Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes
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Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.
KeywordsBiofuel Metabolic engineering Isobutanol Alcohol dehydrogenase
To meet the increasing global demand for energy and reduce the negative environmental impact, biofuels derived from renewable sources have become increasingly important. In particular, increasing attention has been paid to higher alcohols as potential substitutes for gasoline. Those alcohols possess a host of qualities making them more suitable as a liquid fuel than ethanol, including lower vapor pressure, lower hygroscopicity, and higher energy density. However, efficient production of these alcohols is challenging (Atsumi et al. 2008a; Cann and Liao 2008; Connor and Liao 2008; Inui et al. 2008; Shen and Liao 2008).
We previously devised a biosynthetic strategy to produce higher alcohols in Escherichia coli which takes advantage of the amino acid biosynthesis capability to produce various 2-keto acids and the broad substrate range of 2-keto acid decarboxylases (KDCs) and alcoholdehydrogenases (ADHs; Atsumi et al. 2008b). In this work, we investigate the effect of various ADHs which convert isobutyraldehyde to isobutanol in the isobutanol biosynthesis pathway. ADHs are oxidoreductases that catalyze the reversible oxidation of alcohols to aldehydes or ketones, with the corresponding reduction of nicotinamide adenine dinucleotide (NAD) or NAD phosphate (NADP; Jornvall et al. 1987). There is a versatile set of ADHs with distinct structural differences, different substrate specificities, and different use of cofactors and metals (Jornvall et al. 1987).
In a previous experiment (Atsumi et al. 2008b), isobutyraldehyde was detected only in trace amounts with the overexpression of ADH2 from Saccharomyces cerevisiae, indicating sufficient activity of ADH for reducing isobutyraldehyde to isobutanol in the strain (JCL260/pSA55/pSA69; Atsumi et al. 2008b). However, E. coli has six genes (adhE, adhP, eutG, yiaY, yqhD, and yjgB) coding for various ADHs. It was unclear how much these ADHs contributed to the isobutanol production. In particular, YqhD has a preference for alcohols longer than C3, and it does not show detectable enzymatic activity when tested with short-chain alcohols as substrates (Sulzenbacher et al. 2004). However, its activity for isobutyraldehyde has not been reported. In this work, we first investigated whether ADHs of E. coli play a role in the isobutanol production (Perez et al. 2008). To improve the isobutanol production further, we also compared the activities of Adh2 from S. cerevisiae and AdhA from Lactococcus lactis for isobutanol production in E. coli. These ADHs along with YqhD from E. coli were purified and characterized in vitro and in vivo.
Materials and methods
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
Strains and plasmids used in this study
rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78
Datsenko and Wanner 2000
BW25113/F′ [traD36, proAB+, lacIq Z∆M15]
Atsumi et al. 2008a
Same as JCL16 but with ∆adhE,∆frdBC,∆fnr-ldhA,∆pta, ∆pflB
Atsumi et al. 2008a
Same as JCL260 but ∆yqhD
ColE1 ori; AmpR; PLlacO1: kivd-ADH2
Atsumi et al. 2008b
ColE1 ori; AmpR; PLlacO1: kivd-adhA (L. lactis)
p15A ori; KanR ; PLlacO1: alsS-ilvCD
Atsumi et al. 2008b
ColE1 ori; AmpR; PLlacO1: kivd
ColE1 ori; AmpR; PLlacO1: kivd-yqhD
Derivative of pETDuet-1 with yqhD
Derivative of pETDuet-1 with ADH2
Derivative of pETDuet-1 with adhA
Sequence 5′ → 3′
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 H3BO3, 1.81 g MnCl2⋅4H2O, 0.222 g ZnSO4⋅7H2O, 0.39 g Na2MoO4⋅2H2O, 0.079 g CuSO4⋅5H2O, 49.4 mg Co(NO3)2⋅6H2O 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.
Effects of E. coli ADHs on isobutanol production
Elimination of yqhD decreases isobutanol production
Among these ADHs, YqhD is known to have a preference for longer-chain substrates. YqhD is an NADPH/Zn2+-dependent enzyme (Sulzenbacher et al. 2004). To identify the effect of YqhD for isobutyraldehyde reductase activity, the yqhD gene was deleted from the genome. Isobutanol production with ∆yqhD decreased by 80% (Fig. 2a). Isobutyraldehyde was accumulated (Fig. 2b). These results indicate that YqhD contributed significantly to the isobutyraldehyde reductase activity in E. coli.
Overexpression of ADH2, adhA, and yqhD
To study the effects of overexpression of various ADHs, we cloned three adh genes on plasmids. First, overexpression of ADH2 from S. cerevisiae did not increase isobutanol production compared to the strain without overexpression (Fig. 2a). A deletion of the yqhD gene from this strain resulted in a decrease of isobutanol production to 3.8 g/L from 6.8 g/L and the accumulation of isobutyraldehyde (2.7 g/L; Fig. 2b). However, this was higher than the production (1.4 g/L) from the strain (∆yqhD) without overexpression of ADH2 (Fig. 2a). This result suggests that Adh2 has some activities toward isobutyraldehyde but is not as efficient as YqhD in E. coli.
Finally, we tested whether overexpression of yqhD could improve the isobutanol production. In our system, the yqhD gene was cloned into a high copy plasmid (∼70 copies; pSA138, Table 1). Isobutanol production was measured with or without overexpression of yqhD. Unexpectedly, both strains showed the similar productivity (Fig. 3b). To eliminate the possibility for no expression of yqhD from the plasmids, we tested isobutanol production using the strain with ∆yqhD on the genome. The deletion did not change isobutanol production (Fig. 3b), suggesting that YqhD was expressed well from plasmids. The results indicate that chromosomal yqhD expression has saturated the isobutanol production capability of the strain.
Characterization of ADHs
Aldehyde reductase activity in vitro
27.6 ± 12.4
1.1 ± 0.1
0.14 ± 0.02
6.6 ± 0.5
0.5 ± 0.2
10.0 ± 0.3
1.8 ± 1.3
1 ± 0.1
385.1 ± 30.7
0.9 ± 0.1
2.2 × 10−3
9.1 ± 2.9
6.6 ± 0.2
The last step of isobutanol production in E. coli was examined by employing various ADHs. We found that YqhD contributed significantly to the isobutyraldehyde reductase activity in E. coli. We further compared three enzymes for the last step and found that AdhA from L. lactis showed the highest isobutyraldehyde reductase activity. The in vitro enzyme assay showed that AdhA was NADH-dependent, consistent with the prediction based on sequence. AdhA contains a highly conserved sequence Gly–X–Gly–X–X–Gly (where X is any amino acid) in many NADH-binding domains (Scrutton et al. 1990). Adh2 from S. cerevisiae was highly prone to misfolding and aggregation in the heterologous host E. coli.
There is strong incentive to devise a synthetic pathway for the production of valuable metabolites in a user-friendly organism. In this regard, E. coli is a well-characterized microorganism with a set of readily available tools for genetic manipulation, and its physiological regulation is well-studied. However, the expression of heterologous proteins may result in inefficient translation and misfolding (Baneyx and Mujacic 2004). To achieve high productivity of the target products, it is desirable to seek enzymes that are compatible to the host. Nearly all of the overexpressed Adh2 was found in the insoluble fraction of the cell lysate, indicating that Adh2 is highly prone to misfolding and aggregation when expressed in E. coli. Although a number of strategies have been developed to improve the folding of a protein (Fisher et al. 2006; Fowler et al. 2005), the comparison of several enzymes from a broad range of possibilities allowed us to identify an ideal enzyme for our purpose.
We found YqhD and AdhA were active toward isobutyraldehyde, but Adh2 shows a high Kmisobutyraldehyde value. For YqhD, it is consistent with the previous report that YqhD has a preference for longer-chain substrates (Perez et al. 2008; Sulzenbacher et al. 2004). The crystal structure indicates that YqhD has an elongated shape of the active site (Sulzenbacher et al. 2004). The large size of the active site is in agreement with the enzyme's preference for long substrates. For AdhA, in the absence of actual crystallographic data, we cannot discuss the specific mechanisms responsible for the broad-range substrate binding, but it might have a large active site cavity. Possibilities still exist that alternative ADHs from other organisms might show better activity toward isobutyraldehyde in E. coli. The crystal structures of more than 100 ADHs have been elucidated (e.g., Schwarzenbacher et al. 2004; Valencia et al. 2004). It is possible to use the structural information to identify ADHs that have large active sites. The use of the structural data could facilitate such exploration to identify better enzymes.
This work was partially supported by the UCLA-DOE Institute for Genomics and Proteomics. T. W. is supported by a Graduate Students Study Abroad Program from a National Science Council of R.O.C (Taiwan) (NSC-096-2917-I-007-102).
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