Enhanced Production of Tetramethylpyrazine in Bacillus licheniformis BL1 through aldC Over-expression and acetaldehyde Supplementation

Bacillus licheniformis BL1 was used as a starting strain to construct the recombinant tetramethylpyrazine (TMP)-producing strains by over-expression of the α-acetolactate decarboxylase gene (aldC) and α-acetolactate synthase gene (alsS), named BLC, BLS and BLCS, respectively. Then the addition of acetaldehyde was use to enhance the TMP yield in the fermentation process. During microaerobic fermentation, the aldC-overexpressed BLC strain produced 43.75 g TMP/L which was 15.47% higher than the TMP in culture yielded using the initial BL1 strain. Furthermore, the acetoin yield as TMP precursor similarly rose by 23.06% in BLC recombinant strain. In contrast, the 2,3-BD increased by 23.2% in the recombinant BLCS. TMP produced by BL1 could be bolstered via the supplementation of the acetaldehyde in fermentation medium. This method also has the same effect on the BLC strain.

Production comparation of TMP production by different bacteria strains. We selected an individual B. lincheniformis BL1 colony that was then added to 5 mL LB with the corresponding antibiotic used for selection. Bacteria were cultured for 12 h at 37 °C and 200 rpm, then 2% (v/v) of this mixture was collected and combined for 12 h in a 250 mL flask with 50 mL LB containing 10 g/L glucose. For fermentation, a 4% (v/v) inoculum was incubated with this culture at a starting optical density (OD 600 ) of 0.05 with the following parameters: a 500 mL flask containing 200 mL of LB and 70 g/L glucose was steadily mixed over 8 days at 200 rpm, and every 12 h a 5 mL volume of the glucose supplement (1 mg/mL) was added. The pH was maintained at 7.5 using 10 M NaOH solution.
Analytical methods. Fermentation   www.nature.com/scientificreports www.nature.com/scientificreports/ membrane biosensor (SBA-40C, Institute of Biology, Shandong Academy of Sciences, China) with a glucose oxidase-immobilized membrane was utilized for the measurement of glucose levels in the fermentation broth. TMP levels were established via the headspace solid-phase microextraction and gas chromatography-nitrogen, as in past studies 5,15 . Acetoin and 2,3-butanediol (2,3-BD) levels were measured by gas chromatography 16,17 . Statistical analysis. Experiment datas were accompanied by the number of experiments independently performed and expressed as mean ± SD. The differences of the acetaldehyde supplementation and the transformants, were confirmed by the Student's t test when compared with the parental strain. Differences at P < 0.05 were considered to be significant differences in statistics.
Effect of acetaldehyde supplementation in the fermentation process. The effect of supplemented acetaldehyde on TMP and acetoin production in BL1 fermentation process was explored via the addition of 1, 2, 4, and 8 g/L acetaldehyde in the medium. Acetaldehyde supplementation of BL1 media improved TMP yields, with the addition of 1 g/L to 2 g/L of supplemental acetaldehyde impacting cell growth (Table 3), TMP and acetoin, the concentration of residual glucose, and the yield of 2,3-BD (Fig. 4). The yield of TMP will increase with the increase of the dosage of acetaldehyde up to a dose of 2.0 g/L acetaldehyde, after which TMP levels do not  www.nature.com/scientificreports www.nature.com/scientificreports/ rise further (Table 3). Relative to unsupplemented BL1 (no dosage), the maximal TMP and acetoin yield rose by 13.83% (w/w) and 22.27% (w/w), respectively, in BL1R1 (BL1 with 1 g/L acetaldehyde). These increases were detected following 144 h and 168 h of culture, respectively (Fig. 3). These results suggest that a 1 g/L initial acetaldehyde concentration is ideal for maximizing the yield of TMP and acetoin in BL1.
Effect of the acetaldehyde supplemention on the fermentation process. By adding 1 g/L of acetaldehyde to the medium, the effect of acetaldehyde on TMP and acetoin generation by BLC was explored. The TMP and acetoin production were also impacted by acetaldehyde supplementation of the BLC fermentation medium (Fig. 5). Relative to unsupplemented BLC, maximal TMP and acetoin yields were improved from 43.75 g/L to 47.26 g/L (an increase of 8.1% (w/w) and from 18.73 g/L to 20.13 g/L (an increase of 7.5% (w/w) respectively in BLCR1 (BLC with 1 g/L acetaldehyde). When the two recombinant strains were grown for 144 h and 168 h, these increases were observed (Fig. 5). As such, 1 g/L is also an optimal starting acetaldehyde concentration for achieving maximal TMP and acetoin by recomibniant BLC strain. However, there was almost no change in the yield of 2,3-BD.

Discussion
When the recombinant strains were grown for 168 h and 120 h respectively, the yield of TMP and acetoin in recombinant BLC rose by 23.99% (w/w) and 28.98% (w/w) compared with Bacillus lincheniformis BL1. The yield of TMP and acetoin in recombinant BLS increased slightly at the same time periods (Fig. 3). Compared with Bacillus lincheniformis BLC, the yield of TMP and acetoin in Bacillus lincheniformis BLCS decreased slightly with 2,3-BD increased slightly (Fig. 3). This rise was primarily attributable to (i) a lack of de novo early stationary phase acetaldehyde production, and (ii) acetoin precursor accumulation during this same time period. When cultured for 72 h and 120 h, BL1 accumulated over 13.6 g/L of 2,3-BD and 15.2 g/L of acetoin, respectively. The aldC overexpressed mutant strain (BLC) accumulated less than 11.4 g/L 2,3-BD and 18.8 g/L acetoin when cultured for 72 h and 120 h, respectively, in media containing 0.96 g/L residual glucose. Although acetoin and 2,3-BD levels fell during the stationary phase (Fig. 6). A prior study has similarly found that there is a 2,3-BD degradation pathway in which acetoin functions as an intermediate 18 . In contrast, the production of 2,3-BD in recombinant BLS was more substantial than those of BL1 and BLC (Fig. 3).
After 120 h, acetoin levels in BL1 samples began to fall as a consequence of ongoing degradation (Fig. 6). Acetoin can serve as a carbon source or a TMP precursor for BL1 19 . The concentration of acetoin was increased in BLC; resulting in a higher TMP concentration. However, BL1 can metabolize acetoin more rapidly than can BLC 20 .  Table 3. Effect of acetaldehyde addition on TMP production by B. lincheniformis BL1 (n = 3). a Time in hours from inoculation to the maximal Acetoin concentration arrived. b Time in hours from inoculation to the maximal TMP concentration arrived. c T, temperature. d Values of the effect of acetaldehyde addition on production are significantly (Student's t test, P < 0.05, n = 3) different from the parental strain BL1. Data are average values and standard deviations of triplicate experiments. Interestingly, with the acetaldehyde concentration in BL1 media rose from 0 g/L to 1 g/L, TMP and acetoin yields similarly rose from 37.89 g/L to 44.77 g/L and from 15.22 g/L to 17.94 g/L, respectively. Thus, acetaldehyde can facilitate TMP and acetoin production in a dose-dependent fashion. A 1 g/L acetaldehyde concentration was sufficient to achieve maximal TMP and acetoin yields. Nevertheless, the mechanism that the improvement of acetoin with acetaldehyde supplementation is poorly understood. We have hypothesized from the existing acetoin cleavage pathway that degradation of acetoin in microbial cells occurs on two levels. The first is the reversible transformation between acetoin and 2,3-BD. Secondly, acetoin can be used to produce acetyl-CoA and acetaldehyde under the action of the acetoin dehydrogenase complex (Ao DH ES), and acetaldehyde can then be converted into acetic acid or ethanol. Thus, the acetoin dehydrogenase system can catalyze the conversion of acetoin to acetaldehyde, and 2,3-BD dehydrogenase or acetoin reductase likely catalyzes the conversion between 2,3-butanediol (2,3-BD) and acetoin 21 . Therefore, in our study, the initial acetaldehyde may have a feedback inhibition effect on the catalytic conversion of acetoin into acetaldehyde, which raises the concentration of acetoin and 2,3-BD, and also enhances the ability to synthesize TMP (Fig. 4). Alternatively, the initial addition of acetaldehyde could have a feedback inhibition effect on the Ao DH ES, which affects the conversion of acetoin into acetaldehyde and results in the accumulation of acetoin production, enhancing the ability to synthesize TMP in turn. In addition, the initial acetaldehyde can enhance the metabolism of acetaldehyde to acetyl-CoA, which indirectly provides feedback inhibition of pyruvate to the acetyl-CoA metabolic branch, thereby enhancing the pyruvate to acetoin metabolic branch (Fig. 2). So, when the acetaldehyde was added into the medium, there was an accumulation of the precursor acetoin in BL1 or BLC and an increased yield of TMP (Figs. 5 and 7). Acetaldehyde is less favorable for cell growth (Fig. 4). Enzymatic activity is most robust when high levels are carbohydrates are available, and they fall once these carbon sources are exhausted 22 . Thus, the initial suppression of the acetoin dehydrogenase system by acetaldehyde can impede the synthesis of acetaldehyde when remaining glucose levels at the end of fermentation were minimal. There is further evidence suggesting that 2,3-BD can be utilized as a carbon source in order to produce acetoin in the context of low carbon availability 14 . However, cell growth will be adversely affected, and the product synthesed also be inhibited (Table 3) when the specific inhibitory concentration of acetaldehyde reached. The acetaldehyde inhibitory mechanisms is still unclear, suggesting that there may be certain differentially regulated enzymes that can be impacted by acetaldehyde and which are involved in metabolic or synthetic processes, or the transfer process could be induced by different enzymes. Further study of the inhibitory role of acetaldehyde is thus warranted.