Restriction enzymes and Antarctic phosphatase were from New England Biolabs (Ipswich, MA, USA). Rapid DNA ligation kit was from Roche (Mannheim, Germany). KOD DNA polymerase was from EMD Chemicals (San Diego, CA, USA). Oligonucleotides were from Operon (Huntsville, AL, USA).
Strains and plasmids
A list of the strains and plasmids used is given in Table . All oligonucleotides are listed in Table . The yqhD
gene was deleted by P1 transduction from a strain JW2978 (Baba et al. 2006
). The yqhD
deletion was confirmed with polymerase chain reaction (PCR). To clone kivd
which encodes KDC, we used genomic DNA of L. lactis
(ATCC) as PCR template with a pair of primers A96 and A97. PCR products were digested with Acc65I and SphI and cloned into pZE12-luc (Lutz and Bujard 1997
) cut with the same enzyme, creating pSA129. To clone yqhD
, genomic DNA of E. coli
MG1655 was used as PCR template with a pair of primers K453 and A257. PCR products were digested with SphI and XbaI and cloned into pSA129 cut with the same enzyme, creating pSA138. To clone adhA
, genomic DNA of L. lactis
was used as PCR template with a pair of primers A121 and A122. PCR products were digested with SphI and XbaI and cloned into pSA55 cut with the same enzyme, creating pSA65.
Strains and plasmids used in this study
For protein overexpression and purification, ADH2, adhA, and yqhD were amplified with primers A287 and A288, A289 and A290, and A292 and A299, respectively. PCR products were digested with BamHI and SalI and cloned into pETDuet-1 (Novagen (Madison, WI, USA)) cut with the same enzymes, creating pTW2, pTW3, and pTW4.
Medium and culture conditions for isobutanol production
M9 medium containing 36 g/L glucose, 5 g/L yeast extract, 100 μg/ml ampicillin, 30 μg/ml kanamycin, and 1,000th dilution of Trace Metal Mix A5 (2.86 g H3
, 1.81 g MnCl2
O, 0.222 g ZnSO4
O, 0.39 g Na2
O, 0.079 g CuSO4
O, 49.4 mg Co(NO3
O per liter water) was used for cell growth. Preculture in test tubes containing 3 ml of medium was performed at 37°C overnight on a rotary shaker (250 rpm). Overnight culture was diluted 1:100 into 20 ml of fresh medium in a 250-ml screw cap conical flask. Cells were grown at 37°C for 3 h, followed by adding 0.1 mM isopropyl-β-d
-thio-galactoside (IPTG). Production was performed at 30°C on a rotary shaker (250 rpm) for 24 h. Gas chromatography-flame ionization detector analysis is carried out as previously described (Atsumi et al. 2008b
YqhD was overexpressed from pTW2 in E. coli BL21 Star™ (DE3) (Invitrogen (Carlsbad, CA, USA)). Adh2 and AdhA were overexpressed from pTW3 and pTW4, respectively, in BL21-CodonPlus(DE3)-RIL-X (Stratagene, Cedar Creek, TX, USA). Overexpressed proteins were purified with Ni-NTA Spin Columns (Qiagen (Valencia, CA, USA)). Protein concentrations were determined by the Bradford assay (Bio-Rad (Hercules, CA, USA)).
Protein solubility assay
The strains with pTW2, pTW3, and pTW4 were grown to OD600 value of 0.4–0.6 in 5 mL Luria–Bertani medium at 37°C, followed by adding 1 mM IPTG. Protein overexpression was performed at 30°C for 4 h. The cells were centrifuged, resuspended in 250 μl BugBuster Protein Extraction Reagent (Novagen, San Diego, CA, USA), and incubated at room temperature for 20 min for cell lysis. To separate soluble and insoluble proteins, the samples were centrifuged for 50 min (13,000 rpm, 4°C). Supernatant was taken as a soluble sample. The cell pellets were resuspended by 250 μl 8 M urea and centrifuged. Supernatant was taken as an insoluble sample. Soluble and insoluble protein samples were visually assessed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Enzyme activity assay
The reaction mixture contained 50 mM 3-(N-morpholino)propanesulfonic acid buffer, pH 7.0, 0.25 mM NAD(P)H, a defined aldehyde (acetaldehyde or isobutyraldehyde) as substrate, and purified ADH (100 nM). The mixture was incubated at 37°C, and NAD(P)H oxidation was determined at 340 nm using a spectrophotometer (Beckman Coulter DU800). One unit of enzyme activity was defined as the amount of protein that oxidizes 1 pmol of NAD(P)H/min at 37°C. The Km values for different aldehydes and the Vmax were extrapolated after nonlinear regression of the experimental points with Gauss–Newton method using Matlab.