Engineering Corynebacterium glutamicum for isobutanol production
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- Smith, K.M., Cho, K. & Liao, J.C. Appl Microbiol Biotechnol (2010) 87: 1045. doi:10.1007/s00253-010-2522-6
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The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.
The production of fuels from renewable materials has received increased attention due to the limited supply of fossil fuels and increasing environmental concerns. The annual production of ethanol in the USA reached 9.2 billion gallons in 2008 (Seiferlein 2009). Although ethanol production by fermentation has a long history and plays an important role for transition to bio-based fuels, it is not an ideal gasoline replacement due to its low energy density (∼70% of gasoline), high vapor pressure, and high hygroscopicity, making ethanol incompatible with the current infrastructure. In contrast, higher chain alcohols possess elevated energy densities and are compatible with the existing infrastructure. 1-Butanol production by fermentation was first accomplished with Clostridium acetobutylicum as a host about a century ago and has recently received significant attention (Borden and Papoutsakis 2007; Ezeji et al. 2004). In the past few years, the production of this alcohol and several other higher chain alcohols such as 1-propanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol have been demonstrated in Escherichia coli (Atsumi et al. 2008a, b; Atsumi and Liao 2008; Cann and Liao 2008; Connor and Liao 2008; Shen and Liao 2008). Isobutyraldehyde and isobutanol production by photosynthetic CO2 recycling has also been accomplished in Synechococcus elongatus (Atsumi et al. 2009b).
Corynebacterium glutamicum is a rapidly growing, gram-positive soil bacterium and has been the workhorse of industrial amino acid production (Leuchtenberger et al. 2005). The discovery of this organism’s ability to produce glutamate with high efficiency led to the development of amino acid production by fermentation. In 2005, C. glutamicum was used to produce 1.5 million tons of glutamate as monosodium glutamate per year as well as several thousand tons of other amino acids such as lysine, isoleucine, tryptophan, and threonine. These processes have been engineered extensively, with valine and lysine production titers reaching ∼100 g/L (Leuchtenberger et al. 2005). Because these 2-keto acid pathways for higher chain alcohol production share common precursors with amino acids, C. glutamicum shows potential for the production of higher chain alcohols. To our knowledge, this organism has never been used to synthesize any alcohols beyond ethanol (Inui et al. 2004).
Materials and methods
All restriction enzymes and antarctic phosphatase were purchased from New England Biolabs (Ipswich, MA, USA). The DNA ligation kit was supplied by Roche (Manheim, Germany). KOD DNA polymerase was purchased from EMD Chemicals (San Diego, CA, USA). Oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA, USA).
Strains and plasmids
Strains and plasmids used in this study
E. coli BW25113
rrnBT14 DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78
(Datsenko and Wanner 2000)
E. coli XL-1 Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZ∆M15 Tn10 (TetR)]
C. glutamicum ATCC 13032
Wild type (WT)
American Type Culture Collection
KanR; mobilizable (oriT, oriV); sacB; lacZα
(Schäfer et al. 1994)
P15A ori; CmR; PLtetO1::luc
(Lutz and Bujard 1997)
ColE1 ori; AmpR; PLlacO1::kivd-ADH2
(Atsumi et al. 2008b)
p15A ori; KanR; PLlacO1::alsS-ilvC-ilvD
(Atsumi et al. 2008b)
NG2 ori; KanR
(Messerotti et al. 1990)
NG2 ori; KanR; MCS
NG2 ori; KanR; cat
NG2 ori; KanR; Peftu::cat
NG2 ori; KanR; Peftu::alsS
NG2 ori; KanR; Peftu::alsS-ilvCD
NG2 ori; KanR; Peftu::alsS-ilvCD-kivd
NG2 ori; KanR; Peftu::
NG2 ori; KanR; Peftu::alsS-ilvCD-kivd-adhA
As pK19mobsacB but with aceE fragment
As pK19mobsacB but with ilvE fragment
As pK19mobsacB but with ldh fragment
As pK19mobsacB but with pgi fragment
As pK19mobsacB but with ppc fragment
As pK19mobsacB but with pyc fragment
Oligonucleotides used in this study
Medium and cultivation
E. coli cultures for plasmid construction and propagation were grown in Luria–Bertani (LB) media at 37°C and shaken at 250 rpm. For stepwise construction of the isobutanol pathway, the cultures were grown in 20-mL media CGIII (Menkel et al. 1989), but with 40 g/L glucose in a 250-mL screw-cap shake flask. Longer-term production cultures were done in 20 mL CGIII media but with 70 g/L glucose in a 250-mL screw-cap flask. All strains with an aceE knockout were cultured in the same production media but with 4.5 g/L acetate to allow for adequate cell growth. All C. glutamicum production cultures were inoculated to an initial OD600 nm of 0.1 from a 3-mL overnight culture grown in CGIII at 30°C in a rotary shaker (250 rpm). After inoculation, production cultures were shaken at 250 rpm and 30°C. The pH values of long-term production cultures were checked at every sampling time, and 10 M NaOH was added as needed to adjust to pH 6.8. Antibiotics were added appropriately (kanamycin 25 μg/mL).
Cultures of E. coli BW25113 were grown at 30°C and 37°C in LB with 2% glucose (LBG) in a 500-mL baffled shake flask at 250 rpm. C. glutamicum was grown in LBG and CGIII media at 30°C in a 500-mL baffled shake flask at 250 rpm. After an OD of 1.0 was reached, 5-mL aliquots were moved into test tubes, isobutanol was added at varied concentrations (0%, 1.0%, 1.25%, 1.5%, 1.75%, and 2.0%), and the cultures were incubated at 30°C and shaken at 250 rpm. After 4 h, each culture was diluted and spread onto LB plates for colony counting after 24 h of incubation at 30°C. Percent viability was calculated as the number of cells remaining viable after isobutanol exposure normalized with the cell number obtained with no isobutanol exposure.
For crude extract enzyme assays, 5-mL CGIII cultures of C. glutamicum harboring the relevant plasmids were grown overnight at 30°C in 5 mL CGIII media in test tubes. Crude extracts were prepared by concentrating the cultures by 5-fold in 0.1 M phosphate buffer (pH 7.1) and lysing them with 0.1 mM glass beads. The acetolactate synthase (ALS) assay was performed as previously reported (Holtzclaw and Chapman 1975). KDC activity was assayed by measuring the generation of isobutyraldehyde from 2-ketoisovalerate as previously described (Atsumi et al. 2009a). Reductive ADH activity was detected by measuring NADH oxidation at 340 nm in 1 mL of 50 mM phosphate buffer (pH 7.1), 100 mM isobutyraldehyde, 250 μM NADH, and 100 μL crude extract. Oxidative ADH activity was detected by measuring NAD+ reduction at 340 nm in 1 mL of 50 mM phosphate buffer (pH 7.1), 500 mM ethanol or isobutanol, 250 μM NAD+, and 100 μL crude extract. Total protein concentrations were measured by Bradford assay.
The alcohol compounds produced were quantified using gas chromatography equipped with flame ionization as previously described (Atsumi et al. 2008a). All other compounds were measured by applying filtered fermentation broth to an Agilent 1100 high-performance liquid chromatograph equipped with an autosampler (Agilent Technologies) and a Bio-Rad Aminex HPX87 column (5 mM H2SO4; 0.3 mL/min; column temperature, 35°C; Bio-Rad Laboratories, Hercules, CA, USA).
C. glutamicum exhibits better isobutanol tolerance than E. coli
Constructing the isobutanol production pathway
Specific activities of ALS, KDC, and adhA obtained by overexpression in C. glutamicum using Peftu
Specific activity (U mg−1)
6,700 ± 600
110 ± 9
2.3 ± 0.6
0.62 ± 0.06
0.09 ± 0.02
Isobutanol production with C. glutamicum
Strain improvement by gene knockout
Because we did not detect significant organic acid accumulation in WT, we hypothesized that the pyruvate dehydrogenase complex (PDHc) is a major consumer of pyruvate and constructed a ∆aceE strain deficient in PDHc activity. As shown in Fig. 4a, isobutanol production in the ∆aceE background was severely inhibited and only accumulated to 1.4 g/L isobutanol, while cell growth was similar to the other strains tested (Fig. 4e). Moreover, inactivation of the PDHc resulted in a significant accumulation of 8.3 g/L lactate (Fig. 4c), indicating that it is the major consumer of pyruvate. Next, we aimed to direct this accumulated lactate through the isobutanol pathway with the additional inactivation of LDH by constructing a ∆aceE∆ldh strain. The deletion of ldh in the ∆aceE host did direct the carbon flux away toward isobutanol during the first 24 h. Ultimately, however, this strain did not increase isobutanol production over WT (Fig. 4a) and the strain excreted 2.1 g/L acetate (Fig. 4d). This is perhaps due to the activity of pyruvate:quinone oxidoreductase that has been shown to compete for pyruvate to generate acetate (Blombach et al. 2008). At this point, we predicted that isobutanol production in ∆aceE strains might be inhibited due to an insufficient supply of NADPH that may be generated from the TCA cycle. To increase NADPH availability, pgi (encoding phosphoglucose isomerase, PGI) was knocked out to create a ∆aceE∆ldh∆pgi strain. However, isobutanol production with this strain was eliminated (Fig. 4a). Also, the glucose consumption rate of the ∆aceE∆ldh∆pgi strain was strongly inhibited (Fig. 4b), suggesting that PGI is an essential path for glucose metabolism toward isobutanol. Similar to previous studies regarding valine production in C. glutamicum (Blombach et al. 2008) and isobutanol production in E. coli (Atsumi et al. 2008b), we found that all strains produced isobutanol primarily in the stationary phase (Fig. 4e), and valine was never detected above concentrations present in fresh medium (data not shown).
We engineered C. glutamicum for isobutanol production and produced several other higher chain alcohols as byproducts (1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol), highlighting the capacity of this host for the construction of several different higher chain alcohol production platforms.
Despite the promising use of isobutanol as a fuel, its toxic effects on microbes may be an important factor limiting its production (Brynildsen and Liao 2009). Here, we found that C. glutamicum has an increased ability to tolerate isobutanol toxicity in comparison with E. coli at all concentrations and conditions tested (0–20 g/L isobutanol), showing that C. glutamicum has potential to be a higher alcohol producer as the tolerance of E. coli is similar to that of C. acetobutylicum (Atsumi et al. 2008a; Lin and Blaschek 1983), a natural 1-butanol producer. Corynebacteria possess a thick cell wall that helps protect the cell against external stresses such as freeze damage, pressure, and hyperosmotic shock (Marienfeld et al. 1997). Similar to mycobacteria, the cell wall of corynebacteria contains mycolic acids linked to polysaccharides which, in mycobacteria, form a second bilayer surrounding the cell wall and limiting its permeability (Eggeling and Sahm 2001). The higher isobutanol tolerance of C. glutamicum may be the result of these protective characteristics, and this trait may prove to be advantageous for higher chain alcohol production once alcohol titers reach toxic levels.
To begin construction of the isobutanol pathway, the promoter of eftu was chosen for expression of the isobutanol operon. This promoter has been shown to be effective for strong gene expression in C. glutamicum for the production of lysine (Becker et al. 2005). With this promoter, we were able to successfully express ALS of B. subtilis and KDC of L. lactis in C. glutamicum and obtain good activities. C. glutamicum also possesses native acetohydroxyacid synthase (AHAS) enzymes capable of generating 2-acetolactate, one of which has been used for valine production (Radmacher et al. 2002); however, ALS has been shown to be beneficial for isobutanol production in E. coli, and, unlike AHAS (encoded by ilvBN), ALS is not feedback-inhibited by valine (Elisakova et al. 2005; Holtzclaw and Chapman 1975), and it is more specific for acetolactate synthase activity.
Overexpression of alsS-ilvCD without kivd did not produce any isobutanol, indicating that C. glutamicum does not have an endogenous KDC with activity toward KIV. The additional overexpression of kivd and adhA resulted in improved production of isobutanol, and we also detected 1-propanol, 1-butanol, 2-phenylethanol, 2-methyl-1-butanol, and 3-methyl-1-butanol as byproducts. We therefore conclude that the overexpessed KDC is active in C. glutamicum and that this organism may be engineered to produce a wide array of higher alcohols.
Long-term fermentations with WT harboring pKS167 (Peftu::alsS-ilvCD-kivd-adhA) produced 4.0 g/L isobutanol in 96 h and gave a yield that is 19% of the theoretical maximum (isobutanol/glucose). WT produced isobutanol slowly for the first 24 h when the cells are growing, whereas 85% of the isobutanol was produced after the cells reach stationary phase. In an effort to improve isobutanol productivity, we targeted pathways that were potentially consuming several isobutanol precursors: phosphoenolpyruvate, pyruvate, and KIV. The inactivations of PPC and IlvE did not increase isobutanol production, and disruption of PYC only slightly improved the final titer. However, the absence of PYC resulted in the accumulation of 2.6 g/L lactic acid, indicating that PYC actively consumes pyruvate, which might explain why WT does not produce lactate. Our results also indicate that the pyruvate made available in the ∆pyc strain is more effectively consumed by LDH, outcompeting ALS of the isobutanol pathway. This is plausible, as the Km value of ALS for pyruvate is 13.6 mM (Atsumi et al. 2009a), whereas LDH has a lower Km of 7.4 mM (Dietrich et al. 2009), which may explain the increased efficacy of LDH for pyruvate consumption. Our results are in agreement with this argument, as the additional inactivation of the competitive LDH (∆pyc∆ldh) improved isobutanol production by ∼25% to 4.9 g/L isobutanol and increased the yield to 23% of the theoretical maximum.
Previous studies concerning valine production in C. glutamicum have shown that inactivation of the PDHc resulted in significant improvement of valine production and high product yield (Blombach et al. 2008; Blombach et al. 2007). In our system, we also found that inactivation of the PDHc increases pyruvate availability; however, LDH becomes the more dominant enzyme, and lactic acid becomes the primary product, similar to our results in the ∆pyc strain. It seemed that by constructing a ∆aceE∆ldh strain, we may convert this “available” pyruvate to isobutanol. Initially, this double knockout strain produced isobutanol well in comparison with WT but productivity slowed drastically after the cells entered the stationary phase. We hypothesized that TCA cycle activity might be important for isobutanol production in C. glutamicum, as this metabolic pathway supplies ATP and NAD(P)H. More specifically, NADPH has been shown to be a critical cofactor for the production of amino acids in C. glutamicum, including lysine and valine (Kabus et al. 2007; Marx et al. 2003), and in fact, the introduction of a pgi knockout in a strain deficient in PDHc activity has been shown to increase the valine yield from glucose (Blombach et al. 2008). Unfortunately, redirecting the carbon flow through the pentose phosphate pathway by disruption of PGI (∆ldh∆aceE∆pgi) eliminated isobutanol production and severely stunted glucose consumption. This is in contrast with results obtained for valine production (Blombach et al. 2008); however, unlike the valine pathway that ultimately consumes 2 mole of NADPH per mole of valine produced, here, the isobutanol pathway consumes 1 mole of NADPH and 1 mole of NADH per mole of isobutanol produced. This difference in redox cofactor requirement may be one reason for the ineffectiveness of a pgi knockout on isobutanol production. Also, several studies have shown that PGI mutants dramatically disturb NADPH metabolism and retard growth and glucose consumption in C. glutamicum (Marx et al. 2003).
Our initial attempts to produce isobutanol in C. glutamicum resulted in the maximum production of 4.9 g/L in a ∆pyc∆ldh background. This success emphasizes the efficacy of the 2-keto acid based pathways for higher alcohol production, and future studies should be aimed to increase the low isobutanol yield. There have been several cases of generating mutants of C. glutamicum that overproduce valine, as well as several other pertinent amino acids for higher alcohol production such as isoleucine and threonine, by applying random mutagenesis coupled with a selection for growth in the presence of an amino acid analogue (Shimura 1972a, b; Uemura et al. 1972). In addition, other metabolic engineering tools and experiences (Barkovich and Liao 2001; Liao and Delgado 1993) might prove to be useful to help elucidate the effects of the mutations introduced and to direct the metabolic flux to the desirable pathway(s). Perhaps this would be an effective method to develop isobutanol overproducers as valine and isobutanol share the same 2-keto acid precursor, KIV. As a first attempt at isobutanol production in C. glutamicum, our primary challenge was the effective expression of heterologous enzymes (KDC and ALS) that are important for isobutanol production. The success we have had with this more-tolerant host supports future studies to engineer several higher chain alcohol production platforms in C. glutamicum using the 2-keto acid pathways.
We are grateful to Margaret Chen for her experimental assistance and to Dr. Sinskey and Dr. Lessard (MIT–Department of Biology) for the generous donation of pEP2.
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