Background

Renewable chemicals have attracted attention due to increasing interest in environmental concerns and resource utilization. Biobased production of industrial compounds from nonfood biomass has become increasingly important as a sustainable replacement for traditional petroleum-based production processes depending on fossil resources. Both acetoin and succinic acid are C4 chemicals that listed biobased high-value-added chemicals by the United States Department of Energy [1]. Currently, acetoin and succinic acid are building block chemicals used extensively in the food and pharmaceutical industries.

Acetoin is a volatile compound that occurs naturally in certain fruits and dairy products. Commercial acetoin can be used as a plant growth promoter, biological pest control measure, and additive to improve food flavor [2, 3]. At present, acetoin is produced mainly by chemical synthetic routes. Compared with chemical synthesis methods, microbial fermentation methods have the advantages of easy access to feedstock, environmental friendliness and mild process conditions [4, 5]. Therefore, microbial fermentation is considered to be an environmentally friendly method for the production of acetoin, which has made great progress in recent years. Many microorganisms synthesize acetoin during the mixed acid fermentation process, such as Enterobacter, Klebsiella, Lactococcus, Bacillus, Serratia and Saccharomyces species [6]. Acetoin is an intermediate product of the 2,3-butanediol biosynthesis pathway [7]. It is produced from pyruvate through α-acetolactate by two enzymes, including α-acetolactate synthase (budB) and α-acetolactate decarboxylase (budA), and finally converted to 2,3-butanediol by 2,3-butanediol dehydrogenase (budC) with the consumption of NADH [8]. Several studies have reported that the deletion of 2,3-butanediol dehydrogenase (budC) improves the production of acetoin in different species of microorganisms [2, 3, 9, 10].

In a traditional acetoin fermentation process, succinic acid is an undesirable by-product. However, succinic acid, a C4 dicarboxylic acid, has been used as a precursor for various chemicals, ion chelators, and additives in the food and pharmaceutical industries [11]. In addition, succinic acid can be converted into other chemicals, such as γ-butyrolactone, 1,4-butanediol, and tetrahydrofuran, and act as the precursor of polybutylene succinate synthesis. In E. coli, the reductive branch of the tricarboxylic acid (TCA) pathway function is the key pathway for the synthesis of succinic acid. The carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate (OAA) catalyzed by PEP carboxylase (PEPC) is considered the most important reaction. In this step, 1 mol CO2 is assimilated to form OAA [2]. Therefore, CO2 is an essential substrate for succinic acid biosynthesis, and it has been demonstrated that the production of succinic acid can be increased by sodium bicarbonate (NaHCO3) addition to the culture medium [12, 13]. Additionally, when grown under anaerobic conditions, E. coli metabolizes phosphoenolpyruvate (PEP) and pyruvate via the glycolytic pathway to form ethanol, lactic acid, and formic acid [14]. Therefore, changing the carbon flux towards the synthesis of succinic acid by metabolic engineering is very important [15]. Currently, numerous industrially used microorganisms have been metabolically engineered for succinic acid production by fermentation [16,17,18,19].

An earlier study revealed that E. cloacae can produce 40.67 g L−1 2,3-butanediol and 21.79 g L−1 succinic acid from xylose [20]. In this study, to redirect the carbon flux, a double-deletion mutant (strain EC∆budC∆ldhA) of E. cloacae was developed by deleting 2,3-butanediol dehydrogenase (budC) to produce acetoin and by deleting lactate dehydrogenase (ldhA) to improve succinic acid production (Fig. 1). Moreover, the feeding mode and time of glucose and NaHCO3 during the fermentation of the EC∆budC∆ldhA strain were optimized, which further enhanced the production of acetoin and succinic acid. The present findings demonstrated a potential practical strategy for the simultaneous production of two commercial products in a single fermentation step by redirecting the carbon flux and optimizing the culture conditions.

Fig. 1
figure 1

Schematic representation of improved production of acetoin and succinic acid by deletion of budC and ldhA in E. cloacae

Full size image

Results

Construction of the metabolically engineered strains

Enterobacter cloacae has an extraordinary ability to utilize biomass for 2,3-butanediol production, during which intermediary acetoin is formed [10]. Acetoin reductases (also known as 2,3-butanediol dehydrogenase) catalyze the transformation reaction from acetoin to 2,3-butanediol. Knockout of the budC gene would generate the EC∆budC strain, which mainly produces acetoin. A previous study showed that reducing the carbon flux to lactate, ethanol, and acetate by-products can be performed by deleting the ldhA, adhE, and pta genes in K. pneumoniae [7]. In this study, to further reduce lactic acid production in the fermentation process, the ldhA gene was disrupted from the wild type and EC∆budC strains to generate strains EC∆ldhA and EC∆budC∆ldhA, respectively. The budC and ldhA gene knockout of these strains was verified by screening with colony PCR (data not shown).

To determine the effects of deleting budC and ldhA on cell growth, wild type and three deletion mutants, EC∆budC, EC∆ldhA, and EC∆budC∆ldhA, were grown under 60 g L−1 glucose and 5 g L−1 NaHCO3 at 35 °C at 150 rpm, followed by comparison of the growth curves. The initial inoculum of these cultures was the same (OD600 = 0.15). The growth results are shown in Fig. 2. The EC∆budC and EC∆budC∆ldhA strains grew slower than the wild type in the first 12 h. The OD600 values were 5.62, 4.58, and 4.54 for the wild type, EC∆budC, and EC∆budC∆ldhA strains, respectively, after 24 h (Fig. 2). The growth rates of the EC∆budC and EC∆budC∆ldhA strains were reduced by 22.7% and 23.7%, respectively, in comparison with the wild type. In contrast, compared with the wild type, the EC∆ldhA strain exhibited increased cell growth. The OD600 value was 5.77 for the EC∆ldhA strain (Fig. 2a). The glucose concentration in the medium of wild type and EC∆ldhA strains was depleted after 24 h of fermentation. The glucose concentrations of EC∆budC and EC∆budC∆ldhA were depleted at 36 h (Fig. 2b). Jang et al. [9] reported that deletion of budC resulted in reduced cell growth and glucose consumption rate in Enterobacter aerogenes. These results indicated that deletion of the budC gene in E. cloacae cells might reduce the growth rate and glucose consumption.

Fig. 2
figure 2

Effects of knockout budC and ldhA on cell growth and consumed glucose of E. cloacae. The experiments were conducted in 50 mL of fermentation medium containing 60 g L−1 glucose and 5.0 g L−1 NaHCO3 in a 250 mL flask at 35 °C with shaking (150 rpm)

Full size image

Effects of metabolic engineering on enhanced co-production of acetoin and succinic acid

The wild type and EC∆budC strains were grown at 35 °C in 250 mL shake flasks containing 50 mL of fermentation medium supplemented with 90.0 g L−1 glucose and 5.0 g L−1 NaHCO3. The fermentation was finished when glucose was consumed nearly completely. As shown in Table 1, EC∆budC produced various organic acids and ethanol, with acetoin being a major product that accumulated to 18.6 g L−1, resulting in a 0.419 yield (mol mol−1 glucose). The concentrations of succinic acid, 2,3-butanediol, lactic acid, acetic acid and ethanol were 1.05, 7.7, 2.6, 2.75, and 4.75 g L−1, respectively. The succinic acid content of the EC∆budC strain was decreased by 2.24-fold in comparison with that of the wild type strain. Succinic acid was measured as 2.35 and 1.05 for the wild type and EC∆budC strains, respectively. Lactic acid formation was not detected in the wild type strain. However, the lactic acid content of EC∆budC (2.6 g L−1) was increased compared with that of the control and wild type (not detected).

Table 1 Fermentation profiles of the gene knockout strains of E. cloacae
Full size table

d-Lactate dehydrogenase (encoded by ldhA) catalyzes the conversion of pyruvate to d-lactic acid by coupling with the oxidation of NADH in E. cloacae [21]. In this study, to reduce lactic acid production in the fermentation process, the ldhA gene was inactivated in the wild type. The results indicated that the fermentation products of the EC∆ldhA strain were similar to those of the wild type (Table 1). The difference is that the succinic acid content of the EC∆ldhA strain was increased by 19% in comparison with that of the wild type.

To achieve a higher yield of acetoin and succinic acid co-production, the EC∆budC∆ldhA strain was constructed by knocking out ldhA genes in strain EC∆budC. EC∆budC∆ldhA produced various organic acids and ethanol, with acetoin being a major product that accumulated to 17.75 g L−1, resulting in a yield of 0.397 (mol mol−1 glucose). The concentrations of succinic acid, 2,3-butanediol, acetic acid and ethanol were 2.75, 8.15, 1.4, and 5.05 g L−1, respectively. Lactic acid was not observed in the EC∆budC∆ldhA strain. The succinic acid content of the EC∆budC∆ldhA strain was increased by 2.24-fold in comparison with that of the EC∆budC strain. The final concentrations of succinic acid were measured as 1.05 and 2.75 g L−1 for the EC∆budC and EC∆budC∆ldhA strains, respectively. The succinic acid yield was measured as 0.046 and 0.018 (mol mol−1 glucose) for the EC∆budC∆ldhA and EC∆budC strains, respectively.

The results indicated that elimination of 2,3-butanediol and lactic acid formation in the EC∆budC∆ldhA strain led to enhanced acetoin and succinic acid co-production, and the maximum acetoin and succinic acid yields were obtained as 0.397 and 0.046 mol mol−1 glucose, respectively.

Effect of NaHCO3 concentration on metabolite production by the EC∆budC∆ldhA strain

Previous studies have shown that CO2 is a key parameter in batch succinic acid fermentation. The amount of dissolved CO2 can be increased effectively by the addition of NaHCO3 to the medium [20]. To compare the effects of NaHCO3 levels, different concentrations of NaHCO3 (0, 2.5, 5, 7.5, and 10 g L−1) were added to the fermentation medium. As shown in Table 2, when grown in fermentation medium without NaHCO3, the final production of acetoin after 24 h was 16.45 g L−1. The concentrations of succinic acid, 2,3-butanediol and ethanol were 1.15, 3, and 6.25 g L−1, respectively. However, a higher concentration of NaHCO3 led to a negative effect on acetoin production. The concentration of acetoin was decreased by 7, 19, 10.6, and 24.6% for 2.5, 5, 7.5, and 10 g L−1 NaHCO3 addition, respectively. When grown in fermentation medium supplemented with different NaHCO3 levels (0, 2.5, 5, 7.5, and 10 g L−1), the concentrations of succinic acid were slightly enhanced from 1.15 to 1.55 g L−1 within 24 h. The maximum acetoin and succinic acid yields (0.469 and 0.034 mol mol−1 glucose) were obtained when 2.5 g L−1 NaHCO3 was added. Furthermore, the cell growth and acetic acid titer were also improved, while the amount of 2,3-butanediol slightly decreased. Therefore, the optimum NaHCO3 concentration for the combined production of acetoin and succinic acid was 2.5 g L−1.

Table 2 The fermentation performance of EC∆budC∆ldhA strain under different NaHCO3 concentration
Full size table

Fed-batch fermentation for co-production of acetoin and succinic acid

To increase the production of acetoin and succinic acid, fed-batch fermentation was performed using strain EC∆budC∆ldhA with an initial glucose concentration of 57.8 g L−1. NaHCO3 (1 g L−1) was added at fermentation times of 6 and 12 h. Next, glucose (20 g L−1) and NaHCO3 (2 g L−1) were added simultaneously at 24, 36, 48, 60, 72, and 84 h of fermentation.

Figure 3 shows 47.6 g L−1 acetoin and 7.35 g L−1 succinic acid from 171.25 g L−1 glucose obtained in 96 h by the EC∆budC∆ldhA strain. The acetoin and succinic acid yields were 0.568 and 0.065 mol mol−1 glucose, respectively. In fed-batch fermentation, the maximum acetoin and succinic acid yields were obtained as 0.565 and 0.071 mol mol−1 glucose, respectively, after 72 h. Compared with batch fermentation (Table 2), the maximum acetoin and succinic acid yields of fed-batch fermentation were increased by 1.2- and 2-fold, respectively. The results indicated that the acetoin and succinic acid co-production of the EC∆budC∆ldhA strain was improved by fed-batch fermentation.

Fig. 3
figure 3

Time course of fed-batch fermentation of EC∆budC∆ldhA. a Glucose, acetoin, succinic acid, b by-product. The experiments were conducted in 50 mL of fermentation medium containing 60 g L−1 glucose in a 250 mL flask. NaHCO3 (1 g L−1) was added at 6 and 12 h of fermentation. After that, NaHCO3 (2 g L−1) was added at 24, 36, 48, 61, 72, and 84 h of fermentation. Glucose (20 g L−1) was added at 24, 36, 48, 61, 72, and 84 h of fermentation. Samples were withdrawn every 12 h for detection of cell density and concentration of substrates and products

Full size image

Optimization of fed-batch fermentation for co-production of acetoin and succinic acid

A previous study showed that after glucose was depleted, the accumulated products were reused by Klebsiella pneumoniae as a carbon source [2]. Our previous work also found that with a low glucose concentration during cultivation, succinic acid did not accumulate (data not shown). To prevent the exhaustion of glucose, the glucose concentration was increased during fed-batch fermentation to achieve higher acetoin and succinic acid co-production. In this study, the optimal conditions for acetoin and succinic acid fermentation were determined by the glucose and NaHCO3 times and modes of feeding. The initial glucose and NaHCO3 concentrations were 60 g L−1 and 2.5 g L−1, respectively. When the glucose concentration was reduced to approximately 30 g L−1, glucose was added to the fermentation medium. Glucose (25, 35, 35 and 35 g L−1) was added at 12, 24, 36 and 48 h of fermentation, respectively. NaHCO3 (1, 1, 5, 5, 5, and 2 g L−1) was added at 6, 12, 24, 36, 48, and 60 h of fermentation, respectively. The time-course results of the production of succinic acid are shown in Fig. 4. After growth for 72 h, the maximum production of acetoin and succinic acid was measured as 39.5 g L−1 and 20.3 g L−1, respectively, in optimized fed-batch fermentation. The acetoin and succinic acid yields were 0.439 and 0.168 mol mol−1 glucose, respectively. The maximum acetoin and succinic acid yields were obtained as 0.559 and 0.322 mol mol−1 glucose, after 36 h. Compared with fed-batch fermentation (Fig. 3), the succinic acid titer of optimized fed-batch fermentation was increased by 2.8-fold. However, when only the budC gene was deleted, we found that a large amount of lactic acid (15 g L−1) was produced under the same optimized conditions, and caused a decrease in the production of succinic acid (Additional file 1: Fig. S1). This is similar to the results in Table 1. Thus, deletion of the ldhA gene of E. cloacae is required. The results indicated that the succinic acid production of the EC∆budC∆ldhA strain was significantly increased by optimizing the glucose and NaHCO3 feeding mode and time during fermentation, further enhancing the co-production concentrations of acetoin and succinic acid.

Fig. 4
figure 4

Time course of acetoin and succinic acid co-production by fed-batch fermentation using strain EC∆budC∆ldhA under the optimized conditions. a Glucose, acetoin, succinic acid, b by-product. The experiments were conducted in 50 mL of fermentation medium containing 60 g L−1 glucose in a 250 mL flask. NaHCO3 (2.5, 1, 1, 5, 5, 5 and 2 g L−1) was added at 0, 6, 12, 24, 36, 48, and 60 h of fermentation, respectively. Glucose (60, 25, 35, 35, and 35 g L−1) was added at 0, 12, 24, 36, and 48 h of fermentation, respectively. Samples were withdrawn every 12 h for detection of cell density and concentration of substrates and products

Full size image

Fed-batch fermentation for co-production of acetoin and succinic acid in a bioreactor

Previous studies have shown that cultivating the EC∆budC∆ldhA strain in a 250 mL flask through an optimized fed-batch culture method can increase the production of acetoin and succinic acid. Therefore, the same optimized fed-batch fermentation conditions were implemented in a 3-L bioreactor.

As shown in Fig. 5, the initial glucose and NaHCO3 concentrations were 60 g L−1 and 2.5 g L−1, respectively. Glucose (25, 35, 25, 10, and 10 g L−1) was added at 12, 24, 36, 48, and 60 h of fermentation, respectively. NaHCO3 (1, 1, 5, 5, 5, and 2 g L−1) was added at 6, 12, 24, 36, 48, and 60 h of fermentation, respectively. The time-course results are shown in Fig. 5. After growth for 82 h, the maximum production of acetoin and succinic acid was measured as 38 g L−1 and 16.3 g L−1, respectively. The acetoin and succinic acid yields were 0.490 and 0.157 mol mol−1 glucose, respectively.

Fig. 5
figure 5

Time course of acetoin and succinic acid co-production by fed-batch fermentation using strain EC∆budC∆ldhA in bioreactor. a Glucose, acetoin, succinic acid, b by-product. The experiments were conducted in 1.5-L of fermentation medium containing 60 g L−1 glucose under 0.5 vvm air flow in 3-L bioreactor. NaHCO3 (2.5, 1, 1, 5, 5, 5, and 2 g L−1) was added at 0, 6, 12, 24, 36, 48, and 60 h of fermentation, respectively. Glucose (60, 25, 35, 25, 10, and 10 g L−1) was added at 0, 12, 24, 36, 48, and 60 h of fermentation, respectively. Samples were withdrawn every 12 h for detection of cell density and concentration of substrates and products

Full size image

Discussion

Several studies have also shown that the inactivation of budC significantly improves the production of acetoin. Indeed, previous reports have shown that the deletion of the budC gene could decrease 2,3-butanediol. Three butanediol stereoisomers, namely, (2R,3R)-2,3-butanediol, (2S,3S)-2,3-butanediol, and meso-2,3-butanediol, are found in many bacterial species, such as Enterobacter cloacae [10, 22], Klebsiella pneumoniae [23], and Bacillus licheniformis [24], and meso-2,3-butanediol and (2S,3S)-2,3-butanediol are the major forms that accumulate in E. cloacae [25]. However, when the budC gene was deleted, a small amount of 2,3-butanediol could still be detected [3, 22, 23, 26, 27]. In this study, the budC gene was knocked out, and we observed that the production of meso-2,3-butanediol and (2S,3S)-2,3-butanediol decreased (data not shown).

A previous study characterized a budC and glycerol dehydrogenase (encoded by gldA and dhaD)-deficient Klebsiella pneumoniae strain, which removes 2,3-butanediol under conditions wherein glycerol is used as a carbon source. These findings suggested that dhaD and gldA may be involved in 2,3-butanediol formation [22]. Another study reported diacetyl production by inactivating budA, budC, and diacetyl reductases (also known as glycerol dehydrogenase, encoded by gdh) in E. cloacae SDM. When the gdh and budC genes were both inactivated in the strain E. cloacae SDM (∆budA), (2R,3R) 2,3-butanediol could be slightly detected [10]; these results show that there is a third enzyme responsible for 2,3-butanediol production in the E. cloacae strain. In the present work, disruption of the budC gene remarkably decreased the production of 2,3-butanediol by almost 2.7-fold compared to that of the wild type and EC∆ldhA strains (Table 1). However, small amounts of 2,3-butanediol were still detected in a few of the EC∆budC and EC∆budC∆ldhA strains, indicating the presence of other genes encoding enzymes that convert acetoin to 2,3-butanediol in E. cloacae.

Theoretically, the formation of 1 mol succinic acid from glucose requires 1 mol of CO2 [20, 28]. Therefore, CO2 is indispensable for succinic acid biosynthesis, and many studies have demonstrated that succinic acid production can be increased by adding HCO3− to the fermentation medium [12, 28]. Cheng et al. [28] increased succinic acid production in K. pneumoniae by adding HCO3− to the fermentation medium. In another study, Wu et al. [20] reported yields of 40.67 g L−1 2,3-butanediol and 21.79 g L−1 succinic acid by adding NaHCO3 to E. cloacae. In this study, supplying NaHCO3 during batch fermentation may enhance succinic acid production by improving the quantity of dissolved CO2 and by increasing the carbon flux to succinic acid. When grown in fermentation medium without NaHCO3, the final acetoin production (16.45 g L−1) was slightly higher; however, the final amount of succinic acid produced was only 1.15 g L−1. When grown in fermentation medium supplemented with NaHCO3, succinic acid production was 34.8% higher than the amount produced during batch fermentation without NaHCO3 (Table 2).

In general, the production of succinic acid was higher under anaerobic conditions, and bacterial producers of succinic acid can be found among facultative and strictly anaerobic rumen bacteria such as Mannheimia succiniciproducens [29], Actinobacillus succinogenes [30], and Anaerobiospirillum succiniciproducens [31]. E. cloacae is a facultative anaerobe, and when it is cultured under anaerobic conditions, the glucose consumption rate of the ΔbudCΔldhA strain is slower, resulting in lower production concentration of acetoin. In addition, when cultured under anaerobic conditions, the ΔbudCΔldhA strain was found to produce lactic acid (Additional file 1: Fig. S2). Although we only knocked out d-lactate dehydrogenase, this may activate other lactate dehydrogenases under anaerobic conditions, such as l-lactate dehydrogenase, leading to the production of lactic acid.

A previous study showed that reducing the carbon flux to lactate, ethanol, and acetate by-products can be performed by deleting the ldhA, adhE, and pta genes in K. pneumoniae [32]. In this study, by blocking lactic acid synthesis pathways to redirect more carbon sources to succinic acid synthesis in wild type E. cloacae, the engineered EC∆budC significantly increased succinic acid yield. This engineering approach may represent a practical strategy involving the deletion of ldhA and budC genes to reduce carbon flux towards the formation of by-products.

Conclusions

In this study, we engineered an E. cloacae budC and ldhA double-deletion strain (namely, EC∆budC∆ldhA) to produce succinic acid and acetoin. The highest acetoin and succinic acid titers achieved by this engineered strain were 39.5 and 20.3 g L−1, respectively, during optimization of fed-batch fermentation conditions. Our findings demonstrated that the EC∆budC∆ldhA strain would be useful for the simultaneous production of commercial products (acetoin and succinic acid) and the prevention of by-product formation, thus reducing the cost of microbial fermentation in a single step.

Methods

Bacterial strains

The strains used in this study are described in Table 3. Escherichia coli and E. cloacae were grown in LB broth with rotary shaking agitation at 200 rpm at 37 °C and 35 °C, respectively. Ampicillin (100 µg mL−1) and kanamycin (50 µg mL−1) were added to LB broth. E. cloacae (CICC 10011) was purchased from the China Center of Industrial Culture Collection (China). E. coli DH5α was used as the host for all recombinant plasmid constructs. E. coli S17-1 λpir, which is able to host pKR6K and its derivatives, was used for conjugation with E. cloacae.

Table 3 Strains and plasmid used in this study
Full size table

Plasmid construction

Plasmids constructed and used are described in Table 3. The gene replacement vector of E. cloacae was constructed by a previously described method [22]. The R6K origin of replication was amplified with primers (BspHI-oriR6K-F and BsaXI-oriR6K-R) using the plasmid pRL27 as a template. The 0.6 kb oriR6K fragment was ligated to the pGEM-T Easy vector (Promega, Madison, WI, USA) to create the plasmid pToriR6K. The oriR6K fragment (BspHI/BsaXI) was digested from pToriR6K and cloned into plasmid pK18mobsacB to create the suicide plasmid pKR6K. The plasmid pKR6K was used for gene knockout by homologous recombination in E. cloacae.

Gene knockout mutants of E. cloacae were constructed using the suicide vector pKR6K. To construct the budC and ldhA gene replacement vector of E. cloacae, the selected flanks were 510 bp long and homologous to sequences upstream and downstream of the region targeted for deletion. The upstream and downstream flanking sequences of the budC and ldhA genes were amplified with their respective primers (EcoRI-budCup-F/EcoRI-budCup-R, XbaI-budCdown-F/SphI-budCdown-R and EcoRI-ldhAup-F/BamHI-EcoRI-ldhAup-R, XbaI-ldhAdown-F/SphI-SalI-ldhAdown-R) using the total genomic DNA of E. cloacae as a template for PCR and cloned into the pGEM-T Easy vector to generate plasmids pTBCup and pTBCdown. Then, the budC upstream and downstream fragments were digested by EcoRI and XbaI/SphI from plasmids pTBCup and pTBCdown, respectively. The two fragments were ligated to pKR6K digested with EcoRI and XbaI/SphI, producing pKΔbudC. The ldhA upstream and downstream fragments were digested by EcoRI and XbaI/SalI from plasmids pTLAup and pTLAdown, respectively. The two fragments were ligated to pKR6K digested with EcoRI and XbaI/SalI, producing pKΔldhA. Then, the plasmids pKΔbudC and pKΔldhA were transformed into E. coli S17-1. E. coli S17-1 (pKΔbudC and pKΔldhA) was used as the donor in conjugation with E. cloacae. The primer sequences are shown in Table 4.

Table 4 Primers used in this study
Full size table

Gene knockout in the chromosome of E. cloacae

Allelic exchange of E. cloacae was performed as previously described [10] with slight modifications. The constructed strains used are described in Table 3. Strain EC∆budC was constructed by allelic exchange of plasmid pKΔbudC into E. cloacae. Strain EC∆ldhA was constructed by allelic exchange of plasmid pKΔldhA into E. cloacae. Strain EC∆budC∆ldhA was constructed by allelic exchange of plasmid pKΔldhA into E. cloacae strain EC∆budC. Colonies with confirmed deletions were screened by PCR using specific primers. The primer sequences are shown in Table 4.

Batch and fed-batch fermentations

The seed culture, batch fermentation, and fed-batch fermentation were carried out according to the procedure described by Wu et al. [20]. Sterilized glucose was added before fermentation. Wild type and gene knockout strains of E. cloacae were inoculated into flasks (250 mL) containing 50 mL of seed culture medium and cultured overnight at 35 ℃ with continuous shaking at 150 rpm. The fermentation medium contained final concentrations of 5% (v/v) seed medium.

Batch fermentation and fed-batch fermentation were conducted in 250 mL flasks containing 50 mL of medium. Cultivation was carried out at 35 °C with a speed at 150 rpm. The pH was maintained by the addition of NaHCO3. Samples were withdrawn periodically to measure the OD600 and the concentrations of glucose, succinic acid, acetoin, and by-products. Each experiment described in this research was performed in two replicates.

Fed-batch fermentations in the bioreactor

Seed culture (5%, v/v) was inoculated into the fermentation medium, and fed-batch fermentation was carried out in a 3-L stirred-vessel bioreactor (BLBIO-3GC, Bailun, China) containing 1.5-L of fermentation medium under 0.5 vvm air flow. Cultivation was performed at 35 °C with a speed at 300 rpm and an aeration rate of 0.5 vvm. The pH was maintained by the addition of NaHCO3.

Analytical methods

Glucose, succinic acid, acetoin, and by-products were analyzed by the methods described in Wu et al. [20]. Samples were measured by HPLC (LC20, Shimadzu, Japan) using an Aminex HPX-87H column (Bio Rad, USA) with a refractive index detector (RID-20A).