Supporting Information Table S1 Core regions of 18 identified promoters in C. acetobutylicum Gene Core regions of the promoters Reference 35 region 10 region thl TATA TTGATA AAAATAATAATAGTGGG TATAAT TAA (1) pta-ack GATG TTGCAA AAATATTAATAGGTTAA TATAAT TAT (2) adhe AATA TTGGTA CTATTAATTAAAAATTT TATAAT ATA (3) bdhb ATTA TTGTAA TAATTTTATAAAATATA TATAAT GTA (4) bdha TAGT TTGCAT GAAATTTCGTTGTTTAT TCATAT TAG (4) adc AAAA TTTACT TAAAAAAACAATATGTGT TATAAT GTA (5) glna TCAC TTGATT TCTTAAAAAAAAGGGAAG TATAAT TTA (6) GGGG TTCGAT AGAAGTTTATACTTGTC TATTGT GCG (6) dnak AAAG TTGACA AAGATAATGTCAGGTGA TATTTT ATA (7) AATT TTATGA AAATAAGAAAAGTTGAC AAAGAT AAT (7) groesl GATG TTGCTA ATATATTCAGGATTATT TATTAT AAT (8) sinr TACA TTGACA TAGCGAAATATTACAATA TATTAT TAA (9) abrb AGCA TTGACT TTTCAATTATATGGTAG TATATT AAC (9) hyd A ACAT TTTAGA CTTTATTTAAATATGA TATAAT TAT (10) ptb AAAC TTAACT TCATGTGAAAAGTTTGT TAAAAT ATA (11) crt AATA TTGAAA TATAAAATAAATCATTA TATAAT AAT (12) aad ATTT TTGACC TATGCTTTTTATTGAAC TATAAT AAA (13) adhe2 ATGT TTGACA ATCTTTAATTACTGTTA TATAAT AAT (14) 1
Table S2: Strains and plasmids in this study Strains and Plasmids Characteristics References and Strains C.ljungdahlii DSM 13528 (wild-type) C. acetobutylicum 824i ATCC 824 ΔCAC1502 (15) Plasmids Dual-reporter plasmids placzft pimp1-p ptb pimp1-p thl pimp1-ptb-catp-lacz pimp1-thl-catp-lacz LacZ reporter plasmids Amp r, lacz gene from Thermoanaerobacterium thermosulfurogenes EM1 ColE1 ori, pim13 ori, MLS r, Amp r, ptb (cac3076) promoter region of C. acetobutylicum ATCC 824 ColE1 ori, pim13 ori, MLS r, Amp r, thl (cac2873) promoter region of C. acetobutylicum ATCC 824 Derived from pimp1-p ptb ; expressing catp and lacz under the control of P ptb ; ColE1 ori, pim13 ori, Amp r, MLS r Derived from pimp1-ptb-catp-lacz; replacing P ptb with thl promoter pxy1 ColE1 ori, pcb102 ori; Amp r (16) pimp1-p thl -LacZ pxy1-p thl -LacZ pimp1-p 100-1 -LacZ pxy1-p 100-1 -LacZ pimp1-p 200-1 -LacZ pxy1-p 200-1 -LacZ pimp1-p 1200-4 -LacZ pxy1-p1 200-4 -LacZ pimp1-p 1200-9-9 -LacZ pxy1-p 1200-9-9 -LacZ sadhe-expressing plasmids Derived from pimp1-p thl, lacz expression under the control of the thl promoter Derived from pxy1, LacZ expression under the control of the thl promoter Derived from pimp1-p thl -LacZ, lacz expression under control of promoter 100-1 Derived from pxy1-p thl -LacZ, lacz expression under control of promoter 100-1 Derived from pimp1-p thl -LacZ, lacz expression under control of promoter 200-1 Derived from pxy1-p thl -LacZ, lacz expression under control of promoter 200-1 Derived from pimp1-p thl -LacZ, lacz expression under control of promoter 1200-4 Derived from pxy1-p thl -LacZ, lacz expression under control of promoter 1200-4 Derived from pimp1-p thl -LacZ, lacz expression under control of promoter 1200-9-9 Derived from pxy1-p thl -LacZ, lacz expression under control of promoter1200-9-9 Sources DSMZ Provided by Prof Papoutsakis E. T. Provided by Prof Papoutsakis E. T. 2
psadh The plasmid containing the sadhe gene Provided by Prof. Yin Li pimp1-p thl -SadhE Derived from pimp1-p thl, sadhe expression under the control of the thl promoter pimp1-p 100-1 - SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter 100-1 pimp1-p 200-1 - SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter 200-1 pimp1-p 1200-4 - SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter 1200-4 pimp1-p 1200-9-9 - SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter 1200-9-9 dnak-expressing plasmids pxy1-p thl -DnaK Derived from pxy1, dnak expression under the control of the thl promoter pxy1-p 100-1 - DnaK Derived from pxy1, dnak expression under control of promoter 100-1 pxy1-p 200-1 - DnaK Derived from pxy1, dnak expression under control of promoter 200-1 pxy1-p 1200-4 - DnaK Derived from pxy1, dnak expression under control of promoter 1200-4 pxy1-p 1200-9-9 - DnaK Derived from pxy1, dnak expression under control of promoter 1200-9-9 3
Table S3: Oligonucleotides used in this study Oligos Sequence (5' 3') Description thl(pst1)-ps TGGCTGCAGTTTTTAACAAAA For constructing LacZ thl(bamh1)-pr CGCGGATCCTCTAGAGTCGACTCTAACT AAC lacz(bamh1)-ps CGCGGATCCTATGAGAAAGATTATTCCT ATTAATAATA lacz(sma1)-pr TCCCCCGGGAGATGAAATTCTCTTTCTG TTTC cat(bamhi)-ps CGCGGATCCATGAACTTTAATAAAATTG cat-rbs-overlap-a CTCATTCTAACTAACCTCCTATTATAAAA GCCAGTCATTAG rbs-lacz-overlap-s TATAATAGGAGGTTAGTTAGAATGAGAA AGATTATTCCTA lacz(smai)-a TCCCCCGGGAGATGAAATTCTCTTTCTG thl-m GCTTGGCTGCAGTTTTTAACAAAAWNN NTTGVNWNNNNNNNNNNNNNNNNNNT ATAATNWNGTTGTTAGAGAAAACGTAT AAATTAG reporter plasmids. W represents A or T; D represents G, A or T; V represents A, C or G; N represents any base. The restriction enzyme-recognizing sites were underlined. cat GAGTCCAAATACCAGAGAATG pimp1-pr-s GCAAGAGGCAAATGAAATAG thl-7(bamh1)-pr CGCGGATCCACCTCCTAAATTTTGATAC For constructing thl-9(bamh1)-pr CGCGGATCCTAACCTCCTAAATTTTGAT AC thl-11(bamh1)-pr CGCGGATCCACTAACCTCCTAAATTTTG thl-13(bamh1)-pr CGCGGATCCTAACTAACCTCCTAAATTT TG thl-15(bamh1)-pr CGCGGATCCTCTAACTAACCTCCTAAAT TTTG thl-17(bamh1)-pr CGCGGATCCACTCTAACTAACCTCCTAA thl-19(bamh1)-pr CGCGGATCCCGACTCTAACTAACCTCC thl-21(bamh1)-pr CGCGGATCCGTCGACTCTAACTAACCTC thl-23(bamh1)-pr CGCGGATCCGAGTCGACTCTAACTAAC thl-25(bamh1)-pr CGCGGATCCTAGAGTCGACTCTAACTA AC sadhe(bamhi)- Ps CGCGGATCCATGAAAGGTTTTGCAATG CTAGG sadhe(smai)-pr TCCCCCGGGTTATAATATAACTACTGCTT TAATTAAGTC DnaK(BamHI)- Ps CGCGGATCCATGTCAAAAATAATAGGTA TTGA DnaK(SmaI)-Pr TCCCCCGGGTTATTTATCATCATCTACTT TGTAATCC promoters with truncated length between RBS and translational start code. The restriction enzyme-recognizing sites were underlined. For the construction of sadhe- and dnak-expressing plasmids. The restriction enzyme-recognizing sites were underlined. 4
Table S4: Sequence analysis of the core regions of P thl -derived artificial promoters with different activities Promoter Sequence 5' 3 ' Relative strength to P thl 100-1 TCTTTTGATTTGTGTTGTCTGTGGATGGTATAATGTC 0.098869 100-2 TAAGTTGCGATTTAGAGTTCGTGTCGAATATAATTAA 0.139342 100-3 TACATTGAAATCTGCCCAGTCGTTAACTTATAATTAT 0.17806 100-4 AGTCTTGATACCTGGGACGGCGAAAGGCTATAATGAT 0.178891 100-5 AGTTTTGAAAGTTTTTGGAGCACGGTTGTATAATTAA 0.188111 200-1 TATTTTGAGAGACATGGATGCCCTATGCTATAATGTA 0.324946 200-2 AGTGTTGACAAGGTGAGGGAATATCTTGTATAATTAA 0.434886 200-3 ATGGTTGAAATACTGGTTCGGGTGTTGATATAATTTA 0.441969 200-4 TGGTTTGCGTCATTTGTGTAAATTGTGGTATAATAAT 0.455897 200-5 ACCGTTGCGATAATGCAGTTAGGTGTTGTATAATAAA 0.463737 200-6 AGTCTTGCAATCAGAGAATGGGAGATTGTATAATCAA 0.496853 200-7 ATGGTTGAAATACTGGTTCGGGTGTTGATATAATTTA 0.533376 400-1 AGTATGGCAGTTGTACCGAGCT-TGAGTTATAATGAA 0.646019 400-2 TATTTTGATTATTCGGCTCGCGGTAGGGTATAATATG 0.730167 400-3 TATTTTGATTATTCGGCTCGCGGTAGGGTATAATATG 0.742514 400-4 AGTCTTGCTAGCGTGCCGTCTTCTTTGTTATAATTAT 0.759981 400-5 AGATTTGAGATATTACACTAAAGGGTGTTATAATTAA 0.76021 400-6 TAGATTGACAGCCTTGCGTATCGAAGTCTATAATAAT 0.819137 400-7 ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG 0.849344 800-1 TGCTTTGACATTGGATGAGCTGATCAAGTATAATCAA 0.896506 800-2 AGATTTGACATAGGACTTGCAAGTTTCATATAATTAT 0.902469 800-3 AAGATTGAGTTAGGAGGGTGCTACGAGGTATAATTAT 0.912713 800-4 ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG 0.915584 800-5 ACTGTTGATAACTATAGCAGCTTCTTGATATAATTAG 0.937867 800-6 ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG 0.95389 thl TATATTGATAAAAATAATAATAGTGGGTATAATTAA 1 800-7 TATATTGATAAAAATAATAATAGTGGGTATAATTAA 1.0094 1200-1 AAAATTGGGAAACGTAGATTGCTGGTGGTATAATTAT 1.078815 1200-2 AGTCTTGCTAGCGTGCCGTCTTCTTTGTTATAATTAT 1.083593 1200-3 TATCTTGGCAAAGCTCTCGATCTCTGT-TATAATTAG 1.144117 1200-4 AAATTTGACTTCGATTGGCGCATGCCCCTATAATAAA 1.168027 1200-5 TATCTTGGCAAAGCTCTCGATCTCTGT-TATAATTAG 1.184636 1200-6 ATATTTGGCAAGGAGACTAAGAGAATAGTATAATTAC 1.229247 1200-7 AGAATTGCAAAGATCAAATCTCTCGATGTATAATTTT 1.28099 1200-8 TTCCTTGACATACTCAACACAAGGCCCGTATAATTTA 1.389572 1200-9 AGTATTGAAATTTGGTCTACCCAGGTATTATAATGTG 1.404737 5
Table S5: The sequence of the original P thl (containing the synthetic spacer between RBS and the initial code ATG) and its derivative expression parts that were listed in Figure 4A. The bases remaining unchanged in the 35 and 10 region were highlighted in red. Expression parts Sequence 5' 3 ' P thl TTTTTAACAAAATATATTGATAAAAATAATAATAGTGGGTATAATTA AGTTGTTAGAGAAAACGTATAAATTAGGGATAAACTATGGAACTTA TGAAATAGATTGAAATGGTTTATCTGTTACCCCGTATCAAAATTTAG GAGGTTAGTTAGAGTCGACTCTAGAGGATCC 1200-9-27 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCTAGAGGATCC 1200-9-25 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCTAGGATCC 1200-9-23 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCGGATCC 1200-9-21 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACGGATCC 1200-9-19 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGGGATCC 1200-9-17 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTGGATCC 1200-9-15 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGGATCC 1200-9-13 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG 6
GGAGGTTAGTTAGGATCC 1200-9-11 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTGGATCC 1200-9-9 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGGATCC 1200-9-7 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTGGATCC 7
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