Supplementary Methods
Construction of pRCI vector
The lacI promoter was amplified by PCR from pET21a (Novagen, Madison, WI) using
following primers: lacI-F, 5′-GCGGATCCGAATTCAAGGGAGAGCGTCGAGATCC-3′ (BamHI and EcoRI restriction sites are underlined) and lacI-R,
5′-GGTTTCTTTTTTGTGCTCATATTCACCACCCTGAATTGACTC-3′. The gene
encoding the cI857 repressor was amplified using  DNA (Toyobo, Osaka, Japan) as the
template. The oligonucleotide primers of cI857-F (5′-ATGAGCACAAAAAAGAAACCATTAACACAA-3′) and cI857-R (5′-GGAGCGGCCGCTTACTATGTTATGTTCTGAGGGGAGTGAAA-3′, NotI restriction site is underlined) were used for the
amplification. The PCR products were mixed and used as the template for overlapping
PCR to construct the expression cassette of the cI857 repressor. The primer pair of
lacI-F and cI857-R was used for the overlapping PCR. The amplicon was digested with
NotI and BamHI.
A 2,670-kb internal fragment of pBR322 (Toyobo, Osaka, Japan) containing the ColE1
ori and the ampicillin resistance gene was amplified by PCR with the following primers:
pBR-F, 5′-GGAGCGGCCGCGCTACCCTGTGGAACACCTACAT-3′ (NotI restriction
is underlined), and pBR-R, 5′-CGCGGATCCTTCTTGAAGACGAAAGGGCCTC-3′
(BamHI restriction site is underlined). The PCR product was digested with NotI and
BamHI, and ligated with the expression cassette of the cI857 repressor, and the resulting
plasmid was designated as pBR-CI857.
The Pr promoter was amplified from  DNA using the following primers: PR-F,
5′-GGAAGATCTACGTTAAATCTATCACCGCAAGGGATAAATATTTAACACCGTG-3′ (BglII restriction sites are underlined), and PR-R, 5′-CCGCTCGAGGCTCTTCACACCATACAACCTCCTTAGTACATGCAAC-3′ (XhoI and BspQI restriction sites
are underlined). The T7 terminator region of pET21a was amplified using T7T-F
(5′-CCGCTCGAGGCTCTTCATAAGGCTGCTAACAAAGC-3′, XhoI and BspQI
restriction sites are underlined) and T7T-R (5′-GGAGAATTCATCCGGATATAGTTCCTCCTTTCAG-3′, EcoRI restriction site is underlined) primers. The
PCR-amplified Pr promoter and T7 terminator were digested with XhoI and then
tandemly ligated with T4 DNA ligase (Toyobo). The ligation product was further
amplified by PCR using the primer pair of PR-F and T7T-R. The amplified DNA was
digested with BglII and EcoRI and then introduced into the corresponding sites of
pBR-CI857. Finally, a point mutation was introduced into the resulting plasmid to
eliminate the extra-BspQI restriction site derived from pBR322, using the PrimeStar
mutagenesis kit (Takara, Ohtsu, Japan) with the primer pair of MUT-1,
5′-TCAGGCGCTATTCCGCTTC-3′ and MUT-2, 5′-GAAGCGGAATAGCGCCTGA-3′ (the mutated nucleotide is underlined).
NADH oxidase assay
The codon-optimized gene encoding the NADH oxidase from Thermococcus profundus
was synthesized and expressed in E. coli. The synthetic gene was flanked with NdeI and
EcoRI restriction sites at its 5′- and 3′-terminals, respectively. The gene was digested
with these restriction enzymes and introduced into the corresponding sites of pET21a. E.
coli BL21 (DE3) (Novagen) was transformed with the resulting plasmid.
The recombinant E. coli was cultivated at 37°C in LB medium supplemented with 100
μg/ml ampicillin. Gene expression was induced by adding of 0.2 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) at the late log phase. Cells were harvested by
centrifugation, suspended in 50 mM HEPES-NaOH (pH 7.0), and disrupted by
ultrasonication. The crude lysate was heated at 70°C for 30 min and centrifuged to
remove debris and denatured proteins. The resulting supernatant was used as the
enzyme solution.
The enzyme activity was determined by monitoring the oxidation of NADH at 340 nm.
A reaction mixture comprising 50 mM HEPES-NaOH (pH 7.0), 5 mM MgCl2, 0.5 mM
MnCl2, and 0.2 mM NADH was preincubated at 70°C for 2 min and then the reaction
was started initiated by adding an appropriate amount of enzyme.