Table 2 Plasmids and strains used in this study

Plasmids

 pNW33N

Bacillus–E. coli shuttle vector, Cm^R, pBC1 ori for Gram-positive strains, pBR322 ori for E. coli, source of CAT_Sa

Bacillus Genetic Stock Center

 pETDuet-1

pBR322 ori, Amp^R, lacI, T7lac promoter

Novagen

 pET_CAT_Sa

CAT_Sa wild-type encoding gene between BamHI, SacI site, pETDuet-1 backbone, 6× His-tag at N-terminus

This study

 pET_CAT_Sa F97W

F97W site-directed variant, pET_CAT_Sa backbone

This study

 pHS0024

CAT_Sa wild-type gene under C. thermocellum PgapDH promoter, downstream of Clo1313_2927 for the transcription terminator, tdk operon under cbp promoter substituting with the native cat selection marker, pNW33N plasmid backbone

This study

 pHS0024_F97W

CAT_Sa F97W site-directed mutated from pHS0024

This study

Strains

 E. coli Top10

Host for molecular cloning, mcrA, ∆(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ∆lacX74, deoR, recA1, araD139, ∆(ara-leu)7697, galU, galK, rpsL(SmR), endA1, nupG

Invitrogen

 E. coli BL21 (DE3)

E. coli B dcm, ompT, hsdS(rB-mB-), gal

Invitrogen

 M1354

C. thermocellum DSM1313 ∆hpt

[37]

 HSCT0101

M1354 harboring pHS0024

This study

 HSCT0102

M1354 harboring pHS0024_F97W

This study