaBC-hpaBC-F

ATGAAACCAGAAGATTTCCG