Bioresource Technology 174 (2014) 81–87
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Engineering Escherichia coli for fumaric acid production from glycerol Ning Li a,b, Bo Zhang a,b, Zhiwen Wang a,b, Ya-Jie Tang c, Tao Chen a,b,⇑, Xueming Zhao a,b a
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China c Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, PR China b
h i g h l i g h t s Evolved mutant E. coli E2 was engineered for fumaric acid production from glycerol. Deletion of fumarases resulted in fumaric acid accumulation from glycerol. Enhancing the anaplerotic pathways significantly improved fumaric acid production. High titer, yield, and productivity were achieved through fed-batch culture.
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Article history: Received 23 August 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 7 October 2014 Keywords: Fumaric acid Glycerol Escherichia coli Fed-batch culture Acetate accumulation
a b s t r a c t The evolved mutant Escherichia coli E2 previously developed for succinate production from glycerol was engineered in this study for fumaric acid production under aerobic conditions. Through deletion of three fumarases, 3.65 g/L fumaric acid was produced with the yield of 0.25 mol/mol glycerol and a large amount of acetate was accumulated as the main byproduct. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5 g/L fumaric acid from glycerol with 70% of the maximum theoretical yield and an overall productivity of 0.51 g/L/h. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fumaric acid is a dicarboxylic acid which is widely used in many industries such as food, pharmaceutical and chemical industries. It can be used as food and beverage additives, also as starting material for polymerization and esterification to produce paper resin, plasticizer, and polyester (Roa Engel et al., 2008). Nowadays, fumaric acid is mainly produced through petrochemical process. However, due to the environmental friendly nature, bio-based production of fumaric acid has been paying more and more attentions. Rhizopus oryzae and Rhizopus arrhizus have been mainly studied for fermentative production of fumaric acid and high product yields have been achieved (Xu et al., 2012b). However, due to the morphological characteristics of these Rhizopus species, ⇑ Corresponding author at: School of Chemical Engineering and Technology, Tianjin University, 92# Weijin Road, Nankai District, Tianjin 300072, PR China. Tel./fax: +86 22 27406770. E-mail address: [email protected] (T. Chen). http://dx.doi.org/10.1016/j.biortech.2014.09.147 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
industrialization of this process is limited. In addition, Saccharomyces cerevisiae has been engineered for fumaric acid production. Through overexpressing the endogenous pyruvate carboxylase and heterologous malate dehydrogenase and fumarase from R. oryzae in S. cerevisiae, up to 3.18 g/L fumaric acid was produced (Xu et al., 2012a). Furthermore, the simultaneous use of reductive and oxidative routes to produce fumaric acid was successful explored to improve product yield (Xu et al., 2013). Escherichia coli, which is the most characterized and best studied bacterium, was also engineered for fumaric acid production (Song et al., 2013). Under aerobic conditions, the engineered strain CWF812 produced 28.2 g/L fumaric acid with the yield of 0.389 g/g glucose. Glycerol, which is the major by-product of biodiesel, has become an inexpensive and extensive substrate for biochemical industry (Zhu et al., 2013). Many microorganisms can utilize glycerol as the sole carbon source, including Citrobacter freundii, Klebsiella pneumonia, Clostridium pasteurianum, Clostridium butyricum, Enterobacter aerogenes, and Lactobacillus reuteri (da Silva et al., 2009). In recent years, a number of chemicals and fuels have been
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studied for production from glycerol through microbial fermentation, such as 1,3-propanediol, biosurfactants, ethanol, butanol, organic acids, polyhydroxyalcanoates, and so on (Dobson et al., 2012). E. coli can utilize glycerol under aerobic conditions, however, it was always thought that external electron acceptor was required for the anaerobic fermentation of glycerol. Recently, the metabolic pathways for microaerobic and anaerobic utilization of glycerol without external electron acceptors by E. coli were reported (Durnin et al., 2009; Gonzalez et al., 2008). Based on these valuable information, E. coli was engineered to produce many chemicals and fuels from glycerol, such as ethanol, hydrogen, succinate, 1,2-propanediol, lactate and so on (Clomburg and Gonzalez, 2011; Kim et al., 2014; Mazumdar et al., 2010). Rhizopus species have been studied for their capability to produce fumaric acid from glycerol (Kordowska-Wiater et al., 2013; Zhou et al., 2014). However, fumaric acid production by E. coli with glycerol as the sole carbon source has not been reported. In our previous study, directed pathway evolution was employed to engineer E. coli for aerobic succinate production from glycerol (Li et al., 2013). Through deletion of phosphoenolpyruvate (PEP) carboxylase which catalyzed the main anaplerotic pathway and performing adaptive evolution in minimal medium with glycerol as the sole carbon source, the glyoxylate shunt was recruited as the alternative anaplerotic pathway and therefore the flux of this route was enhanced. As a result, succinate production was increased and the main byproduct a-ketoglutarate was reduced. Due to similar pathways for production of succinate and fumaric acid under aerobic conditions, this system might also be suitable for fumaric acid production. Therefore, in this study, the evolved strain E. coli E2 was employed and engineered for fumaric acid production from glycerol. Through increasing the flux of the anaplerotic pathways, the main byproduct acetate was significantly reduced and fumaric acid production was improved. In fed-batch fermentation, high titer and yield of fumaric acid were achieved using glycerol as carbon source and corn steep liquor powder as nitrogen source. 2. Methods 2.1. Strains and plasmids Strains and plasmids used in this work are listed in Table 1. The evolved strain E. coli E2 (Li et al., 2013) was engineered in this study for fumaric acid production from glycerol. E. coli DH5a was used for gene cloning. Luria–Bertani (LB) broth and LB plates were used for strain development. Ampicillin (50 lg/mL), kanamycin (24 lg/mL), and chloramphenicol (4 lg/mL) were added as appropriate. Plasmid pSCTrc and pACYCTrc were constructed in this study for gene overexpression. For pSCTrc construction, DNA fragment amplified from plasmid p5CS with primers PSC1 and PSC2 and DNA fragment amplified from plasmid pTrc99a with primers PSC3 and PSC4 were assembled using CEPC method (Quan and Tian, 2009). For pACYCTrc construction, plasmids pTrc99a and pACYC184 were both digested by BspHI and EcoRV, and then the smaller fragment of pTrc99a and the larger fragment of pACYC184 were ligated by T4 DNA ligase (Fermentas, Thermo Scientific). The primers used in this study are listed in Table S1. Plasmids pSCyodC, pSCppc, pSCBA were constructed by PCR amplifying genes yodC, ppc, aceBA from the genome DNA of Bacillus subtilis (yodC) and E. coli BL21(DE3) (ppc, aceBA) with the respective primers (Table S1) and cloning the acquired DNA fragments into plasmid pSCTrc with respective restriction enzymes (Table S1). Plasmid p15yodC was constructed by cloning gene yodC into plasmid pACYCTrc with the same restriction enzymes as pSCyodC construction. Plasmid pSCBAppc was constructed by cloning gene ppc from E. coli BL21(DE3) into plasmid pSCBA.
2.2. Genetic methods Genes fumB, fumAC and aspA deletion were performed following the method described previously (Datsenko and Wanner, 2000). Promoter replacement of sucAB by trc promoter was carried out as previously reported (Li et al., 2013). For the recovery of gene ppc in strain E. coli EF00, DNA fragment amplified from the genome DNA of strain E2-Dsdh-ppc (Li et al., 2013) with primers Pout5/ Pout6 was electroporated into E. coli EF00 harboring pKD46. For arcA insertional mutation, the coding region of gene arcA was divided into two parts at site 513 which were amplified separately and ligated into pUC18 in sequence to yield plasmid pUCarcA. The tetracycline resistant gene amplified from pACYC184 was then inserted into pUCarcA to yield plasmid pUCarc-tet. This plasmid was then used as the template for amplification of DNA fragment which was needed for chromosome integration. 2.3. Medium and cultivation conditions M9 minimal medium (Li et al., 2013) supplemented with glycerol was used in this study for fumaric acid production. 50 lg/mL ampicillin was supplemented for plasmid maintenance and 0.5 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) was added in the shake flask culture at the beginning of fermentation to induce trc promoter. For flask cultures, 250 lL overnight cultures in 15 mL tubes prepared from single colonies were used to inoculate 25 mL M9 + glycerol medium in 250 mL flasks, and incubated at 37 °C and 220 rpm until OD600 reached about 1.0–2.0. IPTG was added in these cultures to induce the expression of gene ppc and aceBA which were required for growth in minimal medium. Then, 500 lL cultures were transferred into 50 mL M9 + glycerol medium in 500 mL flasks. For fumaric acid production, the cultures were incubated at 37 °C and 220 rpm till glycerol was exhausted. All the fermentation experiments were performed in triplicate. Fed-batch fermentation was carried out in a 1.3 L reactor (New Brunswick Scientific BioFlo 110, USA). M9 medium supplemented with about 50 g/L glycerol, 20 g/L corn steep liquor powder, 50 lg/mL ampicillin, and 0.5 mM IPTG was used. Prior to fermentation, overnight culture of strain EF02(pSCppc) in 15 mL tube prepared from single colony was used to inoculate 50 mL M9 medium with 10 g/L glycerol in 500 mL flask, and incubated at 37 °C and 220 rpm until OD600 of 1.0–2.0. All the culture in flask was then transferred into 500 mL medium in the reactor. For providing aerobic environment, sterile air was aerated at 0.5 L/min and the agitation speed was adjusted between 500 and 800 rpm to maintain the dissolved oxygen above 30% of air saturation. The fermentation was operated at 37 °C. In addition, 30% ammonium hydroxide and 1 M sulfuric acid were added automatically to control the pH at 7.0. The glycerol concentration of the feeding solution was 1000 g/L, and 25 mL feeding solution was supplemented when the glycerol concentration of the culture broth decreased below 20 g/L. 2.4. Analytical methods Cell growth was monitored by measuring the optical density at 600 nm (OD600) with a UV–vis spectrophotometer. The fermentation broth was centrifuged at 13,000 rpm for 5 min and the supernatant was used for metabolite analysis through HPLC which was equipped with a cation exchange column (Aminex HPX 87-H, Bio-Rad, Richmond, USA). Glycerol, fumaric acid and acetate were analyzed by an refractive index (RI) detector (Agilent, HP1047A), while a-ketoglutarate was examined by an UV absorbance detector (Agilent, G1315D). The mobile phase was 5 mM H2SO4 at 0.4 mL/ min, and the column was operated at 65 °C. The intracellular NADH
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N. Li et al. / Bioresource Technology 174 (2014) 81–87 Table 1 Escherichia coli strains and plasmids used in this study. Strains/plasmids
Relevant characteristics
Reference/source
Strains DH5a BL21(DE3) E2 EF00 EF01 EF01(pSCyodC) EF01S(pSCyodC) EF01S(p15yodC) EF01(arcA) EF01(pSCppc) EF01(pSCBA) EF01(pSCBAppc) EF02(pSCppc)
Cloning host F’ ompT gal dcm lon hsdSB(rB- mB-) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Evolved E1 E2, DfumB, DfumAC E2, DfumB, DfumAC, ppc:trc EF01 harboring pSCyodC EF01, sucA:trc, harboring pSCyodC EF01, sucA:trc, harboring p15yodC EF01, arcA:tet EF01 harboring pSCppc EF01 harboring pSCBA EF01 harboring pSCBAppc EF01, DaspA, harboring pSCppc
Lab collection Novagen Li et al. (2013) This study This study This study This study This study This study This study This study This study This study
Plasmids pUC18 pTrc99a pACYC184 p5CS pKD3 pKD4 pCP20 pKD46 pUCarcA pUCarc-tet pSCTrc pACYCTrc pSCyodC p15yodC pSCppc pSCBA pSCBAppc
bla, pBR322 ori bla, pBR322 ori, trc promoter, lacIq cat, tet, p15A ori bla, pSC101 ori bla, FRT-cat-FRT bla, FRT-kan-FRT bla, cat, yeast FLP recombinase bla, k-Red recombinase under araBAD promoter, temperature-conditional replicon Two parts of arcA coding region cloned in pUC18 Two parts of arcA coding region and tet cloned in pUC18 bla, pSC101 ori, trc promoter cat, p15A ori, trc promoter yodC gene from B. subtilis cloned in pSCTrc yodC gene from B. subtilis cloned in pACYCTrc ppc gene from E. coli BL21(DE3) cloned in pSCTrc aceBA gene from E. coli BL21(DE3) cloned in pSCTrc ppc gene and aceBA from E. coli BL21(DE3) cloned in pSCTrc
Lab collection Invitrogen Chang and Cohen (1978) Lab collection Datsenko and Wanner (2000) Datsenko and Wanner (2000) Cherepanov and Wackernagel (1995) Datsenko and Wanner (2000) This study This study This study This study This study This study This study This study This study
and NAD+ concentrations were examined according to the method described previously (Fu et al., 2014). 2.5. Enzyme assays Cells were grown in 500 mL flasks containing M9 medium supplemented with 10 g/L glycerol and 0.5 mM IPTG to the midexponential phase, and were harvested by centrifugation at 8000g for 10 min. After washed twice in 50 mM potassium phosphate buffer (pH 7.0), cells were resuspended in the same buffer and broken to prepare crude extract according to the method described previously (Li et al., 2013). The activity of NADH oxidase was assayed by monitoring the decrease of absorbance of NADH at 340 nm (Reusch and Burger, 1974). The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.07 mM NADH, 0.1 mM FAD. PEP carboxylase and isocitrate lyase were measured as previously reported (Chell et al., 1978; Terada et al., 1991). 2.6. Real time quantitative PCR (RT-qPCR) For total RNA isolation, cells were grown in M9 minimal medium supplemented with 10 g/L glycerol and harvested at the mid-exponential phase. Total RNA was extracted with RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. RT-PCR was performed as described previously (Li et al., 2013). 3. Results and discussion 3.1. Fumarases deletion for accumulation of fumaric acid The evolved mutant E. coli E2 was previously developed for succinate production from glycerol (Li et al., 2013). In this strain, the
glyoxylate shunt was recruited as the primary anaplerotic pathway by employing laboratory adaptive evolution of a ppc deletion strain. Due to the enhanced glyoxylate shunt, succinate yield was improved along with the decreased a-ketoglutarate accumulation. Under aerobic conditions, the pathway for fumaric acid production was almost the same as that of succinate, which suggested that strain E. coli E2 was also suitable for fumaric acid production. Being a part of the TCA cycle, succinate was converted to fumarate by succinate dehydrogenase, and fumarate was then transformed to malate through fumarase (Fig. S1). Therefore, for fumaric acid production, elimination of fumarase activities would be the first step. There are three fumarases in E. coli, encoded by fumA, fumC, and fumB respectively (Tseng et al., 2001). Fumarase C and A were mainly functional under aerobic and microaerobic conditions, whereas fumarase B was the dominant enzyme under anaerobic conditions. It has been reported that fumaric acid was not accumulated in fumAC deletion mutant, whereas simultaneously deletion of fumAC and fumB resulted in fumaric acid production (Song et al., 2013). Therefore, all three fumarases were deleted in E. coli E2 in this work. Similar to the phenomenon resulted from deletion of succinate dehydrogenase in our previous study (Li et al., 2013), deletion of fumarases also abolished the capacity of the resulting strain E. coli EF00 to grow in glycerol minimal medium due to the lack of oxaloacetate required for synthesis of cellular constituents. Therefore, in the same way, ppc was recovered in E. coli EF00 to yield strain EF01. Under aerobic conditions, strain EF01 produced 3.65 g/L fumaric acid from about 11 g/L glycerol in 36 h. In addition, acetate was accumulated to 2.75 g/L as the main byproduct unexpectedly. In our previous study for succinate production, little acetate was produced and it was consumed at the end of fermentation (Li et al., 2013). Moreover, in contrast to the fact that a-ketoglutarate was the main byproduct for succinate production, here, for production of fumaric acid, only
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a small amount of a-ketoglutarate was accumulated. These phenomena were surprising and interesting in view of the almost identical pathway for these two products. 3.2. Improving fumaric acid production by reducing acetate The production of acetate wasted carbon and reduced fumaric acid yield. In order to improve fumaric acid production, decreasing acetate might probably be effective. Therefore, several strategies were attempted to reducing acetate accumulation. 3.2.1. Expression of NADH oxidase from B. subtilis Acetate accumulation was previously reported to strongly correlated to cellular NADH/NAD ratio (redox ratio), and it could be largely decreased through expression of Streptococcus pneumoniae NADH oxidase which could reduce the cellular redox ratio (Vemuri et al., 2006). In comparison to succinate production, one more step catalyzed by succinate dehydrogenase is needed for fumaric acid production. This reaction will transfer electrons to ubiquinone for entry into the electron transport chain while oxidizing succinate to fumarate, which might increase cellular redox ratio and result in acetate accumulation. Therefore, in order to reduce acetate production, NADH oxidase encoded by gene yodC from B. subtilis (Zhang et al., 2014) was exogenously expressed in strain EF01 on a low-copy-number plasmid pSCTrc. The resulting strain EF01(pSCyodC) was examined for fumaric acid production under aerobic conditions from glycerol. As can be seen from Fig. 1, the amount of acetate was successfully reduced by nearly 80% through expressing NADH oxidase. However, fumaric acid production was also reduced by 13% for strain EF01(pSCyodC) as compared to strain EF01. At the same time, a-ketoglutarate was increased to 1.32 g/L. From these results, it could be seen that although carbon flux was directed down into the TCA cycle from the node acetate, it was blocked again at another node a-ketoglutarate. Through expression of NADH oxidase, the product pattern of strain EF01(pSCyodC) was now much more similar to that of succinate producing strain E2-Dsdh-ppc (Li et al., 2013). In our previously study, a-ketoglutarate dehydrogenase was overexpressed, which significantly
decreased a-ketoglutarate production (Li et al., 2013). Therefore, in this study, we also attempted to direct carbon flux from a-ketoglutarate to fumaric acid through overexpressing a-ketoglutarate dehydrogenase. The native promoter of gene sucAB was replaced with trc promoter in strain EF01(pSCyodC). However, the resulting strain EF01S(pSCyodC) accumulated large amount of acetate again, which was the same as strain EF01 (Fig. 1). In addition, fumaric acid and a-ketoglutarate production also changed back to the level of strain EF01. The reaction catalyzed by a-ketoglutarate dehydrogenase oxidizes a-ketoglutarate to succinyl-CoA with reduction of NAD to NADH. Therefore, overexpression of a-ketoglutarate dehydrogenase might result in increasing cellular NADH/NAD ratio which might be the reason for the high level of acetate for strain EF01S(pSCyodC). In order to verify this hypothesis, the expression level of NADH oxidase was further improved by replacing pSCyodC with p15yodC, the copy number of which was higher than that of pSCyodC. The specific activity of NADH oxidase in the resulting strain EF01S(p15yodC) (0.175 ± 0.007 lmol/min/mg protein) was almost 3-fold higher than that of strain EF01S(pSCyodC) (0.057 ± 0.008 lmol/min/mg protein). However, the fermentation results (Fig. 1) showed that acetate was not reduced and fumaric acid was not increased in strain EF01S(p15yodC). NADH/NAD ratios in these strains were examined and a NADH/NAD decrease from 0.32 ± 0.026 in strain EF01S(pSCyodC) to 0.23 ± 0.019 in strain EF01S(p15yodC) was observed. This suggested that the redox ratio might not be the reason for acetate production in strain EF01S(pSCyodC) overexpressing a-ketoglutarate dehydrogenase. Although acetate accumulation was reduced through expression of NADH oxidase, the saving carbon could not be converted to fumaric acid. Therefore, other strategies were attempted to reduce acetate production. 3.2.2. ArcA mutation The accumulation of acetate at the acetyl-CoA node might indicate the decreased flux of the TCA cycle in strain EF01. Therefore, the transcriptional levels of the TCA cycle enzymes of strain E2-Dsdh-ppc (Li et al., 2013) developed for succinate production and strain EF01 were examined and compared. As expected, the
Fig. 1. Fumaric acid and byproduct production, cell growth and fumaric acid yield for different strains. EF01, fumarases deleting strain; EF01(pSCyodC), overexpressing yodC from Bacillus subtilis; EF01S(pSCyodC), overexpressing yodC and sucAB; EF01S(p15yodC), overexpressing yodC on a plasmid with higher copy number and overexpressing sucAB; EF01(arcA), mutating gene arcA. All the strains were cultured in M9 minimal medium with about 11 g/L glycerol in 500 mL flasks at 37 °C and 220 rpm.
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Table 2 Activities of PEP carboxylase and isocitrate lyase expressed from relevant plasmids. Strain
PEP carboxylase activity (lmol/min/mg protein)
Isocitrate lyase activity (lmol/min/mg protein)
EF01 EF01(pSCppc) EF01(pSCBA)
0.169 ± 0.030 0.356 ± 0.071 –
0.343 ± 0.030 – 0.694 ± 0.067
levels of enzymes involved in the TCA cycle was not the main reason for acetate accumulation.
Fig. 2. The transcriptional levels of genes involved in the TCA cycle for strains E2Dsdh-ppc (succinate producing strain) and EF01 (fumaric acid producing strain). The values of strain EF01were all normalized to that of strain E2-Dsdh-ppc.
transcriptional levels of all genes investigated in strain E2-Dsdh-ppc were higher than strain EF01 (Fig. 2). Therefore, we attempted to reduce acetate by increasing the transcriptional levels of enzymes involved in the TCA cycle. ArcAB two-component regulatory system, which consists of ArcB sensor kinase and ArcA response regulator, regulates the expression of hundreds of genes in E. coli in response to respiratory conditions (Salmon et al., 2005). It has been reported that deletion of arcA and arcB could further activate the TCA cycle under aerobic conditions (Nizam et al., 2009). In addition, acetate accumulation could be reduced in arcA deletion mutants (Peebo et al., 2014; Vemuri et al., 2006). Therefore, in order to derepress the TCA cycle and reduce acetate production, gene arcA was mutated by insertion of the tetracycline resistant gene in the coding region as previously reported (Nikel et al., 2006). However, the resulting strain EF01(arcA) produced similar acetate and less fumaric acid than strain EF01 (Fig. 1). This phenomenon was not in accordance with the results previously reported (Peebo et al., 2014; Vemuri et al., 2006). The reason for this result was not clear. This suggested that the transcriptional
3.2.3. Increasing the flux of the anaplerotic pathways Oxaloacetate is a very important intermediate which is not only a part of the TCA cycle but also the precursor of cellular components. In addition, the velocity of citrate synthase to some extent depends on the concentration of oxaloacetate which is one of the substrates of citrate synthase (Robinson et al., 1983). To explore whether oxaloacetate was insufficient which might limit the flux of the TCA cycle and result in acetate accumulation, the fluxes of the anaplerotic pathways would be enhanced to increase the oxaloacetate pool. The reaction catalyzed by PEP carboxylase and the glyoxylate cycle were both manipulated by overexpression of ppc and aceBA in plasmid pSCTrc respectively. The specific activities of PEP carboxylase and isocitrate lyase were both increased about twofold (Table 2). Fumaric acid production of the two resulting strains EF01(pSCppc) and EF01(pSCBA) was examined under aerobic conditions. As shown in Fig. 3, acetate was reduced by more than 80% and fumaric acid was increased by 26.5% for strain EF01(pSCppc) as compared to the control strain EF01. Similar effects were also exerted by overexpression of the glyoxylate cycle operon. Although acetate was not so much reduced in strain EF01(pSCBA), fumaric acid was improved with almost the same degree as strain EF01(pSCppc). Fumaric acid yields from glycerol for these strains were 63% and 59% of the maximum theoretical yield respectively. However, a-ketoglutarate was largely accumulated to 1.37 g/L in strain EF01(pSCppc), while that of strain EF01(pSCBA) was a little increased. This difference might probably be resulted from the fact that the glyoxylate shunt could redirect carbon flow away from the oxidative arm of the TCA cycle and thus decrease a-ketoglutarate accumulation.
Fig. 3. Effects of overexpression of ppc and aceBA on products concentration, cell growth and fumaric acid yield. EF01(pSCppc), overexpressing gene ppc; EF01(pSCBA), overexpressing aceBA; EF01(pSCBAppc), overexpressing ppc and aceBA. All the strains were cultured in M9 minimal medium with about 11 g/L glycerol in 500 mL flasks at 37 °C and 220 rpm.
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In addition, biomass yield was increased though overexpression of the glyoxylate cycle operon, whereas overexpression of PEP carboxylase had no effect on biomass yield (Fig. 3). However, cell growth of strain EF01(pSCBA) was severely retarded, while that of strain EF01(pSCppc) was not affected as compared to EF01. The specific growth rate of strain EF01(pSCBA) (0.102 h 1) was much lower than that of strain EF01 (0.261 h 1) and strain EF01(pSCppc) (0.254 h 1). Given that fumaric acid production could be improved through overexpression of either PEP carboxylase or the glyoxylate shunt operon, they were coexpressed in strain EF01 through inserting ppc gene on plasmid pSCBA for further improving fumaric acid production. The results (Fig. 3) showed that acetate was reduced by 56% for the resulting strain EF01(pSCBAppc) as compared to EF01, which was higher than that of EF01(pSCBA) and lower than that of strain EF01(pSCppc). The amount of a-ketoglutarate produced by strain EF01(pSCBAppc) was as low as strain EF01(pSCBA). Fumaric acid yield was just increased a little as compared with strain EF01(pSCppc) and achieved 67% of the maximum theoretical yield. Biomass yield was not that high as strain EF01(pSCBA) and cell growth was faster with the specific growth rate of 0.15 h 1.
Fig. 4. Fed-batch fermentation of strain EF02(pSCppc) for fumaric acid production under aerobic conditions in the M9 medium supplemented with 20 g/L corn steep liquor powder. Lower triangle, OD600; square, glycerol; circle, fumaric acid; upper triangle, acetate.
3.3. Fed-batch production for fumaric acid from glycerol Although strain EF01(pSCppc) produced a little less fumaric acid than strain EF01(pSCBAppc), cell growth of the former was much faster than that of the latter. Therefore, strain EF01(pSCppc) was further explored for fumaric acid production performance in fed-batch culture. However, when cultured in 1.3 L bioreactor containing M9 minimal medium supplemented with glycerol, fumaric acid was initially produced and gradually consumed at the late stage of fermentation (data not shown). This phenomenon was not observed in the shake flask culture. Due to the fact that the conditions between flask culture and bioreactor culture were not completely consistent, some particular pathways might be triggered in bioreactor culture. It was reported that aspartase (encoded by aspA) reversibly converted fumarate to aspartate (Rudolph and Fromm, 1971). Therefore, aspA was deleted in strain EF01(pSCppc) to figure out whether fumaric acid was consumed through this reaction. As expected, fumaric acid was not consumed anymore for the resulting strain EF02(pSCppc) cultured in bioreactor (Fig. 4). For better cell growth, 20 g/L corn steep liquor powder, which was much cheaper than peptone and yeast extract, was supplemented in M9 minimal medium as nitrogen source in 1.3 L fermenter. At the end of fed-batch culture, 94 g/L glycerol was consumed and 41.5 g/L fumaric acid was produced by strain EF02(pSCppc) in 82 h (Fig. 4). The main byproduct acetate was accumulated to 8.7 g/L. The yields of fumaric acid and acetate were 0.44 g/g glycerol and 0.09 g/g glycerol respectively, both of which were higher than those of the flask culture (0.398 and 0.04 g/g respectively). Fumaric acid yield was approximate 70% of the maximum theoretical yield. Cell growth was rapid and reached the maximum OD600 of 66 in 40 h. The maximum and average glycerol consumption rates were 2.31 and 1.15 g/L/h, respectively. And the maximum and average fumaric acid productivities were 1.14 and 0.51 g/L/h. To the best of our knowledge, the titer, yield and productivity of fumaric acid achieved in this study are all the highest values described in literature for fumaric acid production by engineered E. coli. As compared to succinate production in our previous work (Li et al., 2013), production of fumaric acid from glycerol by the engineered E. coli here achieved higher yield in fed-batch fermentation (70% and 57% of the maximum theoretical yield for fumaric acid and succinate respectively). However, the average productivity of fumaric acid was lower than that of succinate (0.51 and
0.77 g/L/h respectively), which was mainly resulted from the longer fermentation time for fumaric acid production. In addition, the average glycerol consumption rate of fumaric acid production process (1.15 g/L/h) was much lower than that of succinate production (2.1 g/L/h). These results indicated different cell states of the two strains which might be partly attributed to the toxicity of fumaric acid on cell growth (Song et al., 2013). However, the fact that only one reaction difference resulted in great metabolic changes prompted us to perform further work and explore the underlying influence. Currently, the microbial fermentation process was less favorable economically than the petrochemical process due to the lower product yield of the former. However, if the raw material price would be lower it could compensate the low yield of the fermentation process (Roa Engel et al., 2008). Crude glycerol, which was much cheaper than glycerol and glucose, was one of the candidates. Here, the results demonstrated the promising potential of E. coli for fumaric acid production from glycerol, which provided vital platform for directly utilizing crude glycerol for production of fumaric acid. 4. Conclusions The evolved strain E. coli E2 with enhanced flux of the glyoxylate cycle was engineered in this study for fumaric acid production from glycerol under aerobic conditions. Deletion of fumarases resulted in accumulation of fumaric acid with the yield of 0.25 mol/mol glycerol and acetate was produced as the main byproduct. Overexpression of PEP carboxylase or the glyoxylate shunt operon effectively increased fumaric acid production and reduced acetate accumulation. In fed-batch culture, 41.5 g/L fumaric acid was produced from glycerol with approximate 70% of the maximum theoretical yield and an overall productivity of 0.51 g/ L/h. Acknowledgements This work was supported by National 973 Project (2011CBA00804, 2012CB725203); National Natural Science Foundation of China (NSFC-21176182, NSFC-21206112, NSFC21390201); National High-tech R&D Program of China (2012AA02A702, 2012AA022103).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Engineering Escherichia coli for fumaric acid production from glycerol Ning Li a,b, Bo Zhang a,b, Zhiwen Wang a,b, Ya-Jie Tang c, Tao Chen a,b,⇑, Xueming Zhao a,b a
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China c Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, PR China b
h i g h l i g h t s Evolved mutant E. coli E2 was engineered for fumaric acid production from glycerol. Deletion of fumarases resulted in fumaric acid accumulation from glycerol. Enhancing the anaplerotic pathways significantly improved fumaric acid production. High titer, yield, and productivity were achieved through fed-batch culture.
a r t i c l e
i n f o
Article history: Received 23 August 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 7 October 2014 Keywords: Fumaric acid Glycerol Escherichia coli Fed-batch culture Acetate accumulation
a b s t r a c t The evolved mutant Escherichia coli E2 previously developed for succinate production from glycerol was engineered in this study for fumaric acid production under aerobic conditions. Through deletion of three fumarases, 3.65 g/L fumaric acid was produced with the yield of 0.25 mol/mol glycerol and a large amount of acetate was accumulated as the main byproduct. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5 g/L fumaric acid from glycerol with 70% of the maximum theoretical yield and an overall productivity of 0.51 g/L/h. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fumaric acid is a dicarboxylic acid which is widely used in many industries such as food, pharmaceutical and chemical industries. It can be used as food and beverage additives, also as starting material for polymerization and esterification to produce paper resin, plasticizer, and polyester (Roa Engel et al., 2008). Nowadays, fumaric acid is mainly produced through petrochemical process. However, due to the environmental friendly nature, bio-based production of fumaric acid has been paying more and more attentions. Rhizopus oryzae and Rhizopus arrhizus have been mainly studied for fermentative production of fumaric acid and high product yields have been achieved (Xu et al., 2012b). However, due to the morphological characteristics of these Rhizopus species, ⇑ Corresponding author at: School of Chemical Engineering and Technology, Tianjin University, 92# Weijin Road, Nankai District, Tianjin 300072, PR China. Tel./fax: +86 22 27406770. E-mail address: [email protected] (T. Chen). http://dx.doi.org/10.1016/j.biortech.2014.09.147 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
industrialization of this process is limited. In addition, Saccharomyces cerevisiae has been engineered for fumaric acid production. Through overexpressing the endogenous pyruvate carboxylase and heterologous malate dehydrogenase and fumarase from R. oryzae in S. cerevisiae, up to 3.18 g/L fumaric acid was produced (Xu et al., 2012a). Furthermore, the simultaneous use of reductive and oxidative routes to produce fumaric acid was successful explored to improve product yield (Xu et al., 2013). Escherichia coli, which is the most characterized and best studied bacterium, was also engineered for fumaric acid production (Song et al., 2013). Under aerobic conditions, the engineered strain CWF812 produced 28.2 g/L fumaric acid with the yield of 0.389 g/g glucose. Glycerol, which is the major by-product of biodiesel, has become an inexpensive and extensive substrate for biochemical industry (Zhu et al., 2013). Many microorganisms can utilize glycerol as the sole carbon source, including Citrobacter freundii, Klebsiella pneumonia, Clostridium pasteurianum, Clostridium butyricum, Enterobacter aerogenes, and Lactobacillus reuteri (da Silva et al., 2009). In recent years, a number of chemicals and fuels have been
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studied for production from glycerol through microbial fermentation, such as 1,3-propanediol, biosurfactants, ethanol, butanol, organic acids, polyhydroxyalcanoates, and so on (Dobson et al., 2012). E. coli can utilize glycerol under aerobic conditions, however, it was always thought that external electron acceptor was required for the anaerobic fermentation of glycerol. Recently, the metabolic pathways for microaerobic and anaerobic utilization of glycerol without external electron acceptors by E. coli were reported (Durnin et al., 2009; Gonzalez et al., 2008). Based on these valuable information, E. coli was engineered to produce many chemicals and fuels from glycerol, such as ethanol, hydrogen, succinate, 1,2-propanediol, lactate and so on (Clomburg and Gonzalez, 2011; Kim et al., 2014; Mazumdar et al., 2010). Rhizopus species have been studied for their capability to produce fumaric acid from glycerol (Kordowska-Wiater et al., 2013; Zhou et al., 2014). However, fumaric acid production by E. coli with glycerol as the sole carbon source has not been reported. In our previous study, directed pathway evolution was employed to engineer E. coli for aerobic succinate production from glycerol (Li et al., 2013). Through deletion of phosphoenolpyruvate (PEP) carboxylase which catalyzed the main anaplerotic pathway and performing adaptive evolution in minimal medium with glycerol as the sole carbon source, the glyoxylate shunt was recruited as the alternative anaplerotic pathway and therefore the flux of this route was enhanced. As a result, succinate production was increased and the main byproduct a-ketoglutarate was reduced. Due to similar pathways for production of succinate and fumaric acid under aerobic conditions, this system might also be suitable for fumaric acid production. Therefore, in this study, the evolved strain E. coli E2 was employed and engineered for fumaric acid production from glycerol. Through increasing the flux of the anaplerotic pathways, the main byproduct acetate was significantly reduced and fumaric acid production was improved. In fed-batch fermentation, high titer and yield of fumaric acid were achieved using glycerol as carbon source and corn steep liquor powder as nitrogen source. 2. Methods 2.1. Strains and plasmids Strains and plasmids used in this work are listed in Table 1. The evolved strain E. coli E2 (Li et al., 2013) was engineered in this study for fumaric acid production from glycerol. E. coli DH5a was used for gene cloning. Luria–Bertani (LB) broth and LB plates were used for strain development. Ampicillin (50 lg/mL), kanamycin (24 lg/mL), and chloramphenicol (4 lg/mL) were added as appropriate. Plasmid pSCTrc and pACYCTrc were constructed in this study for gene overexpression. For pSCTrc construction, DNA fragment amplified from plasmid p5CS with primers PSC1 and PSC2 and DNA fragment amplified from plasmid pTrc99a with primers PSC3 and PSC4 were assembled using CEPC method (Quan and Tian, 2009). For pACYCTrc construction, plasmids pTrc99a and pACYC184 were both digested by BspHI and EcoRV, and then the smaller fragment of pTrc99a and the larger fragment of pACYC184 were ligated by T4 DNA ligase (Fermentas, Thermo Scientific). The primers used in this study are listed in Table S1. Plasmids pSCyodC, pSCppc, pSCBA were constructed by PCR amplifying genes yodC, ppc, aceBA from the genome DNA of Bacillus subtilis (yodC) and E. coli BL21(DE3) (ppc, aceBA) with the respective primers (Table S1) and cloning the acquired DNA fragments into plasmid pSCTrc with respective restriction enzymes (Table S1). Plasmid p15yodC was constructed by cloning gene yodC into plasmid pACYCTrc with the same restriction enzymes as pSCyodC construction. Plasmid pSCBAppc was constructed by cloning gene ppc from E. coli BL21(DE3) into plasmid pSCBA.
2.2. Genetic methods Genes fumB, fumAC and aspA deletion were performed following the method described previously (Datsenko and Wanner, 2000). Promoter replacement of sucAB by trc promoter was carried out as previously reported (Li et al., 2013). For the recovery of gene ppc in strain E. coli EF00, DNA fragment amplified from the genome DNA of strain E2-Dsdh-ppc (Li et al., 2013) with primers Pout5/ Pout6 was electroporated into E. coli EF00 harboring pKD46. For arcA insertional mutation, the coding region of gene arcA was divided into two parts at site 513 which were amplified separately and ligated into pUC18 in sequence to yield plasmid pUCarcA. The tetracycline resistant gene amplified from pACYC184 was then inserted into pUCarcA to yield plasmid pUCarc-tet. This plasmid was then used as the template for amplification of DNA fragment which was needed for chromosome integration. 2.3. Medium and cultivation conditions M9 minimal medium (Li et al., 2013) supplemented with glycerol was used in this study for fumaric acid production. 50 lg/mL ampicillin was supplemented for plasmid maintenance and 0.5 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) was added in the shake flask culture at the beginning of fermentation to induce trc promoter. For flask cultures, 250 lL overnight cultures in 15 mL tubes prepared from single colonies were used to inoculate 25 mL M9 + glycerol medium in 250 mL flasks, and incubated at 37 °C and 220 rpm until OD600 reached about 1.0–2.0. IPTG was added in these cultures to induce the expression of gene ppc and aceBA which were required for growth in minimal medium. Then, 500 lL cultures were transferred into 50 mL M9 + glycerol medium in 500 mL flasks. For fumaric acid production, the cultures were incubated at 37 °C and 220 rpm till glycerol was exhausted. All the fermentation experiments were performed in triplicate. Fed-batch fermentation was carried out in a 1.3 L reactor (New Brunswick Scientific BioFlo 110, USA). M9 medium supplemented with about 50 g/L glycerol, 20 g/L corn steep liquor powder, 50 lg/mL ampicillin, and 0.5 mM IPTG was used. Prior to fermentation, overnight culture of strain EF02(pSCppc) in 15 mL tube prepared from single colony was used to inoculate 50 mL M9 medium with 10 g/L glycerol in 500 mL flask, and incubated at 37 °C and 220 rpm until OD600 of 1.0–2.0. All the culture in flask was then transferred into 500 mL medium in the reactor. For providing aerobic environment, sterile air was aerated at 0.5 L/min and the agitation speed was adjusted between 500 and 800 rpm to maintain the dissolved oxygen above 30% of air saturation. The fermentation was operated at 37 °C. In addition, 30% ammonium hydroxide and 1 M sulfuric acid were added automatically to control the pH at 7.0. The glycerol concentration of the feeding solution was 1000 g/L, and 25 mL feeding solution was supplemented when the glycerol concentration of the culture broth decreased below 20 g/L. 2.4. Analytical methods Cell growth was monitored by measuring the optical density at 600 nm (OD600) with a UV–vis spectrophotometer. The fermentation broth was centrifuged at 13,000 rpm for 5 min and the supernatant was used for metabolite analysis through HPLC which was equipped with a cation exchange column (Aminex HPX 87-H, Bio-Rad, Richmond, USA). Glycerol, fumaric acid and acetate were analyzed by an refractive index (RI) detector (Agilent, HP1047A), while a-ketoglutarate was examined by an UV absorbance detector (Agilent, G1315D). The mobile phase was 5 mM H2SO4 at 0.4 mL/ min, and the column was operated at 65 °C. The intracellular NADH
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N. Li et al. / Bioresource Technology 174 (2014) 81–87 Table 1 Escherichia coli strains and plasmids used in this study. Strains/plasmids
Relevant characteristics
Reference/source
Strains DH5a BL21(DE3) E2 EF00 EF01 EF01(pSCyodC) EF01S(pSCyodC) EF01S(p15yodC) EF01(arcA) EF01(pSCppc) EF01(pSCBA) EF01(pSCBAppc) EF02(pSCppc)
Cloning host F’ ompT gal dcm lon hsdSB(rB- mB-) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Evolved E1 E2, DfumB, DfumAC E2, DfumB, DfumAC, ppc:trc EF01 harboring pSCyodC EF01, sucA:trc, harboring pSCyodC EF01, sucA:trc, harboring p15yodC EF01, arcA:tet EF01 harboring pSCppc EF01 harboring pSCBA EF01 harboring pSCBAppc EF01, DaspA, harboring pSCppc
Lab collection Novagen Li et al. (2013) This study This study This study This study This study This study This study This study This study This study
Plasmids pUC18 pTrc99a pACYC184 p5CS pKD3 pKD4 pCP20 pKD46 pUCarcA pUCarc-tet pSCTrc pACYCTrc pSCyodC p15yodC pSCppc pSCBA pSCBAppc
bla, pBR322 ori bla, pBR322 ori, trc promoter, lacIq cat, tet, p15A ori bla, pSC101 ori bla, FRT-cat-FRT bla, FRT-kan-FRT bla, cat, yeast FLP recombinase bla, k-Red recombinase under araBAD promoter, temperature-conditional replicon Two parts of arcA coding region cloned in pUC18 Two parts of arcA coding region and tet cloned in pUC18 bla, pSC101 ori, trc promoter cat, p15A ori, trc promoter yodC gene from B. subtilis cloned in pSCTrc yodC gene from B. subtilis cloned in pACYCTrc ppc gene from E. coli BL21(DE3) cloned in pSCTrc aceBA gene from E. coli BL21(DE3) cloned in pSCTrc ppc gene and aceBA from E. coli BL21(DE3) cloned in pSCTrc
Lab collection Invitrogen Chang and Cohen (1978) Lab collection Datsenko and Wanner (2000) Datsenko and Wanner (2000) Cherepanov and Wackernagel (1995) Datsenko and Wanner (2000) This study This study This study This study This study This study This study This study This study
and NAD+ concentrations were examined according to the method described previously (Fu et al., 2014). 2.5. Enzyme assays Cells were grown in 500 mL flasks containing M9 medium supplemented with 10 g/L glycerol and 0.5 mM IPTG to the midexponential phase, and were harvested by centrifugation at 8000g for 10 min. After washed twice in 50 mM potassium phosphate buffer (pH 7.0), cells were resuspended in the same buffer and broken to prepare crude extract according to the method described previously (Li et al., 2013). The activity of NADH oxidase was assayed by monitoring the decrease of absorbance of NADH at 340 nm (Reusch and Burger, 1974). The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.07 mM NADH, 0.1 mM FAD. PEP carboxylase and isocitrate lyase were measured as previously reported (Chell et al., 1978; Terada et al., 1991). 2.6. Real time quantitative PCR (RT-qPCR) For total RNA isolation, cells were grown in M9 minimal medium supplemented with 10 g/L glycerol and harvested at the mid-exponential phase. Total RNA was extracted with RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. RT-PCR was performed as described previously (Li et al., 2013). 3. Results and discussion 3.1. Fumarases deletion for accumulation of fumaric acid The evolved mutant E. coli E2 was previously developed for succinate production from glycerol (Li et al., 2013). In this strain, the
glyoxylate shunt was recruited as the primary anaplerotic pathway by employing laboratory adaptive evolution of a ppc deletion strain. Due to the enhanced glyoxylate shunt, succinate yield was improved along with the decreased a-ketoglutarate accumulation. Under aerobic conditions, the pathway for fumaric acid production was almost the same as that of succinate, which suggested that strain E. coli E2 was also suitable for fumaric acid production. Being a part of the TCA cycle, succinate was converted to fumarate by succinate dehydrogenase, and fumarate was then transformed to malate through fumarase (Fig. S1). Therefore, for fumaric acid production, elimination of fumarase activities would be the first step. There are three fumarases in E. coli, encoded by fumA, fumC, and fumB respectively (Tseng et al., 2001). Fumarase C and A were mainly functional under aerobic and microaerobic conditions, whereas fumarase B was the dominant enzyme under anaerobic conditions. It has been reported that fumaric acid was not accumulated in fumAC deletion mutant, whereas simultaneously deletion of fumAC and fumB resulted in fumaric acid production (Song et al., 2013). Therefore, all three fumarases were deleted in E. coli E2 in this work. Similar to the phenomenon resulted from deletion of succinate dehydrogenase in our previous study (Li et al., 2013), deletion of fumarases also abolished the capacity of the resulting strain E. coli EF00 to grow in glycerol minimal medium due to the lack of oxaloacetate required for synthesis of cellular constituents. Therefore, in the same way, ppc was recovered in E. coli EF00 to yield strain EF01. Under aerobic conditions, strain EF01 produced 3.65 g/L fumaric acid from about 11 g/L glycerol in 36 h. In addition, acetate was accumulated to 2.75 g/L as the main byproduct unexpectedly. In our previous study for succinate production, little acetate was produced and it was consumed at the end of fermentation (Li et al., 2013). Moreover, in contrast to the fact that a-ketoglutarate was the main byproduct for succinate production, here, for production of fumaric acid, only
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a small amount of a-ketoglutarate was accumulated. These phenomena were surprising and interesting in view of the almost identical pathway for these two products. 3.2. Improving fumaric acid production by reducing acetate The production of acetate wasted carbon and reduced fumaric acid yield. In order to improve fumaric acid production, decreasing acetate might probably be effective. Therefore, several strategies were attempted to reducing acetate accumulation. 3.2.1. Expression of NADH oxidase from B. subtilis Acetate accumulation was previously reported to strongly correlated to cellular NADH/NAD ratio (redox ratio), and it could be largely decreased through expression of Streptococcus pneumoniae NADH oxidase which could reduce the cellular redox ratio (Vemuri et al., 2006). In comparison to succinate production, one more step catalyzed by succinate dehydrogenase is needed for fumaric acid production. This reaction will transfer electrons to ubiquinone for entry into the electron transport chain while oxidizing succinate to fumarate, which might increase cellular redox ratio and result in acetate accumulation. Therefore, in order to reduce acetate production, NADH oxidase encoded by gene yodC from B. subtilis (Zhang et al., 2014) was exogenously expressed in strain EF01 on a low-copy-number plasmid pSCTrc. The resulting strain EF01(pSCyodC) was examined for fumaric acid production under aerobic conditions from glycerol. As can be seen from Fig. 1, the amount of acetate was successfully reduced by nearly 80% through expressing NADH oxidase. However, fumaric acid production was also reduced by 13% for strain EF01(pSCyodC) as compared to strain EF01. At the same time, a-ketoglutarate was increased to 1.32 g/L. From these results, it could be seen that although carbon flux was directed down into the TCA cycle from the node acetate, it was blocked again at another node a-ketoglutarate. Through expression of NADH oxidase, the product pattern of strain EF01(pSCyodC) was now much more similar to that of succinate producing strain E2-Dsdh-ppc (Li et al., 2013). In our previously study, a-ketoglutarate dehydrogenase was overexpressed, which significantly
decreased a-ketoglutarate production (Li et al., 2013). Therefore, in this study, we also attempted to direct carbon flux from a-ketoglutarate to fumaric acid through overexpressing a-ketoglutarate dehydrogenase. The native promoter of gene sucAB was replaced with trc promoter in strain EF01(pSCyodC). However, the resulting strain EF01S(pSCyodC) accumulated large amount of acetate again, which was the same as strain EF01 (Fig. 1). In addition, fumaric acid and a-ketoglutarate production also changed back to the level of strain EF01. The reaction catalyzed by a-ketoglutarate dehydrogenase oxidizes a-ketoglutarate to succinyl-CoA with reduction of NAD to NADH. Therefore, overexpression of a-ketoglutarate dehydrogenase might result in increasing cellular NADH/NAD ratio which might be the reason for the high level of acetate for strain EF01S(pSCyodC). In order to verify this hypothesis, the expression level of NADH oxidase was further improved by replacing pSCyodC with p15yodC, the copy number of which was higher than that of pSCyodC. The specific activity of NADH oxidase in the resulting strain EF01S(p15yodC) (0.175 ± 0.007 lmol/min/mg protein) was almost 3-fold higher than that of strain EF01S(pSCyodC) (0.057 ± 0.008 lmol/min/mg protein). However, the fermentation results (Fig. 1) showed that acetate was not reduced and fumaric acid was not increased in strain EF01S(p15yodC). NADH/NAD ratios in these strains were examined and a NADH/NAD decrease from 0.32 ± 0.026 in strain EF01S(pSCyodC) to 0.23 ± 0.019 in strain EF01S(p15yodC) was observed. This suggested that the redox ratio might not be the reason for acetate production in strain EF01S(pSCyodC) overexpressing a-ketoglutarate dehydrogenase. Although acetate accumulation was reduced through expression of NADH oxidase, the saving carbon could not be converted to fumaric acid. Therefore, other strategies were attempted to reduce acetate production. 3.2.2. ArcA mutation The accumulation of acetate at the acetyl-CoA node might indicate the decreased flux of the TCA cycle in strain EF01. Therefore, the transcriptional levels of the TCA cycle enzymes of strain E2-Dsdh-ppc (Li et al., 2013) developed for succinate production and strain EF01 were examined and compared. As expected, the
Fig. 1. Fumaric acid and byproduct production, cell growth and fumaric acid yield for different strains. EF01, fumarases deleting strain; EF01(pSCyodC), overexpressing yodC from Bacillus subtilis; EF01S(pSCyodC), overexpressing yodC and sucAB; EF01S(p15yodC), overexpressing yodC on a plasmid with higher copy number and overexpressing sucAB; EF01(arcA), mutating gene arcA. All the strains were cultured in M9 minimal medium with about 11 g/L glycerol in 500 mL flasks at 37 °C and 220 rpm.
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Table 2 Activities of PEP carboxylase and isocitrate lyase expressed from relevant plasmids. Strain
PEP carboxylase activity (lmol/min/mg protein)
Isocitrate lyase activity (lmol/min/mg protein)
EF01 EF01(pSCppc) EF01(pSCBA)
0.169 ± 0.030 0.356 ± 0.071 –
0.343 ± 0.030 – 0.694 ± 0.067
levels of enzymes involved in the TCA cycle was not the main reason for acetate accumulation.
Fig. 2. The transcriptional levels of genes involved in the TCA cycle for strains E2Dsdh-ppc (succinate producing strain) and EF01 (fumaric acid producing strain). The values of strain EF01were all normalized to that of strain E2-Dsdh-ppc.
transcriptional levels of all genes investigated in strain E2-Dsdh-ppc were higher than strain EF01 (Fig. 2). Therefore, we attempted to reduce acetate by increasing the transcriptional levels of enzymes involved in the TCA cycle. ArcAB two-component regulatory system, which consists of ArcB sensor kinase and ArcA response regulator, regulates the expression of hundreds of genes in E. coli in response to respiratory conditions (Salmon et al., 2005). It has been reported that deletion of arcA and arcB could further activate the TCA cycle under aerobic conditions (Nizam et al., 2009). In addition, acetate accumulation could be reduced in arcA deletion mutants (Peebo et al., 2014; Vemuri et al., 2006). Therefore, in order to derepress the TCA cycle and reduce acetate production, gene arcA was mutated by insertion of the tetracycline resistant gene in the coding region as previously reported (Nikel et al., 2006). However, the resulting strain EF01(arcA) produced similar acetate and less fumaric acid than strain EF01 (Fig. 1). This phenomenon was not in accordance with the results previously reported (Peebo et al., 2014; Vemuri et al., 2006). The reason for this result was not clear. This suggested that the transcriptional
3.2.3. Increasing the flux of the anaplerotic pathways Oxaloacetate is a very important intermediate which is not only a part of the TCA cycle but also the precursor of cellular components. In addition, the velocity of citrate synthase to some extent depends on the concentration of oxaloacetate which is one of the substrates of citrate synthase (Robinson et al., 1983). To explore whether oxaloacetate was insufficient which might limit the flux of the TCA cycle and result in acetate accumulation, the fluxes of the anaplerotic pathways would be enhanced to increase the oxaloacetate pool. The reaction catalyzed by PEP carboxylase and the glyoxylate cycle were both manipulated by overexpression of ppc and aceBA in plasmid pSCTrc respectively. The specific activities of PEP carboxylase and isocitrate lyase were both increased about twofold (Table 2). Fumaric acid production of the two resulting strains EF01(pSCppc) and EF01(pSCBA) was examined under aerobic conditions. As shown in Fig. 3, acetate was reduced by more than 80% and fumaric acid was increased by 26.5% for strain EF01(pSCppc) as compared to the control strain EF01. Similar effects were also exerted by overexpression of the glyoxylate cycle operon. Although acetate was not so much reduced in strain EF01(pSCBA), fumaric acid was improved with almost the same degree as strain EF01(pSCppc). Fumaric acid yields from glycerol for these strains were 63% and 59% of the maximum theoretical yield respectively. However, a-ketoglutarate was largely accumulated to 1.37 g/L in strain EF01(pSCppc), while that of strain EF01(pSCBA) was a little increased. This difference might probably be resulted from the fact that the glyoxylate shunt could redirect carbon flow away from the oxidative arm of the TCA cycle and thus decrease a-ketoglutarate accumulation.
Fig. 3. Effects of overexpression of ppc and aceBA on products concentration, cell growth and fumaric acid yield. EF01(pSCppc), overexpressing gene ppc; EF01(pSCBA), overexpressing aceBA; EF01(pSCBAppc), overexpressing ppc and aceBA. All the strains were cultured in M9 minimal medium with about 11 g/L glycerol in 500 mL flasks at 37 °C and 220 rpm.
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In addition, biomass yield was increased though overexpression of the glyoxylate cycle operon, whereas overexpression of PEP carboxylase had no effect on biomass yield (Fig. 3). However, cell growth of strain EF01(pSCBA) was severely retarded, while that of strain EF01(pSCppc) was not affected as compared to EF01. The specific growth rate of strain EF01(pSCBA) (0.102 h 1) was much lower than that of strain EF01 (0.261 h 1) and strain EF01(pSCppc) (0.254 h 1). Given that fumaric acid production could be improved through overexpression of either PEP carboxylase or the glyoxylate shunt operon, they were coexpressed in strain EF01 through inserting ppc gene on plasmid pSCBA for further improving fumaric acid production. The results (Fig. 3) showed that acetate was reduced by 56% for the resulting strain EF01(pSCBAppc) as compared to EF01, which was higher than that of EF01(pSCBA) and lower than that of strain EF01(pSCppc). The amount of a-ketoglutarate produced by strain EF01(pSCBAppc) was as low as strain EF01(pSCBA). Fumaric acid yield was just increased a little as compared with strain EF01(pSCppc) and achieved 67% of the maximum theoretical yield. Biomass yield was not that high as strain EF01(pSCBA) and cell growth was faster with the specific growth rate of 0.15 h 1.
Fig. 4. Fed-batch fermentation of strain EF02(pSCppc) for fumaric acid production under aerobic conditions in the M9 medium supplemented with 20 g/L corn steep liquor powder. Lower triangle, OD600; square, glycerol; circle, fumaric acid; upper triangle, acetate.
3.3. Fed-batch production for fumaric acid from glycerol Although strain EF01(pSCppc) produced a little less fumaric acid than strain EF01(pSCBAppc), cell growth of the former was much faster than that of the latter. Therefore, strain EF01(pSCppc) was further explored for fumaric acid production performance in fed-batch culture. However, when cultured in 1.3 L bioreactor containing M9 minimal medium supplemented with glycerol, fumaric acid was initially produced and gradually consumed at the late stage of fermentation (data not shown). This phenomenon was not observed in the shake flask culture. Due to the fact that the conditions between flask culture and bioreactor culture were not completely consistent, some particular pathways might be triggered in bioreactor culture. It was reported that aspartase (encoded by aspA) reversibly converted fumarate to aspartate (Rudolph and Fromm, 1971). Therefore, aspA was deleted in strain EF01(pSCppc) to figure out whether fumaric acid was consumed through this reaction. As expected, fumaric acid was not consumed anymore for the resulting strain EF02(pSCppc) cultured in bioreactor (Fig. 4). For better cell growth, 20 g/L corn steep liquor powder, which was much cheaper than peptone and yeast extract, was supplemented in M9 minimal medium as nitrogen source in 1.3 L fermenter. At the end of fed-batch culture, 94 g/L glycerol was consumed and 41.5 g/L fumaric acid was produced by strain EF02(pSCppc) in 82 h (Fig. 4). The main byproduct acetate was accumulated to 8.7 g/L. The yields of fumaric acid and acetate were 0.44 g/g glycerol and 0.09 g/g glycerol respectively, both of which were higher than those of the flask culture (0.398 and 0.04 g/g respectively). Fumaric acid yield was approximate 70% of the maximum theoretical yield. Cell growth was rapid and reached the maximum OD600 of 66 in 40 h. The maximum and average glycerol consumption rates were 2.31 and 1.15 g/L/h, respectively. And the maximum and average fumaric acid productivities were 1.14 and 0.51 g/L/h. To the best of our knowledge, the titer, yield and productivity of fumaric acid achieved in this study are all the highest values described in literature for fumaric acid production by engineered E. coli. As compared to succinate production in our previous work (Li et al., 2013), production of fumaric acid from glycerol by the engineered E. coli here achieved higher yield in fed-batch fermentation (70% and 57% of the maximum theoretical yield for fumaric acid and succinate respectively). However, the average productivity of fumaric acid was lower than that of succinate (0.51 and
0.77 g/L/h respectively), which was mainly resulted from the longer fermentation time for fumaric acid production. In addition, the average glycerol consumption rate of fumaric acid production process (1.15 g/L/h) was much lower than that of succinate production (2.1 g/L/h). These results indicated different cell states of the two strains which might be partly attributed to the toxicity of fumaric acid on cell growth (Song et al., 2013). However, the fact that only one reaction difference resulted in great metabolic changes prompted us to perform further work and explore the underlying influence. Currently, the microbial fermentation process was less favorable economically than the petrochemical process due to the lower product yield of the former. However, if the raw material price would be lower it could compensate the low yield of the fermentation process (Roa Engel et al., 2008). Crude glycerol, which was much cheaper than glycerol and glucose, was one of the candidates. Here, the results demonstrated the promising potential of E. coli for fumaric acid production from glycerol, which provided vital platform for directly utilizing crude glycerol for production of fumaric acid. 4. Conclusions The evolved strain E. coli E2 with enhanced flux of the glyoxylate cycle was engineered in this study for fumaric acid production from glycerol under aerobic conditions. Deletion of fumarases resulted in accumulation of fumaric acid with the yield of 0.25 mol/mol glycerol and acetate was produced as the main byproduct. Overexpression of PEP carboxylase or the glyoxylate shunt operon effectively increased fumaric acid production and reduced acetate accumulation. In fed-batch culture, 41.5 g/L fumaric acid was produced from glycerol with approximate 70% of the maximum theoretical yield and an overall productivity of 0.51 g/ L/h. Acknowledgements This work was supported by National 973 Project (2011CBA00804, 2012CB725203); National Natural Science Foundation of China (NSFC-21176182, NSFC-21206112, NSFC21390201); National High-tech R&D Program of China (2012AA02A702, 2012AA022103).
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