Construction of (2R,3R)-2,3-butanediol generation pathway and optimization of the expression level of butanediol dehydrogenases
CGS9 is a (3R)-acetoin overproducing strain with three copies of the alsSD operon in the genome, in which biosynthesis pathways of the major by-products were disrupted and the TCA cycle was weakened by downregulating the expression of the gltA gene. In order to convert (3R)-acetoin to (2R,3R)-2,3-butanediol, the gene bdhA encoding 2,3-butanediol dehydrogenase from B. subtilis 168, under control of the constitutive promoter Ptrc (without lacO sequence), was inserted into the genome of CGS9 at the ∆ldh locus, generating the strain CGK1. Strains were cultivated in CGXIIP medium containing 40 g/L glucose. As shown in Fig.3A, the final (2R,3R)-2,3-butanediol production by strain CGK1 reached 12.38 g/L, with a yield of 0.341 g/g glucose at 24 h. As expected, the by-products acetate (0.16 g/L), lactic acid (0.35 g/L), glycerin (0.14 g/L) and succinate (<0.01 g/L) were all at low concentrations at 24 h, benefiting from the deletion of relevant genes. It was noticed that (3R)-acetoin was the main by-product and its titer reached 3.50 g/L at 24 h, which may be caused by higher gene dosage of alsSD (3 copies) and the relative lower gene dosage of bdhA (1 copy). Consequently, the enzyme activity of BDH could be not sufficient for converting all the (3R)-acetoin into (2R,3R)-2,3-butanediol.
The expression level of heterologous genes was affected by several factors, including gene dosage, promoter strength, secondary structure of mRNA and RBS sequence. To improve the expression level of bdhA, its promoter Ptrc and RBS in genome of CGK1 were replaced by the strong promoter Psod and RBS-10, which was designed for bdhA with the highest translation initiation rate (TIR). The resulting strain CGK2 showed a significant increase in BDH enzyme activity and (2R,3R)-2,3-butanediol production. As shown in Fig. 2. the BDH activity of CGK2 was 1.95-fold higher than that of CGK1 at 12 h (exponential phase) and still remained at a high level at 24 h (stable phase). Strain CGK2 produced 16.58 g/L (2R,3R)-2,3-butanediol with a yield of 0.405 g/g glucose, which was 18.8% higher than that of CGK1. Meanwhile, (3R)-acetoin titer was decreased to 2.43 g/L (Fig. 3B) but was still at a higher level, most likely due to the insufficient of reducing power in the form of NADH. The total yield of (2R,3R)-2,3-butanediol and (3R)-acetoin of CGK2 reached 0.455 g/g glucose at 24 h, which was 5.8% higher than that of CGK1. These results suggested that carbon flux was further directed toward (2R,3R)-2,3-butanediol synthesis by improving the expression level of bdhA.
Improvement of NADH supply for reducing the accumulation of (3R)-acetoin byproduct
The conversion between (2R,3R)-2,3-butanediol and (3R)-acetoin could regulate the redox balance. The udhA gene from E. coli W1485 encodes a transhydrogenase that partially converts NADPH to NADH, and the latter is beneficial to the conversion of (3R)-acetoin to (2R,3R)-2,3-butanediol. The artificial operon driven by the strong promoter Psod, consisting of the genes bdhA and udhA with RBS-10, was introduced into the chromosome of CGS9 at the ∆ldh locus to generate strain CGK3.
As expected, comparing the reducing power in strains CGK2 and CGK3 at 12 h, the NADPH/NADP+ ratio significantly reduced from 1.19 to 0.74, and the NADH/NAD+ ratio increased from 0.72 to 0.94 (Fig. 4), which demonstrated the ability of udhA to regulate NADPH/NADP+ ratio and provided a strategy for the regulation of cofactor. As shown in Fig.3C, strain CGK3 accumulated 16.47 g/L (2R,3R)-2,3-butanediol and its yield increased by 6.2%, reaching 0.430 g/g glucose at 24 h. The (3R)-acetoin titer of CGK3 was 1.04 g/L at 24 h, which decreased by 57.2% compared with that of CGK2. In addition, there was no significant difference in the activities of BDH between CGK2 and CGK3 (Fig. 2). It was founded that the total yield of (2R,3R)-2,3-butanediol and (3R)-acetoin decreased slightly. Meanwhile, the glucose consumption rate of CGK3 decreased by 6.6% compared with that of CGK2 (Table 2), which might due to that the growth of CGK3 was inhibited by the change of reducing power. Thus, the glucose consumption rate needs to be improved, which can lead to increase in (2R,3R)-2,3-butanediol productivity.
Decrease in ATP content to increase glucose consumption rate
It was observed that microorganisms could increase the glycolytic flux to compensate for the lack of ATP, which improved the consumption of glucose. Therefore, a strategy for reducing the biosynthesis of ATP was taken to increase the glucose consumption rate of strain CGK3. It was generally considered that the bacterial atp operon structure consisted of the gene atpI, atpB, atpE, atpF, atpH, atpA, atpG, atpD and atpC, and the γ subunit of H+-ATPase encoded by the gene atpG worked as the main shaft for the rotation of the H+-ATPase rotor. According to a previous report, the activity of H+-ATPase was reduced to 70% of the original by replacing T to C at 817bp and C to T at 818bp of atpG in C. glutamicum ATCC14067, which resulted in an increase of 24% in specific glucose consumption during the exponential phase. Thus, the mutations were introduced into strain CGK3 to test the effect on (2R,3R)-2,3-butanediol production, yielding strain CGK4.
As shown in Fig. 3D, the glucose consumption of per cell of strain CGK4 increased by 11.8% and reached 1.05 g/L/OD during the exponential phase (0-12h), which was lower than that described in the report. It might result from the different original strain ATCC13032, instead of ATCC14067, used in this study. Another reason might be the different genotype of CGK3, in which several pathways for byproducts synthesis were blocked and the TCA cycle was weakened. The whole glucose consumption rate of CGK4 was 10.5% higher than that of CGK3 (42.28 vs 38.26 g/L, Table 2). Correspondingly, the (2R,3R)-2,3-butanediol titer of CGK4 reached 18.27 g/L with an increase of about 10.9% in productivity at 24 h, and the yield of (2R,3R)-2,3-butanediol was almost the same as that of CGK3 (0.432 g/g glucose), which was about 86% of the theoretical yield. The titer of acetoin was 1.2 g/L, which was still at a low level.
Effect on (2R,3R)-2,3-butanediol production by overexpressing bdhA and udhA
To test if the increase of the expression level of bdhA and udhA can further enhance the production and yield of (2R,3R)-2,3-butanediol, an episomal plamid pECK1 with additional copy of bdhA-udhA was constructed and introduced into strain CGK4, generating strain CGK5. As shown in Fig. 3E, the growth and glucose consumption rate of CGK5 were obviously decreased compared with that of CGK4, and decreased (2R,3R)-2,3-butanediol production was obtained with a titer of 14.41 g/L at 24 h. When glucose was depleted at 30 h, the final (2R,3R)-2,3-butanediol titer reached 18.81 g/L. Although strain CGK5 showed a litter higher (2R,3R)-2,3-butanediol titer than CGK4 (18.65 g/L at 26h, Fig 3D), its productivity decreased by 12.7% compared with strain CGK4. It was presumed that the metabolic burden exerted by the expression plasmid and the imbalance of NADH resulted in the decrease of growth and glucose consumption rate (Table 2). The BDH activities of CGK5 were 1.72-fold and 1.66-fold higher than those of CGK4 at 12 h and 24 h, respectively, but the final yield of (2R,3R)-2,3-butanediol was similar when the glucose was depleted, which indicated the expression level of bdhA and udhA in CGK4 was sufficient to convert (3R)-acetoin to (2R,3R)-2,3-butanediol. Considering the stability of the production strain and bio-safety, strain CGK4 without plasmid and marker gene (antibiotic resistance gene) was chosen to further produce (2R,3R)-2,3-butanediol in fed-batch fermentation.
Fed‑batch fermentation of CGK4 to produce (2R,3R)-2,3-butanediol
To evaluate potential of CGK4 for further industrial application, strain CGK4 was cultured in LBRC medium in a 5-L fermenter without the addition of antibiotics for fed-batch fermentation. As shown in Fig. 5A, a titer of 124.2 g/L (2R,3R)-2,3-butanediol was obtained at 119 h with a productivity of 1.04 g/L/h. It was found that (2R,3R)-2,3-butanediol concentration no longer increased after 119 h, and there was still 15 g/L of glucose in the fermenter. The (2R,3R)-2,3-butanediol yield was 0.410 g/g glucose, which was 82% of the theoretical yield. During the first 24 hours of fermentation, the biomass grew rapidly to an OD600 of 70, and dissolved oxygen also dropped to less than 2%, and then fluctuated between 1% to 2%. The dissolved oxygen gradually increased to 70% from 72 h to 130h. Meanwhile, the (3R)-acetoin production increased significantly during this stage and reached a final concentration of 24.50 g/L at 130 h. It can be inferred that the increased dissolved oxygen during 72-130h resulted in an increase in NADH consumption by respiration, and a shortage in NADH supply for the conversion of acetoin to 2,3-butanediol.
It is well known that control of oxygen supply is the most critical factor for efficient production of 2,3-butanediol. The anaerobic conversion of glucose to 2,3-butanediol is not feasible due to redox imbalance (one NADH is produced in excess). Therefore, the oxygen supply was adjusted to maintain a microaerobic condition during the fermentation to reduce the NADH consumption by respiratory. When the titer of (3R)-acetoin reached about 15 g/L (about 96 hours), the aeration and rotational speed were adjusted to keep the dissolved oxygen between 0.5% to 1%. As shown in Fig. 5B, a final titer of 144.9 g/L (2R,3R)-2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h was obtained by strain CGK4, and the optical purity of (2R,3R)-2,3-butanediol was over 99% (Additional file 1: Fig. S1). In addition, the final concentrations of the by-products α-ketoglutarate, glycerin, acetate and acetoin were 1.61, 1.49, 1.75 and 1.93 g/L, respectively. No succinate and lactate were detected. To the best of our knowledge, this is the highest titer of (2R,3R)-2,3-butanediol production achieved by GRAS strains.
However, there is still much room to improve the (2R,3R)-2,3-butanediol productivity of C. glutamicum compared with other over-producing strains (Table 3). It was reported that productivity of the target product is closely related to the carbohydrate uptake capacity.In our future study, further improvement may be achieved by constructing an ATP futile cycle system  or enhancing the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), which is the major carbohydrates uptake system in C. glutamicum.