Metabolic Engineering of Bacillus licheniformis for Production of Acetoin

Acetoin is a potential platform compound for a variety of chemicals. Bacillus licheniformis MW3, a thermophilic and generally regarded as safe (GRAS) microorganism, can produce 2,3-butanediol with a high concentration, yield, and productivity. In this study, B. licheniformis MW3 was metabolic engineered for acetoin production. After deleting two 2,3-butanediol dehydrogenases encoding genes budC and gdh, an engineered strain B. licheniformis MW3 (ΔbudCΔgdh) was constructed. Using fed-batch fermentation of B. licheniformis MW3 (ΔbudCΔgdh), 64.2 g/L acetoin was produced at a productivity of 2.378 g/[L h] and a yield of 0.412 g/g from 156 g/L glucose in 27 h. The fermentation process exhibited rather high productivity and yield of acetoin, indicating that B. licheniformis MW3 (ΔbudCΔgdh) might be a promising acetoin producer.


INTRODUCTION
Acetoin (3-hydroxy-2-butanone) is widely used as a common additive in food industry and a building block for various chemicals (Liu et al., 2011;Xiao and Lu, 2014;Yang et al., 2017). It was listed as one of 30 platforms compound whose development and utilization was given priority by the US Department of Energy in 2004 (Xiao and Lu, 2014). Currently, commercially available acetoin is primarily produced from fossil feedstocks by chemical methods involving radical reactions and severe environmental pollution. With increasing concerns about production cost and environment protection, microbial fermentation production of acetoin has attracted extensive attention in recent years (Xiao and Lu, 2014;Yang et al., 2017).
Many native or recombinant microorganisms, including the genera Serratia, Klebsiella, Saccharomyces, Gluconobacter, and Bacillus, have abilities to produce acetoin (Sun et al., 2012;Wang et al., 2013Wang et al., , 2017Bae et al., 2016;Jia et al., 2017). Among these species, Bacillus species which can produce various industrial products have become the research hotspot for acetoin production (Zhang Y. et al., 2013;Fan et al., 2018;Liu et al., 2019). Acetoin is an intermediate of 2,3-butanediol fermentation-pathway (Ji et al., 2011;Yang and Zhang, 2019). Except a few native acetoin producers, most of the Bacillus strains produce 2,3-butanediol but not acetoin as their major product Dai et al., 2019). Thus, different metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin in various Bacillus strains. For example, Zhang et al. successfully improved acetoin production by Bacillus subtilis through disruption of acetoin reductase (also called as 2,3-butanediol dehydrogenase) and expression of a water-forming NADH oxidase. Acetoin at a concentration of 56.7 g/L was produced with a yield of 0.378 g/g and a productivity of 0.639 g/[L h] (Zhang et al., 2014b).
Bacillus licheniformis, a generally regarded as safe (GRAS) strain with the characteristics of strong sugar consumption and fast growth rate, is a promising chassis strain for acetoin production. Natural B. licheniformis strains usually produce a mixture of (2R,3R)-2,3-butanediol and meso-2,3-butanediol isomers Qi et al., 2014;Qiu et al., 2016). In this study, two 2,3-butanediol dehydrogenases encoding genes in B. licheniformis MW3 were deleted and an engineered strain B. licheniformis MW3 ( budC gdh) was constructed. The recombinant strain B. licheniformis MW3 ( budC gdh) can produce 64.2 g/L acetoin within 27 h with a productivity of 2.378 g/[L h] and a yield of 0.412 g/g. Considering its desirable characteristics, B. licheniformis MW3 ( budC gdh) may be a promising alternative for fermentative production of acetoin.

Bacterial Strains, Media, and Plasmids
All the strains and plasmids used in this study are listed in Supplementary Table S1. The plasmids pKVM1-budC and pKVM1-gdh based on the temperature sensitive plasmid pKVM1 were used for gene knockout in B. licheniformis MW3 (Waschkau et al., 2008;Rachinger et al., 2013;Ge et al., 2016). Lysogenic broth (LB) medium was used for cultivation of Escherichia coli and B. licheniformis. NB medium (8 g/L Nutrient Broth, Difco) supplemented with 50 mM glucose was used for selection of B. licheniformis containing pKVM1. Ampicillin was used at a concentration of 100 µg/mL for the selection of E. coli. Erythromycin (5 µg/mL) and polymyxin B (40 µg/mL) were used for the selection of B. licheniformis. X-Gal was added at a concentration of 40 µg/mL for blue-white screening.
Gene Knockout in B. licheniformis MW3 E. coli S17-1 λpir was used as donor strain to allow the conjugal transfer of plasmids pKVM1-budC and pKVM1gdh into B. licheniformis MW3. After growth in liquid NB/glucose medium with erythromycin at 30 • C, serial dilutions and spreading on NB/glucose plates with erythromycin and X-gal and incubation at 42 • C, one of blue colonies of B. licheniformis MW3 with plasmid integration by homologous recombination was picked, inoculated in NB/glucose medium without antibiotic, and cultivated at 30 • C for at least two passages. After dilutions and spreading on NB/glucose with X-gal, white colonies in which plasmids used to knockout were cured resulting from a second recombination event were picked. PCR was used to verify the disruption event of gene budC and gdh using primer pairs budC-f/ budC-r and gdh-f/ gdh-r (Supplementary Table S2), respectively.

Enzyme Activity Assays
To measure 2,3-butanediol dehydrogenase activities in B. licheniformis MW3 and B. licheniformis MW3 ( budC gdh), the cells of the strains were grown for 12 h, harvested, washed twice and resuspended in 67 mM phosphate buffer (pH 7.4), and disrupted by sonication with a Sonics sonicator (500 W; 20 kHz). Cell debris was removed through centrifugation (12,000 g, 15 min) and the resulting supernatants were used as the crude extracts for enzyme activity assays.
Activities of 2,3-butanediol dehydrogenase were spectrophotometrically assayed by measuring the change in absorbance at 340 nm corresponding to the oxidation of NADH or reduction of NAD + at 30 • C using a UV/visible spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, United States). For the reduction reaction, the reaction solution contains 0.2 mM of NADH and 5 mM of acetoin or diacetyl in 67 mM phosphate buffer (pH 7.4). For oxidation reactions, the reaction solution contains 10 mM (2R,3R)-2,3-butanediol, (2S,3S)-2,3-butanediol, and meso-2,3-butanediol and 1 mM NAD + in 67 mM phosphate buffer (pH 7.4). One unit of activity was defined as the amount of enzyme that consumed or formed 1 µmol of NADH per min. The protein concentration in crude extract was measured by the Lowry method, with bovine serum albumin as the standard (Hartree, 1972).

Batch and Fed-Batch Fermentations
B. licheniformis MW3 was maintained on LB agar slants at 4 • C. A loop of cells from the fully grown slant was inoculated into 100 mL of LB in 500-mL Erlenmeyer flasks and incubated at 50 • C on a rotary shaker at 180 r/min for 12 h to prepare the seed culture. Then, the seed culture was inoculated (5%, v/v) into the bioreactors for acetoin production.
Batch and fed-batch fermentations were conducted in a 1-L bioreactor (Infors AG, Bottmingen, Switzerland) with 0.8 L initial medium and a 5-L bioreactor (BIOSTAT B, B. Braun Biotech International GmbH, Germany) with 4 L initial medium, respectively. The seed culture was inoculated into the fermentation medium containing about 70 g/L glucose; 1 g/L triammonium citrate; 12 g/L yeast extract; 2 g/L K 2 HPO 4 ·3H 2 O; 6.5 g/L sodium acetate; 0.25 g/L MgSO 4 ·7H 2 O; 0.0225 g/L FeSO 4 ·7H 2 O; 0.0075 g/L ZnSO 4 ·7H 2 O; and 0.0038 g/L MnSO 4 ·H 2 O. The cultivation was carried out at 50 • C, stirring at 500 r/min, and airflow at 1.0 vvm (volume air per volume broth per minute). The pH was maintained at 7.0 by automatic addition of 6 M acetic acid and 6 M NaOH using a program-controlled peristaltic pump. Samples were collected periodically to determine the biomass, concentrations of glucose, 2,3-butanediol, and acetoin.

Analytical Methods
Samples were centrifuged at 12,000 g for 10 min and the concentration of glucose was measured enzymatically using a bioanalyzer (SBA-40D, Shandong Academy of Sciences, China) after diluting to an appropriate concentration. The concentrations of 2,3-butanediol and acetoin were analyzed by gas chromatography (GC; GC2014c, Shimadzu) with capillary GC columns (AT. SE-54, inside diameter, 0.32 mm; length, 30 m; Chromatographic Technology Center, Lanzhou Institute of Chemical Physics, China) as described previously .

Batch Fermentation of Acetoin by B. licheniformis MW3 ( budC gdh)
Batch fermentation was conducted in a 5-L bioreactor with 4 L initial medium to analyze acetoin production by B. licheniformis MW3 ( budC gdh). As shown in Figure 2, inactivation of both budC and gdh led to a high acetoin accumulation in B. licheniformis MW3 ( budC gdh). This strain consumed 63 ± 1 g/L glucose in 14 h and produced 27.4 ± 1.45 g/L acetoin, with a yield of 0.435 ± 0.023 g/g (Figure 2). Although the growth of B. licheniformis MW3 ( budC gdh) was slightly lower than that of B. licheniformis MW3, this strain can produce acetoin instead of 2,3-butanediol as its major product (Supplementary Table S4).

Fed-Batch Fermentation of Acetoin by B. licheniformis MW3 ( budC gdh)
To achieve higher acetoin concentrations, fed-batch fermentation was carried out with an initial glucose concentration of about 70 g/L using B. licheniformis MW3 ( budC gdh). As shown in Figure 3, 64.2 g/L acetoin from 156 g/L glucose was obtained in 27 h by B. licheniformis MW3 ( budC gdh). The yield of acetoin was 84.1% of the theoretical value and the average productivity was 2.378 g/[L h]. The carbon flux channeled into the acetoin biosynthetic might be further enhanced since there were still 2,3butanediol accumulated during the fermentation (Figure 3 and Supplementary Figure S1). Toxicity of acetoin can hinder its higher production by various microorganisms. It was reported that an approximate 30% cell growth of B. licheniformis will be inhibited with 40 g/L exogenous acetoin (Yuan et al., 2019). Thus, production of 2,3-butanediol may be due to induction of an unidentified acetoin reductase or meso-2,3-butanediol dehydrogenase to resistant the toxicity of acetoin at high concentrations. Higher production of acetoin by B. licheniformis may be accomplished by searching the undiscovered acetoin reductase or overexpressing NADH oxidase, which could lead to prevention of NADH-dependent reduction of acetoin.
FIGURE 2 | Time-course of batch fermentation by B. licheniformis MW3 ( budC gdh). , Glucose; , OD 620nm ; , 2,3-butanediol; , acetoin. The experiment was conducted in a 1-L bioreactor (Infors AG, Bottmingen, Switzerland) with 0.8 L initial medium with an initial glucose concentration of 70 g/L approximately. Cultivation was carried out at 50 • C and an initial pH of 7.0. The pH was maintained at 7.0 by automatic addition of 6 M acetic acid and 6 M NaOH. The agitation speed was 500 r/min and the aeration rate was 1 vvm. Data were the means ± SDs from three parallel experiments. strategy during which 2,3-butanediol was first accumulated and then converted to acetoin using B. licheniformis WX-02 budC acoR can produce 78.79 g/L acetoin with a yield of 0.31 g/g and a productivity of 0.58 g/[L h] at 37 • C (Li et al., 2017). B. licheniformis MW3 is a thermophilic strain which can produce 2,3-butanediol as its major fermentation product with higher yield and productivity than that of B. licheniformis WX-02 at 50 • C, implying a latent capacity of strain MW3 for efficient acetoin production Qiu et al., 2016).
Thus, B. licheniformis MW3 ( budC gdh) was constructed and the recombinant strain was able to produce 64.2 g/L acetoin with a relatively high productivity (2.378 g/[L h]) through a simple fermentation process. The values of productivity and yield of acetoin produced using B. licheniformis MW3 ( budC gdh) were the highest ever obtained in acetoin production by recombinant Bacillus species. B. licheniformis MW3 ( budC gdh) might be useful for the production of acetoin on a commercial scale. . , Glucose; , OD 620nm ; , 2,3-butanediol; , acetoin. The experiment was conducted in a 5-L bioreactor (BIOSTAT B, B. Braun Biotech International GmbH, Germany) with 4 L initial medium with an initial glucose concentration of 70 g/L approximately. Cultivation was carried out at 50 • C and an initial pH of 7.0. The pH was maintained at 7.0 by automatic addition of 6 M acetic acid and 6 M NaOH. The agitation speed was 500 r/min and the aeration rate was 1 vvm. The fed-batch fermentation was conducted by feeding glucose solution when the residual glucose in the fermentation was lower than 10 g/L. Data were from a representative fed-batch fermentation repeated for three times.

CONCLUSION
We constructed a recombinant strain B. licheniformis MW3 ( budC gdh) for acetoin production. Acetoin at a high concentration (64.2 g/L) was produced with a productivity of 2.378 g/[L h] and a yield of 0.412 g/g through fed-batch fermentation by B. licheniformis MW3 ( budC gdh). Because of its GRAS characteristic and ability to produce acetoin with