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To produce meso-2,3-BD with high optical purity in B. licheniformis, we proposed to block the synthesis of D-2,3-BD by knocking out the gene encoding the enzyme catalyzing the conversion of D-AC to D-2,3-BD, so that the carbon flux from D-AC would be diverted into meso-2,3-BD (Fig. 1). It was hypothesized that the reversible conversion of D-AC to D-2,3-BD was catalyzed by a putative D-2,3-butanediol dehydrogenase (D-BDH) enzyme (Fig. 1). Although gene bdhA encoding the D-BDH enzyme had been identified in B. subtilis ), the analysis of the B. licheniformis genome did not reveal the existence of any homologs to bdhA. Alternatively, researchers have reported that the glycerol dehydrogenase (GDH) in B. licheniformis is capable of catalyzing the in vitro conversion of D-AC to D-2,3-BD . Similar activities for GDHs (DhaD and GldA) were also observed in K. pneumonia . Based on these results, the gdh gene in B. licheniformis was studied for its role in D-2,3-BD biosynthesis.
A 1104-bp putative GDH encoding gene (gdh) was identified in B. licheniformis WX-02 . The deduced amino acid sequence of the protein (WX-02 GDH) was compared with GDHs reported in other strains via sequence alignments through ClustalW. Comparative analysis revealed that WX-02 GDH had nearly all the conserved residues of the iron-dependent alcohol dehydrogenase (Fe-ADH), including three glycine residues that are predicted to interact with the NAD(H) cofactor and three histidine residues that coordinate an iron cofactor. This suggests that the WX-02 GDH is a member of the Fe-ADH superfamily. In addition, a strong similarity (>48 %) was found between WX-02 GDH and GDHs, exhibiting D-BDH activity from other organisms, such as Serratia marcescens , Klebsiella pneumoniae (DhaD, KJ206474.1), Citrobacter freundii (DhaD, P45511.1), Klebsiella oxytoca (DhaD, YP_005016612), and B. licheniformis 10-1-A (GldA, AHW84748.1).
As shown in Fig. 2a, the His-tagged recombinant GDH protein was efficiently expressed in soluble form after IPTG induction. The purified GDH protein was observed as a single band on SDS-PAGE with an approximate molecular weight consistent with that predicted from its amino acid sequence (39.5 kDa). The activity of purified GDH from WX-02 was further investigated for its catalytic activities on various substrates with coenzymes. Figure 2b showed that WX-02 GDH accepted D-AC, D-2,3-BD, meso-2,3-BD, and glycerol as substrates. However, this enzyme exhibited the highest activity towards D-2,3-BD among various substrates. Its catalytic activity on glycerol was only around 5 % of that for D-2,3-BD. The preference on D-2,3-BD as the substrate by GDH was also reported in Hansenula polymorpha . These results indicated that the GDH from B. licheniformis WX-02 possessed a substrate specific catalytic activity towards D-2,3-BD. Based on these observations, we concluded that the GDH enzyme was the best candidate enzyme for catalyzing D-2,3-BD synthesis in B. licheniformis WX-02. Consequently, we decided to delete the gdh gene, so the conversion of D-AC to 2,3-BD isomers could be diverted solely towards meso-2,3-BD production.Fig. 2
Expression and catalytic activity of GDH from B. licheniformis WX-02 (WX-02 GDH). a SDS-PAGE of the expression and purification of WX-02 GDH. The recombinant E. coli BL21(DE3)/pET-gdh was grown to the appropriate density and induced with IPTG for the production of the GDH protein. The total-cell extracts from the induced cells were separated into soluble and insoluble fractions. Proteins in the soluble fractions were purified by the Ni–NTA purification kits, and the GDH protein was purified. Lane 1 sediment of cell extracts; lane 2 supernatant of cell extracts; lane 3 purified GDH protein in 50-fold dilution; lane 4 purified GDH protein in tenfold dilution; lane 5 purified GDH protein. b Activities of purified WX-02 GDH on different substrates with corresponding coenzymes
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To investigate the role of GDH in the biosynthesis of 2,3-BD isomers, we constructed WX-02Δgdh, a gdh-deficient strain of B. licheniformis WX-02. As shown in Fig. 3, WX-02 produced both meso-2,3-BD and D-2,3-BD, while no D-2,3-BD was produced by WX-02Δgdh. Complementation of gdh in WX-02Δgdh (strain WX-02Δgdh/pHY-gdh) restored its ability to produce D-2,3-BD. Figure 3 also showed dramatic differences in the production of 2,3-BD and D-AC between the wild-type and the engineered strain. Compared to WX-02, production of meso-2,3-BD and D-AC increased by 66.3 and 37.8 %, respectively, in WX-02Δgdh. The results indicate that the deletion of gdh gene from the wild-type strain eliminates the synthesis of D-2,3-BD, leading to an accumulation of its precursor D-AC, which in turn promoted the conversion of D-AC to meso-2,3-BD. Meso-2,3-BD was the only 2,3-BD isomer detected in the medium, resulting in a product of high purity. It was also found that the mutant WX-02Δgdh/pHY-gdh not only restored the production of D-2,3-BD but produced an even higher titer of total 2,3-BD isomers (D-2,3-BD and meso-2,3-BD) compared to that of wild-type. Correspondingly, the D-AC produced by the complementation strain was lower than the wild-type WX-02. The distribution profile of these metabolites indicates that the high expression of gdh gene is likely a result of the strong promoter P43 and multiple copies of pHY300PLK vector.Fig. 3
Production of acetoin and 2,3-BD isomers by B. licheniformis WX-02 and the gdh mutant strains. WX-02 wild-type strain, WX-02Δgdh the mutant strain with gdh gene knocked out from the genome of WX-02, WX-02Δgdh/PHY-gdh the mutant strain with gdh gene complemented to the knock-out strain WX-02Δgdh. The cells were grown under shake-flask culture conditions for 24 h. Data are means of three replicates, and error bars show standard deviations
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In addition to serving as the precursor for 2,3-BD synthesis, acetoin has also been reported to be used as a carbon source by B. licheniformis. This is regulated by AoDH ES (Fig. 1) when glucose was depleted [17, 24]. Acetoin dissimilation would reduce its availability for meso-2,3-BD synthesis resulting in an adverse effect on 2,3-BD production. In this work, the acoR gene encoding transcriptional activator of AoDH ES was disrupted to block the degradation of acetoin. Two separate experiments were then performed to confirm the effect of the acoR knock-out on acetoin metabolism. First, the acoR-disrupted strain WX-02ΔacoR was grown in the medium containing acetoin as the sole carbon source. The wild-type strain WX-02 was also grown in this medium as a control. As shown in Fig. 4a, the mutant WX-02ΔacoR had a very poor growth compared to that of the wild-type WX-02. The poor growth caused by the acetoin utilization deficiency was also observed in B. subtilis and B. licheniformis strains with inactivated acoABCL operons [17, 25, 26]. The results of the acetoin consumption study presented in Fig. 4a also indicated the inability of utilizing acetoin by the acoR-disrupted mutant. Second, WX-02ΔacoR and WX-02 were grown in 2,3-BD production medium containing glucose as the carbon source. As shown in Fig. 4b, the mutant WX-02ΔacoR produced 15.7 and 4.3 % more D-AC and total 2,3-BD isomers than the wild-type WX-02, respectively. The results clearly demonstrate the role of acoR in D-AC metabolism, and the deficiency of acoR contributed to D-AC accumulation, thus favoring 2,3-BD production.Fig. 4
The effect of acoR knock-out on acetoin metabolism of B. licheniformis. a Time course of the growth (squares) and acetoin consumption (triangles) by the acoR-disrupted strain (WX-02ΔacoR) (open symbols) and wild-type strain (WX-02) (solid symbols) in medium with acetoin (2.3 g/L) as the sole carbon source. b The accumulation of acetoin and 2,3-BD isomers by the mutant strain (WX-02ΔacoR) and wild-type strain (WX-02) (at 24-h of culture) in medium with glucose (120 g/L) as carbon source. The cells were grown under the shake-flask culture with minerals in 2,3-BD production medium were used for each cases. Data are means of three replicates, and error bars show standard deviations
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The above results demonstrated that single deletions of gdh or acoR gene were beneficial for the production of meso-2,3-BD production in terms of both titer and purity. Compared to the wild-type strain WX-02, a gdh gene deletion resulted in the sole production of meso-2,3-BD, while deletion of the acoR gene led to an accumulation of D-AC, the precursor for 2,3-BD synthesis. To maximize meso-2,3-BD biosynthesis, we engineered a strain with both gdh and acoR genes deleted. The metabolites produced by the double-gene-deletion mutant WX-02ΔgdhΔacoR were compared to those produced by the wild-type and the single-gene deficient strains. As shown in Table 1, the mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD, 50.3 % higher than that of the wild-type WX-02. The meso-2,3-BD yield and productivity of the double-deletion strain were also significantly higher with respect to the wild-type. Disruption of the gdh and acoR genes also affected the synthesis of other metabolites, such as lactic acid, acetic acid, and ethanol. Table 1 shows that the mutant strains, particularly WX-02ΔgdhΔacoR, produced lower amounts of these by-products, indicating the benefit of metabolic engineering of B. licheniformis for meso-2,3-BD production.Table 1 Production of various metabolites by different B. licheniformis strains
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To this point, the previously described results demonstrated the capability of the mutant WX-02ΔgdhΔacoR to produce meso-2,3-BD with a high purity and titer. A bench-scale fermentation was further developed in both batch and fed-batch modes to explore the potential of commercial production of meso-2,3-BD by this double-deletion mutant.
Figure 5 shows the batch fermentation profiles. The growth (Fig. 5a) and glucose consumption (Fig. 5b) of the mutant WX-02ΔgdhΔacoR were similar to the wild-type WX-02 strain. The WX-02ΔgdhΔacoR strain accumulated higher acetoin (Fig. 5c), but negligible D-2,3-BD isomer (Fig. 5d) compared to the wild-type. The meso-2,3-BD isomer produced from WX-02ΔgdhΔacoR strain was double that of the wild-type strain (Fig. 5e). The yield of meso-2,3-BD from glucose, Y meso-2,3-BD/glucose, was 0.35 g/g for WX-02ΔgdhΔacoR, also much higher than wild-type (0.16 g/g). Finally, lactic acid was still produced as the main by-product (Fig. 5f), while acetic acid and ethanol were less than 1.0 g/L throughout the fermentation process (data not shown). It should be noted that the lactic acid produced by the mutant WX-02ΔgdhΔacoR was similar to that produced by the wide type strain under fermenter culture conditions (Fig. 5f). This trend is different from that in flask culture (Table 1), where WX-02ΔgdhΔacoR produced less lactic acid than WX-02. The reason may be due to the different operation conditions between the flask culture and the fermenter culture.Fig. 5
Batch fermentation profile of B. licheniformis strains WX-02 and WX-02ΔgdhΔacoR in a bench top (5-L) fermenter. a Biomass density; b residual glucose; c acetoin production; d D-2,3-BD production; e meso-2,3-BD production; f lactic acid production
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Compared to the shake-flask culture results (Table 1), both the wild-type and WX-02ΔgdhΔacoR strains produced more meso-2,3-BD with less accumulation of acetoin in the fermenter culture. This was due to the use of the two-stage agitation/aeration, and thus the controlled dissolved oxygen (DO) levels, during the fermenter operation. It has been reported that DO levels play a crucial role in the reversible conversion of acetoin to 2,3-BD [14–16, 19]. In this work, the relatively high DO level generated by high aeration and agitation during the initial fermentation stage produced an increased amount of acetoin, resulting in the favorable conversion to meso-2,3-BD during the second stage when DO was intentionally reduced to a lower level.
A fed-batch fermentation using WX-02ΔgdhΔacoR was further developed to improve the meso-2,3-BD isomer titer. As shown in Fig. 6, glucose was maintained at 10-20 g/L throughout the entire culture period through periodic feedings. Meso-2,3-BD titer reached up to 98.0 g/L with a yield Y meso-2,3-BD/glucose of 0.40 g/g and productivity of 0.94 g/L–h, which was the highest meso-2,3-BD yield reported in Bacillus species (Table 2).Fig. 6
Fed-batch fermentation profiles of B. licheniformis mutant WX-02ΔgdhΔacoR in a bench top (5-L) fermenter with pH control. The tank was stirred at 350 rpm with 3-L/min aeration for first 16 h, and then decreased to 200 rpm and 1.5 mL/min for the remaining period. Glucose was fed to the medium from 24–70 h to maintain the residual glucose concentration between 10–20 g/L. Filled square Biomass; filled circle residual glucose; filled triangle meso-2,3-BD; ❊, Acetoin; ☆, Lactic acid; circle acetic acid; square ethanol. The arrow indicates the start of feeding the reactor with a concentrated glucose solution (650 g/L)
Full size imageTable 2 2,3-Butanediol (2,3-BD) production by native or engineered Bacillus strains
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