Supporting Information Table S1 Core regions of 18 identified promoters in C. acetobutylicum

Transkriptio

1 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

2 Table S2: Strains and plasmids in this study Strains and Plasmids Characteristics References and Strains C.ljungdahlii DSM (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 LacZ pxy1-p LacZ pimp1-p LacZ pxy1-p LacZ pimp1-p LacZ pxy1-p LacZ pimp1-p LacZ pxy1-p 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 Derived from pxy1-p thl -LacZ, lacz expression under control of promoter Derived from pimp1-p thl -LacZ, lacz expression under control of promoter Derived from pxy1-p thl -LacZ, lacz expression under control of promoter Derived from pimp1-p thl -LacZ, lacz expression under control of promoter Derived from pxy1-p thl -LacZ, lacz expression under control of promoter Derived from pimp1-p thl -LacZ, lacz expression under control of promoter Derived from pxy1-p thl -LacZ, lacz expression under control of promoter Sources DSMZ Provided by Prof Papoutsakis E. T. Provided by Prof Papoutsakis E. T. 2

3 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 SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter pimp1-p SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter pimp1-p SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter pimp1-p SadhE Derived from pimp1-p thl -LacZ, sadhe expression under control of promoter dnak-expressing plasmids pxy1-p thl -DnaK Derived from pxy1, dnak expression under the control of the thl promoter pxy1-p DnaK Derived from pxy1, dnak expression under control of promoter pxy1-p DnaK Derived from pxy1, dnak expression under control of promoter pxy1-p DnaK Derived from pxy1, dnak expression under control of promoter pxy1-p DnaK Derived from pxy1, dnak expression under control of promoter

4 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

5 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 TCTTTTGATTTGTGTTGTCTGTGGATGGTATAATGTC TAAGTTGCGATTTAGAGTTCGTGTCGAATATAATTAA TACATTGAAATCTGCCCAGTCGTTAACTTATAATTAT AGTCTTGATACCTGGGACGGCGAAAGGCTATAATGAT AGTTTTGAAAGTTTTTGGAGCACGGTTGTATAATTAA TATTTTGAGAGACATGGATGCCCTATGCTATAATGTA AGTGTTGACAAGGTGAGGGAATATCTTGTATAATTAA ATGGTTGAAATACTGGTTCGGGTGTTGATATAATTTA TGGTTTGCGTCATTTGTGTAAATTGTGGTATAATAAT ACCGTTGCGATAATGCAGTTAGGTGTTGTATAATAAA AGTCTTGCAATCAGAGAATGGGAGATTGTATAATCAA ATGGTTGAAATACTGGTTCGGGTGTTGATATAATTTA AGTATGGCAGTTGTACCGAGCT-TGAGTTATAATGAA TATTTTGATTATTCGGCTCGCGGTAGGGTATAATATG TATTTTGATTATTCGGCTCGCGGTAGGGTATAATATG AGTCTTGCTAGCGTGCCGTCTTCTTTGTTATAATTAT AGATTTGAGATATTACACTAAAGGGTGTTATAATTAA TAGATTGACAGCCTTGCGTATCGAAGTCTATAATAAT ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG TGCTTTGACATTGGATGAGCTGATCAAGTATAATCAA AGATTTGACATAGGACTTGCAAGTTTCATATAATTAT AAGATTGAGTTAGGAGGGTGCTACGAGGTATAATTAT ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG ACTGTTGATAACTATAGCAGCTTCTTGATATAATTAG ATTCTTGCTTTTCGACAAGCAGCGGTGCTATAATGAG thl TATATTGATAAAAATAATAATAGTGGGTATAATTAA TATATTGATAAAAATAATAATAGTGGGTATAATTAA AAAATTGGGAAACGTAGATTGCTGGTGGTATAATTAT AGTCTTGCTAGCGTGCCGTCTTCTTTGTTATAATTAT TATCTTGGCAAAGCTCTCGATCTCTGT-TATAATTAG AAATTTGACTTCGATTGGCGCATGCCCCTATAATAAA TATCTTGGCAAAGCTCTCGATCTCTGT-TATAATTAG ATATTTGGCAAGGAGACTAAGAGAATAGTATAATTAC AGAATTGCAAAGATCAAATCTCTCGATGTATAATTTT TTCCTTGACATACTCAACACAAGGCCCGTATAATTTA AGTATTGAAATTTGGTCTACCCAGGTATTATAATGTG

6 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 TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCTAGAGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCTAGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACTCGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGACGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTCGGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGTGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTTAGAGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG 6

7 GGAGGTTAGTTAGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGTGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTTAGGATCC TTTTTAACAAAAAGTATTGAAATTTGGTCTACCCAGGTATTATAATG GGAGGTGGATCC 7

8 References for supplementary materials: 1. Stim-herndon, K. P., Petersen, D. J., Bennett, G. N. (1995) Characterization of an acetyl-coa C-acetyltransferase (thiolase) gene from Clostridium acetobutylicum ATCC 824, Gene 154 (1), Boynton, Z. L., Bennett, G. N., Rudolph, F. B. (1996). Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 62 (8), Fischer, R. J., Helms, J. A. N., Durre, P., Mikrobiologie, I., Gcttingen, G. U. (1993). Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. J. Bacteriol. 175 (21), Walter, K. A., Bennetti, G. N., Papoutsakisl, E. T. (1992) Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J. Bacteriol. 174 (22), Gerischer, U., Dürre P. (1992) mrna analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J. Bacteriol. 174 (2), Janssen, P. J., Jones, D. T., Woods, D. R. (1990) Studies on Clostridium acetobutylicum gina promoters and antisense RNA. Mol. Microbiol. 4 (9), Narberhaus, F., Giebeler K., Bahl, H. (1992) Molecular characterization of the dnak gene region of Clostridium acetobutylicum, including grpe, dnaj, and a new 8

9 heat shock gene. J. Bacteriol. 174 (10), Narberhaus, F., Bahl., H. (1992) Cloning, sequencing, and molecular analysis of the groesl operon of Clostridium acetobutylicum. J. Bacteriol. 174 (10), Scotcher, M. C., Rudolph, F. B., Bennett, G. N. (2005) Expression of abrb310 and SinR, and effects of decreased abrb310 expression on the transition from acidogenesis to solventogenesis, in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 71 (4), Gorwa, M. F., Croux, C., Soucaille, P. (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824, J. Bacteriol. 178 (9), Walter, K. A., Nair, R. V, Caryb, J. W., Bennettc, G. N., Papoutsakis, E. T. (1993) Sequence and arrangement of two genes of the butyrate-synthesis of Clostridium acetobutylicum ATCC 824. Gene 134 (1), Boynton, Z. L., Bennett, G. N., Rudolph, F. B. (1996) Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-coa dehydrogenase from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 178 (11), Nair, R. V, Bennett, G. N., Papoutsakis, E. T. (1994) Molecular Characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 176 (3), Fontaine, L., Meynial-salles, I., Girbal, L., Yang, X., Croux, C., Soucaille, P. (2002) Molecular Characterization and transcriptional analysis of adhe2, the gene 9

10 encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. J. Bacteriol. 184 (3), Dong, H., Zhang Y. P., Dai Z., Li Y. (2010) Engineering clostridium strain to accept unmethylated DNA. PLoS One, 5 (2), e Zhang, N., Shao, L., Jiang, Y., Gu, Y., Li, Q., Liu, J., Jiang, W., and Yang, S. (2015) I-SceI-mediated scarless gene modification via allelic exchange in Clostridium. J. Microbiol. Methods 108,