Table 2.

Plasmids and oligonucleotides used in this study

Plasmid or oligonucleotideRelevant characteristic(s) or sequenceSource, reference, or purpose
Plasmids
    pK19mobsacBKmr, mobilizable (carrying oriT gene), carrying oriV gene44
    pK19mobsacBldhApK19mobsacB carrying a truncated ldhA geneThis work
    pK19mobsacBmdhpK19mobsacB carrying a truncated mdh geneThis work
    pK19mobsacBmalEpK19mobsacB carrying a truncated malE geneThis work
    pJC4ilvBNCDKanr; plasmid carrying the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and dihydroxyacid dehydratase40
    pJC4ilvBNCD-pntABKanr; plasmid pJC4 carrying the ilvBNCD genes and additionally carrying the pntAB genes from E. coli, encoding the membrane-bound transhydrogenase PntAB; carrying pntAB genes under the control of PtacThis work
    pBB1Cmr; pBB1 is compatible to pJC4ilvBNCD and harbors the Ptac promoter and the Ttrp terminator; lacI negative29
    pEKEx2-pntABPlasmid pEKEx2 carrying the pntAB genes from E. coli25
    pBB1pntABCmr; plasmid pBB1 carrying the pntAB genes from E. coli; pntAB under the control of PtacThis work
    pSA55Plasmid for expression of the adh2 gene (encoding alcohol dehydrogenase 2) from Saccharomyces cerevisiae and the kivd gene (encoding 2-ketoacid decarboxylase) from Lactococcus lactis2
    pBB1kivdCmr; plasmid pBB1 expressing the kivd gene from L. lactis; carrying kivd gene under the control of PtacThis work
    pBB1adh2Cmr; plasmid pBB1 expressing the adh2 gene from S. cerevisiae; carrying adh2 gene under the control of PtacThis work
    pBB1kivd-adh2Cmr; plasmid pBB1 expressing the kivd gene from L. lactis and the adh2 gene from S. cerevisiae; carrying kivd and adh2 genes under the control of PtacThis work
    pBB1kivd-adhACmr; plasmid pBB1 expressing the kivd gene from L. lactis and the adhA gene from C. glutamicum; carrying kivd gene under the control of Ptac and adhA gene under the control of the native promoterThis work
Oligonucleotides
    adh4fow5′-AACTGCAGAACCAATGCATTGGAGGAGACACAACATGTCTATTCCAGAAACTCAAAAAG-3′Amplification of the adh2 gene
    adh2rev5′-CCGCTCGAGCGGTTATTTAGAAGTGTCAACAACGTAT-3′Amplification of the adh2 gene
    kivdfow5′-AACTGCAGAACCAATGCATTGGAGGAGACACAACATGTATACAGTAGGAGATTACCTAT-3′Amplification of the kivd gene
    kivd2rev5′-CCAATGCATTGGTTCTGCAGTTTTATGATTTATTTTGTTCAGCAAAT-3′Amplification of the kivd gene
    Ptaccheck5′-CACTCCCGTTCTGGATAATG-3′Primer to verify orientation of the kivd gene
    kivdchkrevec5′-CTGAGAGTGTACCATTATAG-3′Primer to verify orientation of the kivd gene
    adhAfowsalI5′-ACGCGTCGACGGGAATTGTGTGAATCTTGAAAAG-3′Amplification of adhA gene; primer to verify orientation of adhA gene
    adhArevsalI5′-GCTATGGCCGACGTCGACCAAAGGTCATGCCTTAAGCAGC-3′Amplification of the adhA gene
    pMM36rev5′-ACTACCGGAAGCAGTGTG-3′Primer to verify orientation of the adhA gene
    pntABfow5′-CATGCCTGCAGTCATCAATAAAACCG-3′Amplification of the pntAB genes
    pntABrev5′-GTACGCTGCAGTCTTACAGAGCTTTCAGG-3′Amplification of the pntAB genes
    transfow25′-CTAACATGTATACCCCGCGAATTGCAAGCTGATCCGGGC-3′Amplification of the pntAB genes
    transrev25′-CTAACATGTATACAAAAAAAAGCCCGCTCATTAGGCGGGCTGGATGCTCTTACAGAGCTTTCAGGATTGCATCC-3′Amplification of the pntAB genes
    ldhA15′-CGCCCGGGTTCGGCAACAATGACGGCGAGA-3′Primer for deletion of the ldhA gene
    ldhA25′-CCCATCCACTAAACTTAAACAGACGGTTTCTTTCATTTTCGATCC-3′Primer for deletion of the ldhA gene
    ldhA35′-TGTTTAAGTTTAGTGGATGGGAAGCAGTTCTTCTAAATCTTTGGCG-3′Primer for deletion of the ldhA gene
    ldhA45′-CGCCCGGGGGCATCGACGACATCTGAG-3′Primer for deletion of the ldhA gene
    ldhfow5′-TGATGGCACCAGTTGCGATGT-3′Primer to verify deletion of the ldhA gene
    ldhrev5′-CCATGATGCAGGATGGAGTA-3′Primer to verify deletion of the ldhA gene
    mdh15′-CCCAAGCTTGTTGCCAGGTCCAGACCTCG-3′Primer for deletion of the mdh gene
    mdh25′-CGTCACCGGCGCAGCTGGTCCGAATGCTCAGGAATTGCAGG-3′Primer for deletion of the mdh gene
    mdh35′-GACCAGCTGCGCCGGTGACGGTGACCTTCTTGGTGGAGACG-3′Primer for deletion of the mdh gene
    mdh45′-CGCGGATCCCGCTTGGACATGCCAGATGC-3′Primer for deletion of the mdh gene
    mdhcheckfow5′-CCTGATTCCAGGAACGCATC-3′Primer to verify deletion of the mdh gene
    mdhcheckrev5′-CCTAACATCTTGCAGGTGAG-3′Primer to verify deletion of the mdh gene
    malE15′-CGGGATCCTTGCTGCCTACACCTACCTTG-3′Primer for deletion of the malE gene
    malE25′-CCCATCCACTAAACTTAAACACTGCAGGTCGATGGTCATATC-3′Primer for deletion of the malE gene
    malE35′-TGTTTAAGTTTAGTGGATGGGGTCGCCGAAGCGCAAAACGCTTAA-3′Primer for deletion of the malE gene
    malE45′-CGGGATCCGAAGTGCTGATCCGCGAACC-3′Primer for deletion of the malE gene
    Co-malE15′-CTTCCAGACACGGAATCAGAG-3′Primer to verify deletion of the malE gene
    Co-malE25′-GTGATCCTTCCGAGCGTTCC-3′Primer to verify deletion of the malE gene