Supplementary material

Title:

Genetic modifications and introduction of heterologous pdc genes in Enterococcus faecalis for its use in production of bioethanol

Journal name :

Biotechnology Letters

Names of the authors:

N.F. Rana, S. Gente, A. Rincé, Y. Auffray and J.M. Laplace*

* Corresponding author : Jean-Marie Laplace - Unité de Recherche Aliments Bioprocédés Toxicologie Environnements (UR ABTE) EA 4651, Université de Caen Basse-Normandie, Bd Maréchal Juin, F 14032 Caen, France Phone : (+33) 231 56 70 31/ Fax : (+33) 231 56 71 65. Email:

Supplementary tables

Supplementary table 1. Bacterial strains and plasmids used in the current study.

Bacterial stains / Characteristic feature / Source or Reference
Enterococcus faecalis JH 2-2 / FusR, RifR, plasmid free wildtype strain / Yagi and Clewell (1980)
Escherichia coli Top10F’ / F’[lacIq Tn10 (TetR)] mcrA ∆(mrr-hsdRMS-mcrBC) f80lacZ∆M15∆ lacX74 recA l araD139 ∆(ara-leu) 7697galU galK rpsL (StrR) endA1 nupG / Invitrogen Corp., Carlsbad, CA, USA
Clostridium acetobutylicum ATCC 824 / Source for the Pyruvate decarboxylase gene / Collection ATCC
Sarcina ventriculi Goodsir 1824AL / Source for the Pyruvate decarboxylase gene / Talarico et al. (2005)

Plasmid Genotype or Remarks Source or Reference

pGEM-T / AmpR lac Z’ / Promega, Madison, WI, USA
pMAD / 9.66 kb ori PE194ts, EryR AmpR bgaB / Arnaud et al. (2004)
pMSP3535 / 8.35 kb EryR nisR nisK PnisA (promoter inducible with nisin) / Bryan et al. (2000)

Supplementary table 2. Primers used for the construction of mutant strains

Primers / Sequence (5’-3’) / Restriction site/ Utilisations
ldh down 1 / CATGTTTCCATGGTACACTGTCGCTAAAGCG / (NcoI) / ldh1 replacement with pdc
ldh down 2 / CATGTTTGGATCCAATAGTCAAAAATTAAAAACAACC / (BamHI) / ldh1 replacement in pdc
ldh up 1 / CATGGTTTGGTACCATTCCTTCCTTCCTCTATATTC / (KpnI) / deletion or replacement of ldh1
ldh up 2 / CATGTTTGTCGACCTGTTGTCATTAGTTGACTGC / (SalI) / deletion or replacement of ldh1
ldh1Bam1 / GCGGGATCCCACATGCGAATGTCGCTGGC / (BamHI) / ldh1 deletion
ldh1Bam2 / GCGGGATCCCATCATGGCAATCGTCATAA / (BamHI) / ldh1 deletion
ldh 0255 / TTCTGATTCAGGAAATTATATGTTGG / Deletion or replacement verification
ldh1sal / GCTACGGTCGACGGCTCAACAGAGGTGGGGTAC / Deletion or replacement verification
EF0641 UP / GCTACGGATCCAATCAAGCCCTTTGATGTCC / (BamHI) / ldh2 deletion
EF0641 Down / GCTACGGTCGACGCTTTCGCTAAATCGTTTTCC / (SalI) / ldh2 deletion
EF0641 inside 1 / gcaaacaagcgacctattatg / ldh2 deletion
EF0641 inside 2 / GACAGGATTTGAGGCAATCAC / ldh2 deletion
EF0641 interne 1 / CTTTTTCAGCGGCATTCAGATTC / ldh2 deletion
EF0641 interne 2 / CAAGAAAATGTAAACGTCTGGGC / ldh2 deletion
ldh2 perifor / TCCACCAATTCTTGGCTTTT / Deletion verification
ldh2 perirev / ATTTCCGATGAGATCCCTTG / Deletion verification
pdcCa Bam / CATGTTTGGATCCTTACTAATTATTTTGATTTGCAAAAC / (BamHI) / pdcCa cloning, pGEM-T
pdcCa Kpn / CATGTTTGGTACCGTGACAATTTTGAAGAGTGAATAC / (KpnI) / pdcCa cloning, pGEM-T
pdcSv Bam / CATGTTTGGATCCTTAGATGTGTTATTTTGAGAAC / (BamHI) / pdcSv cloning, pGEM-T
pdcSv Kpn / CATGTTTGGTACCATGAAAATAACAATTGCAGAATAC / (KpnI) / pdcSv cloning, pGEM-T
pdcCap3535Bam / CGCGGATCCATAATAGGGAGGTGACAATTATGAAGAGTG / (BamHI) / pdcCa cloning,pMSP3535
pdcCap3535PstI / CGCCTGCAGCTAATTATTTTGATTTGCAAAACG / (PstI) / pdcCa cloning, pMSP3535
PU / gtaaacgacggccagt / pGEMT
PR
MAD 1F / GGAAACAGCTATGACCATG
TCTAGCTAATGTTACGTTAACAC / pGEMT
pMADprimer/cloning verification
MAD 2R / TCATAATGGGGAAGGCCATC / pMAD primer/cloning verification
MAD 8F / TCTAGCTAATGTTACGTTACAC / pMAD primer
MAD 9R / TCATAATGGGGAAGGCCATC / pMAD primer
pMSP3535 5’ / TTAGATACAATGATTTCGTTC / pdcCa cloning in pMSP3535
pMSP3535 3’ / CTCGAGTCTAGAAGCGCTGTC / pdcCa cloning in pMSP3535

The underlined nucleotides indicate the restriction site in the primer sequence inserted during primer designing.

Supplementary table 3. Primers used for the transcriptional analysis with qPCR.

Primers / Sequence (5’-3’)
EF0255For / }ldh1 / GCTTCCCGAAAGAACGAGTA
EF0255Rev / ACATTTCGTGCATCAACGTC
EF0641For / }ldh2 / GCCTTCAGATTTGGCTTTGT
EF0641Rev / ACTGGTTTTGTTGGCACAAGT
EF0900For / }adhE / TCCGCAAACGGACATATAAAA
EF0900Rev / AACGGCAAATGGTGTAACTTC
EF0949For / }pta / AGCCGTACAAGTGGTGCTTT
EF0949Rev / TTCTTGTGCAGTTGGGTTCA
EF1213For / } alsS / GAGGCACGTAATTGGAACG
EF1213Rev / TTAGCTCTTCTTCCGGTTGC
EF1354For / } pdh / GTAACAACGGTGGGGTTTTC
EF1354Rev / CCACCGATACCAGATTCAGC
EF1613For / } pflB / TCGGAGCGATTGGTTCTAAG
EF1613Rev / GGCGACAAATGGAGAACAAG
EF1826For / } adhA / TCGCCAAAATCTGCATTCTT
EF1826Rev / AGAATCGCGTCCATTGAAAG
EF2654For / } adhZn / AAGCAATGATCTTCGGAACTG
EF2654Rev / ACAATAATGGGGGCTTCTTTG
EF3140For / } adhFe / GTAAAAAGGGTTCCCCTTGC
EF3140Rev / CGTGATTATTTTGTCGCTGGT
EF23SFor / } 23S / GTTCCCTCAGAATGGTTGGA
EF23SRev / CACTAAGCCCCGACTTTCGTC
PDCCaFor / } pdcCa / CATACGCTGAGCAAGTTCCA
PDCCaRev / CTTCCGTCACCTAATGTGTGG
PDCSvFor / } pdcSv / CAGCAGATGGATATGCAAGACT
PDCSvRev / AATCCACGGACCTGAAAGAG