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icum. The full-length gdh glutamicum was amplified by PCR. For subsequent cloning steps, EcoRI and

CGC GTC GAC TTA AT GAC GCC CTG TGC C-3’). After restriction of the PCR product and the expression 1 (Degussa AG, Halle) with EcoRI and SalI, the gdh gene was ligated to

ZgdhEC:

is an expression vector of gdh from E. coli. The full-length gdh gene of E.

-GCG CGC GAA TTC TG GAT CAG ACA TAT TCT CTG G-3’ / 5’-GCG CGC GGA TCC TTA TCA CAC CCT After restriction of the PCR product and the expression vector pZ8-1

gdh deletion strain LN∆GDH and GDH-activity was measured. The cloned gdh

ene was not sequenced.

g a single point mutation (K92L). The vector was derived from Zgdh by site-directed mutagenesis using the primers 5’-GCA CT TGG ACC ATA CCT 3’ and 5’-GAA GCG CAG GCC GCC CAG GTA TGG

his plasmid is an expression vector for GDH from C. glutamicum with a histag at its N- terminus. The full-length gdh gene of C. glutamicum was amplified by PCR. For subsequent cloning steps, SphI and HindIII restriction sites (shown in bold) were introduced in the primer sequences (5’-GCG CGC GCA TGC ATG GAT CAG ACA TAT TCT CTG G-3’ / 5’-GCG CGC AAG CTT TTA GAT GAC GCC CTG TGC C-3’). After restriction of the PCR product and the expression vector pQE30Xa (Qiagen, Hilden) This plasmid is an expression vector of gdh from C. glutam

gene of C.

SalI restriction sites (shown in bold) were introduced in the primer sequences (5’-GCG

CGC GAA TTC ATG ACA GTT GAT GAG CAG GTC-3’ / 5’-GCG G

vector pZ8-

vector pZ8-1 leading to plasmid pZgdh. The cloned gdh gene was sequenced.

p

This plasmid

coli was amplified by PCR. For subsequent cloning steps, EcoRI and BamHI restriction

sites (shown in bold) were introduced in the primer sequences (5’ A

GCG CCA G-3’).

(Degussa AG, Halle) with EcoRI and BamHI, the gdh gene of E. coli was ligated to vector pZ8-1 leading to plasmid pZgdhEC. For verification, the plasmid was transformed

into the g

pZgdh-K92L:

This plasmid is an expression vector for an enzymatic inactive mutant of GDH from C.

glutamicum harbourin

p

GGG CGG CCT GCG CTT C-

TCC AAG TGC-3’. The mutated gdh gene was sequenced.

pQE30Xagdh:

with SphI and HindIII, the gdh gene was ligated to vector pQE30Xa leading to plasmid agdh. The cloned gdh gene was sequenced.

ading to JCgdhlacZ.

UCwhiH:

an expression vector of whiH from C. glutamicum. The full-length whiH

gene was ligated to pUC18 ading to plasmid pUCwhiH. The cloned whiH gene was sequenced.

pQE30X

pJCgdhlacZ:

This plasmid harbours a fusion of the gdh promoter region of C. glutamicum and the

lacZ gene coding for β-galactosidase and can be used for reporter gene assays. The

vector pK18gdh-lacZ (Nolden, 2001) was restricted with EcoRI and PstI, The resulting insert, which harbours the fusion of the gdh promoter region and the lacZ gene, was ligated to the EcoRI-PstI-restricted vector pJC1 (Cremer et al., 1990) le

p

pUCfarR:

This plasmid is an expression vector of farR from C. glutamicum. The full-length farR gene of C. glutamicum was amplified by PCR. For subsequent cloning steps, BamHI restriction sites (shown in bold) were introduced in the primer sequences (5’-GCG CGC

GGA TCC TTG CTT TTT ACT AGG CGC TCC-3’ / 5’-GCG CGC GGA TCC CGT CAG

AGA TCT TCG GAG-3’). After restriction of the PCR product and the expression vector pUC18 (Viera & Messing, 1982) with BamHI, the farR gene was ligated to pUC18 leading to plasmid pUCfarR. The cloned farR gene was sequenced.

p

This plasmid is

gene of C. glutamicum was amplified by PCR. For subsequent cloning steps, EcoRI restriction sites (shown in bold) were introduced in the primer sequences (5’-GCG CGC

GAA TTC ATG ACC CCA GCA AAC GAA AG-3’ / 5’-GCG CGC GAA TTC TTA GTT

CAG CGT GCC CCA GC-3’). After restriction of the PCR product and the expression vector pUC18 (Viera & Messing, 1982) with EcoRI, the whiH

le

pUCoxyR:

This plasmid is an expression vector of oxyR from C. glutamicum. The full-length oxyR gene of C. glutamicum was amplified by PCR. For subsequent cloning steps, EcoRI restriction sites (shown in bold) were introduced in the primer sequences (5’-GCG CGC

GAA TTC ATG AGC AAT AAA GAG TAC CGG-3’ / 5’-GCG CGC GAA TTC CGT TAC

pUC18 (Viera & Messing, 1982) with EcoRI, the oxyR gene was ligated to pUC18 leading to plasmid pUCoxyR. The cloned oxyR gene was sequenced.

s amplified by PCR. For subsequent cloning steps, XbaI and XmaI striction sites (shown in bold) were introduced in the primer sequences (5’-GCG CGC GAC GTG AAC CCA TTT TGG TG -3’ / 5’-GCG CGC CCC GGG ACA CCA

was ligated to the nearized vector pUC18-∆FarR-unten leading to pUC-∆FarR-gesamt. This plasmid as 8mobsacB (Schäfer et al., 1994) were restricted with HindIII and XmaI, and the insert of pUC- FarR-gesamt was ligated to pK18mobsacB leading to pK18 farR.

pK18∆whiH:

This plasmid is a deletion vector for whiH from C. glutamicum. By SOE-PCR, a fusion of the 800 bp sequences upstream and downstream of the whiH gene of C. glutamicum was synthesized. For subsequent cloning steps, EcoRI restriction sites (shown in bold) were introduced in the outer primer sequences (5’-GCG CGC GAA TTC ACA GGT CTC AAA CTG GGC C-3’ / 5’-CGC AGT GCG CGT ATC ACG GGT GCC TCT TTA ATG GGC C-3’ / 5’-GGC CCA TTA AAG AGG CAC CCG TGA TAC GCG CAC TGC G-3’ / 5’- GCG CGC GAA TTC GCA GCT GAA GCT GTG CGC G-3’). After restriction of the SOE-PCR product and the vector pK18mobsacB (Schäfer et al., 1994) with EcoRI, the SOE-PCR product was ligated to vector pK18mobsacB leading to plasmid pK18∆whiH, which was sequenced.

pK18∆farR:

This plasmid is a deletion vector for farR from C. glutamicum. The 800 bp sequence upstream of the farR gene of C. glutamicum was amplified by PCR (5’-TCA ATG ATT TCG TCC TTG TGG-3’ / 5’-GTG GTT TTG GTG ACT GAA GC-3’) and ligated to the vector pDrive (Qiagen, Hilden) by T/A-cloning. The resulting plasmid pDrive-∆FarR- oben-neu was sequenced. The 800 bp sequence downstream of the gdh gene of C.

glutamicum wa

re

TCT AGA

AGG TTG ACT G-3’). The PCR product and the vector pUC18 (Viera & Messing, 1982) were restricted with XbaI and XmaI and ligated to form the plasmid pUC18-∆FarR- unten, which was sequenced. After that, the plasmid pDrive-∆FarR-oben-neu was linearized by restriction with BamHI and the plasmid pUC18-∆FarR-unten was linearized with XbaI. Subsequently, both were treated the large Klenow fragment of DNA polymerase I for fill-in of 5’ overhangs to form blunt ends and subsequently restricted with HindIII. The resulting 800 bp insert of pDrive-∆FarR-oben-neu

li

well as pK1

pK18∆oxyR:

This plasmid is a deletion vector for oxyR from C. glutamicum. By SOE-PCR, a fusion of e 800 bp sequences upstream and downstream of the oxyR gene of C. glutamicum zed. For subsequent cloning steps, EcoRI restriction sites (shown in bold) th

was synthesi

were introduced in the outer primer sequences (5’-GCG CGC GAA TTC GTT GTG CGC GAC ATG ATC G-3’ / 5’-CTC TGG AAA ACC TCT AGA AAA ATG CCT ATA ACT ATA ACG GTG-3’ / 5’-CAC CGT TAT AGT TAT AGG CAT TTT TCT AGA GGT TTT CCA GAG-3’ / 5’-GCG CGC GAA TTC GCA GCT GAA GCT GTG CGC G-3’). After restriction of the SOE-PCR product and the vector pK18mobsacB (Schäfer et al., 1994) with EcoRI, the SOE-PCR product was ligated to vector pK18mobsacB leading to plasmid pK18∆oxyR, which was sequenced.