Supporting Information

Transcription

1 1 Supporting Information Sequences: E. coli lysine riboswitch (ECRS): GTACTACCTGCGCTAGCGCAGGCCAGAAGAGGCGCGTTGCCCAAGTAACGGTGTTGGAGGAGCCAG TCCTGTGATAACACCTGAGGGGGTGCATCGCCGAGGTGATTGAACGGCTGGCCACGTTCATCATCGG CTACAGGGGCTGAATCCCCTGGGTTGTCACCAGAAGCGTTCGCAGTCGGGCGTTTCGCAAGTGGTG GAGCACTTCTGGGTGAAAATAGTAGCGAAGTATCGCTCTGCGCCCACCCGTCTTCCGCTCTTCCCTTG TGCCAAGGCTGAAAATGGATCCCCTGACACGAGGTAGTT B. subtilis lysine riboswitch (BSRS): GTGAACTAATTTCATAGTTAGATCGTGTTATATGGTGAAGATAGAGGTGCGAACTTCAAGAGTATGC CTTTGGAGAAAGATGGATTCTGTGAAAAAGGCTGAAAGGGGAGCGTCGCCGAAGCAAATAAAACCC CATCGGTATTATTTGCTGGCCGTGCATTGAATAAATGTAAGGCTGTCAAGAAATCATTTTCTTGGAGG GCTATCTCGTTGTTCATAATCATTTATGATGATTAATTGATAAGCAATGAGAGTATTCCTCTCATTGCT TTTTTTATTGTGGACAAAGCGCTCTTTCTCCTCACCCGCACGAACCAAAATGTAAAGGGTGGTAATAC Underslined text, lysine-binding aptamer Blue text, expression platform Table S1. Bactrial strains and plasmids Strains or plasmids Major characteristics a Source or reference Strains E. coli MG1655 F -, λ -, rph-1 DSMZ b TOP10 F - mcraδ(mrr-hsdrms-mcrbcφ80laczδm15δ lacx7 reca1 arad19(araleu)7697galu galk rpsl(strr) enda1 nupg Invitrogen DH5α MCR Host for suicide plasmid construction S1

2 DL Lysine producing strain S DLECRS ECRS-gltA, derived from DL This study DLBSRS BSRS-gltA, derived from DL This study DLG1C ECRS(G1C)-gltA, derived from DLECRS This study C. glutamicum ATCC c 10 Wild-type strain S CgECRSlacZ ATCC 10 carrying pecrs-lacz This study CgBSRSlacZ ATCC 10 carrying pbsrs-lacz This study LP917 lysc(q98g), ppc(r917g), derived from ATCC 10 S LPECRS ECRS-gltA, derived from LP917 This study LPBSRS BSRS-gltA, derived from LP917 This study Plasmids pjet1. Cloning vector Thermo pkd FRT-Kan R -FRT Lab collection pkd6 λ Red recombination Lab collection pk18mobsacb Kan R, Sucrose S, sucide vector S pec-xk99e E. coli-c. glutamicum shuttle expression vector S5 pjetecrs ECRS inserted into pjet1. This study pjetbsrs BSRS inserted into pjet1. This study pecrs-lacz ECRS-lacZ, derived from pec-xk99e This study pbsrs-lacz BSRS-lacZ, derived from pec-xk99e This study pbba-lacz Consititutive promoter BBa_J100 fused with lacz This study

3 pk18ecrs ECRS-lacZ, derived from pk18mobsacb This study pk18bsrs BSRS-lacZ, derived from pk18mobsacb This study 1 a Abbreviations: Kan R, Kanamycin resistance; Sucrose S, Sucrose sensitive. b DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen. c ATCC, American Type Culture Collection Table S. Primers used in the study Primer Sequence Description ECRS-F1 CGGTCTGCGTTGGATTGATG For ECRS ECRS-R1 CCACTGTGCCTGAACATCGC BSRS-F1 AGCGGCGGCTCAGCTCCTTTAC For BSRS BSRS-R1 Backbone-F1 Backbone-R1 ECRS-F ECRS-R lacz-f lacz-r BSRS-F BSRS-R Backbone-R lacz-f BBa-R DECgltA-F AACCGAAGTGTCGATATCTG TAATAACCGGGCAGGCCATGTCTGCCTCGAC CTGCAGGCATGCAAGCTTGGC GCTAGCACTGTACCTAGGACTGAGCTAGCCG TCAAGCCTCGCGCGTTTCGGTGATGACGG CCGTCATCACCGAAACGCGCGAGGCTTGACG GCTAGCTCAGTCCTAGGTACAGTGCTAGCGT ACTACCTGCGCTAGCGCAGGCCAG CCAGTGAATCCGTAATCATGGTCATAACTACC TCGTGTCAGGGGATCC GGATCCCCTGACACGAGGTAGTTATGACCAT GATTACGGATTCACTGG GCCAAGCTTGCATGCCTGCAGGTCGAGGCAG ACATGGCCTGCCCGGTTATTA CCGTCATCACCGAAACGCGCGAGGCTTGACG GCTAGCTCAGTCCTAGGTACAGTGCTAGCAA TTTCATAGTTAGATCGTGTTATATGG CATGGTCATAGCTGTTTCCTGTGTGATTTTGG TTCGTGCGGGTGAGGAGAAAGAGC GCTCTTTCTCCTCACCCGCACGAACCAAAATC ACACAGGAAACAGCTATGACCATG TTTCACACAGGAAACAGCTATGACC ATGATTACGGATTCACTGGCCGTCG CGCTAGCACTGTACCTAGGACTGAGCTAGCC GTCAAG CATATAGTTTGTCGGGTTTTATCCTGAACAGT GATCCAGGTCACGATAACGAGCGATTGTGTA GGCTGGAG For backbone of pecrs-lacz For insert (ECRS) of pecrs-lacz For insert (lacz) of pecrs-lacz or pbsrs-lacz For insert (BSRS) of pbsrs-lacz For backbone of pbsrs-lacz with Backbone-F1 For pbba-lacz For E. coli glta deletion cassette (Kan R )

4 DECgltA-R ECRS-F ECRS-R ECgltA-F1 ECgltA-R G1C-F G1C-R BSRS-F BSRS-R ECgltA-F BBK18-F1 BBK18-R1 InUpgltA-F1 InUpgltA-R1 ECRS-F ECRS-R glta-f1 glta-r1 InUpgltA-R CCCGCCATATGAACGGCGGGTTAAAATATTT ACAACTTAGCAATCAACCATTAACGGCTGAC ATGGGAATTAG CAAAGTTGTTACAAACATTACCAGGAAAAGC ATATAATGCGTAAAAGTTAGTACTACCTGCG CTAGCGCAGGCCA GGTGAGTTTTGCTTTTGTATCAGCCATAACTA CCTCGTGTCAGGGGATCC GGATCCCCTGACACGAGGTAGTTATGGCTGA TACAAAAGCAAAACTCACC CCCGCCATATGAACGGCGGGTTAAAATATTT ACAACTTAGCAATCAACCA GCGCAGGCCAGAAGACGCGCGTTGCCCAAG TAACGGTGTTGGAG CTTGGGCAACGCGCGTCTTCTGGCCTGCGCT AGCGCAGGTAGTAC CAAAGTTGTTACAAACATTACCAGGAAAAGC ATATAATGCGTAAAAGTTAAATTTCATAGTTA GATCGTGTTATATGG CAGCCATTTAAGGTCTCCTTAGCGCCATTTTG GTTCGTGCGGGTGAGGAGAAAGAGC GCTCTTTCTCCTCACCCGCACGAACCAAAATG GCGCTAAGGAGACCTTAAATGGCTG GGTTCCTCGCGAGGAGCGCTAAAGGCATGCA AGCTTGGCACTGGCCGTC CAACGCTTCCGATCGCTTCATCCCTGAGTAAT CATGTCATAGCTGTTTCC GGAAACAGCTATGACATGATTACTCAGGGAT GAAGCGATCGGAAGCGTTG CTGGCCTGCGCTAGCGCAGGTAGTACGGTTA ACACGCTATACCGATAAATG CATTTATCGGTATAGCGTGTTAACCGTACTAC CTGCGCTAGCGCAGGCCAG CAGTAGCCACGATATCCCTTTCAAACATAACT ACCTCGTGTCAGGGGATCC GGATCCCCTGACACGAGGTAGTTATGTTTGA AAGGGATATCGTGGCTAC CGACGGCCAGTGCCAAGCTTGCATGCCTTTA GCGCTCCTCGCGAGGAACC CCATATAACACGATCTAACTATGAAATTGGTT AACACGCTATACCGATAAATG For ECRS crossover with E. coli glta For E. coli glta crossover with ECRS For site-mutagenesis of lysine riboswitch For BSRS crossover with E. coli glta Paired with ECgltA-R, used for E. coli glta crossover with BSRS For backbone of pk18ecrs For insert (upstream of C. glutamicum glta) of pk18ecrs For insert (ECRS) of pk18ecrs For insert (C. glutamicum glta) of pk18ecrs Paired with InUpgltA-F1, used for insert (upstream of C. glutamicum glta) of pk18bsrs

5 1 BSRS-F BSRS-R glta-f CATTTATCGGTATAGCGTGTTAACCAATTTCA TAGTTAGATCGTGTTATATGG CGATATCCCTTTCAAACATATTTGTTCGGATT TTGGTTCGTGCGGGTGAGGAGAAAGAGC GCTCTTTCTCCTCACCCGCACGAACCAAAATC CGAACAAATATGTTTGAAAGGGATATCG For insert (BSRS) of pk18bsrs Paired with glta-r1, used for insert (C. glutamicum glta) of pk18ecrs 5 Figure S1. HPLC analysis of lysine concentration. HPLC was carried out with a C18 column (Phenomenex, Germany). The solvents were 10 mm sodium acetate (0.1 % acetonitrile, ph 5.9) and 60 % acetonitrile (vol/vol). Detection of peaks was with UV-detector at 5 nm. mau, milli-absorbance units at 5 nm References S1. Grant, S. G., Jessee, J., Bloom, F. R., and Hanahan, D. (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. U.S.A. 87, S. Geng, F., Chen, Z., Zheng, P., Sun, J., and Zeng, A. P. (01) Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production. Appl. Microbiol. Biotechnol. 97, S. Chen, Z., Bommareddy, R. R., Frank, D., Rappert, S., and Zeng, A. P. (01) Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum. Appl. Environ. Microbiol. 80,

6 1 5 S. Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G., and Pühler, A. (199) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pk18 and pk19: Selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 15, S5. Kirchner, O., and Tauch, A. (00) Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J. Biotechnol. 10,