Biosynthesis of 4-hydroxybenzylideneacetone by Whole-Cell Escherichia coli

Abstract

4-Hydroxy benzylideneacetone (4-HBA) is an organic synthesis intermediate and can be used as a precursor for the synthesis of raspberry ketone. Herein, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) was overexpressed in E. coli BL21 (DE3) as an attractive catalyst for enzymatic aldol reactions. The aldol reaction between 4-hydroxybenzaldehyde (4-HBD) and acetone to biosynthesize 4-HBA was catalyzed by whole-cell E. coli BL21 (DE3) (pRSF-Deoc). The yield and 4-HBA concentration were 92.8% and 111.35 mM, respectively, when using 120 mM 4-HBD and acetone as substrates. When the concentration of 4-HBD was increased to 480 mM, 376.4 mM 4-HBA was obtained by a fed-batch strategy with a yield of 78.4%, which was about a 28% improvement compared to the one-time addition strategy. E. coli BL21 (DE3) (pRSF-Deoc) cells were further immobilized with K-carrageenan, and the immobilized cells still maintained a residual activity of above 90% after 10 repeated uses. Our study provides a promising method of biosynthesizing 4-HBA.

1. Introduction

4-Hydroxybenzylideneacetone (4-HBA) is an important organic synthesis intermediate and fine chemical raw material, which can be used as a precursor for the synthesis of raspberry ketone. The hydrogenation product of 4-HBA is raspberry ketone, which is the second-most fragrant compound after vanillin with a total potential market value between EUR 6 and 10 million [1]. There are also related studies showing that 4-HBA has anti-cancer and anti-inflammatory functions, and can be used in the health care industry [2]. 4-HBA is a structural analog of dehydrogingerone. Its synthesis mainly consists of an aldol condensation reaction of substituted aromatic aldehyde and acetone under the catalysis of a basic substance or Lewis acid. At present, the main method of 4-HBA production is the condensation reaction of 4-hydroxybenzaldehyde (4-HBD) and acetone catalyzed by alkali, which easily causes equipment corrosion and environmental pollution. In recent years, the production of flavors and fragrances by microorganisms has received significant attention [3,4]. Microorganisms are usually gentler and more efficient, which is more in line with the concept of green chemistry [5].

Aldol addition is one of the most useful methods for carbon–carbon bond formation in organic synthesis [6]. A mutant of lipase from candida antarctica (CAL-B) was first reported to catalyze aldol addition with low activities by Berglund et al. [7]. Several lipases have demonstrated catalytic activities for asymmetric aldol reactions, in which lipase from porcine pancreas (PPL) showed the activity to catalyze asymmetric aldol condensation of acetone and substituted benzaldehyde [8]. Unfortunately, these reactions are accompanied by the formation of alcohol–ketone by-products due to the lack of specificity of lipase. Alternatively, aldolase can also catalyze the reversible formation of C-C bonds via aldol addition. DERAs (EC4.1.2.4) are widely used in industry due to their broad substrate spectrum [9,10,11,12]. For example, DERA has been successfully employed in whole-cell biotransformation to produce chiral lactol intermediates, which are useful for the synthesis of optically pure superstatins such as rosuvastatin and pitavastatin [7]. Biosynthesis of 4-HBA is rarely reported; G. Feron et al. successfully synthesized 4-HBA by fermentation with Escherichia coli ATCC 886963, Bacillus subtilis ATCC 31324, and Bacillus cereus. Although the highest 4-HBA concentration was only 0.9 mM, the results showed that DERA_Ec catalyzed the condensation of 4-HBD with acetone, and subsequently, 4-HBA was prepared by β-elimination dehydration [13]. As a result, finding a better process to boost 4-HBA production by DERA_Ec is critical.

Whole-cell catalysis has the advantages of low catalytic cost and easy separation of substrate products [14]. Compared with single-enzyme catalysis, it has higher catalytic stability and substrate tolerance because of its protective structure-bearing cell wall and cell membrane [15,16]. Compared to other natural microorganisms, Escherichia coli is easy to genetically manipulate and is a commonly used whole-cell catalyst [17]. More importantly, DERA_Ec has the ability to catalyze the aldol condensation reaction of 4-HBD and acetone [18]. In addition, whole-cell catalysts are beneficial for attenuating the inactivation of DERA by aldehydes [19]. Since DERA prefers aldehydes as acceptors for the aldol condensation reaction, the acetone concentration in the reaction becomes particularly critical. DERA_Ec has a good acetone tolerance [20]. In this study, the E coli BL21 (DE3) overexpressing DERA was used as the whole-cell catalyst to condense 4-HBD and acetone (Figure 1), and a fed-batch strategy was used with immobilized cells evaluated for improved operational efficiency.

2. Results and Discussion

2.1. Effect of Reaction Media on the Biosynthesis of 4-HBA

A high-copy plasmid pRSF-duet-1 was used to overexpress the Deoc gene derived from E. coli BL21 (DE3), and the engineered strain E. coli BL21 (DE3)/pRSF-Deoc was obtained with a significantly high amount of soluble DERA_Ec (Figure S1). The engineered strain was then used as a whole-cell catalyst for condensation of 4-HBD and acetone to produce 4-HBA. According to Figure 2, at the same 4-HBD concentration, the higher the ratio of acetone to 4-HBD, the higher the concentration of 4-HBA was achieved. The highest 4-HBA concentration was obtained when pure acetone was used as the reaction medium, indicating acetone concentration has a great influence on the reaction. S. Milker et al. investigated the aldolase activity of PPL for catalyzing 4-nitrobenzaldehyde (4-NBA) and acetone in different deep eutectic solvents and a pure acetone system, and they found that the fastest and most effective biotransformation was performed in the co-solvent acetone [21]. This seems to be similar to our result.

Although acetone is not the preferred donor in aldol addition by DERA [19], in the reaction of transforming 4-HBD to 4-HBA, acetone was used not only as a substrate but also as a reaction medium for improving the solubility of the substrate 4-HBD and promoting the dehydration of β-aldol. Therefore, the pure acetone reaction system was selected for subsequent optimization of bio-synthesis of 4-HBA.

2.2. Biosythesis of 4-HBA from 4-HBD

The effects of reaction temperature and substrate 4-HBD concentration on whole-cell catalysis were investigated under a pure acetone system (Figure 3). Since the thermostability of aldolases increased with the increase in optimal growth temperature of the source organism [22], the catalysis was carried out at a temperature ranging from 30 °C to 70 °C. According to Figure 3A, the product yield gradually increased with the increase in temperature and reached the maximum value at 60 °C. As a result, 60 °C was chosen as the reaction temperature.

The effect of 4-HBD concentration varying from 120 mM to 480 mM on 4-HBA production was investigated under the reaction temperature of 60 °C in order to overcome the problem of substrate toxicity, and the results are shown in Figure 3B. The time curve of 4-HBA production showed that the initial concentration of 4-HBA increased with the increasing 4-HBD concentration. When the 4-HBD concentration was 360 mM, 4-HBA concentration reached a maximum value of 227.16 mM. However, a further increase in 4-HBD concentration can hardly increase the product concentration. The product yields dropped sharply with the increase in 4-HBD concentration. When the 4-HBD concentration was 120 mM, the yield and concentration of 4-HBA were 92.8% and 111.3 mM, and when it increased to 480 mM, the yield decreased to 49.6% (Figure 3C). Since benzaldehyde organics have a general inhibitory effect on cells and enzymes, this result also verifies our conjecture that high concentrations of 4-HBD have an obvious inhibitory effect on the whole-cell catalyst. In order to confirm that the cells remained intact after the reaction, we provided SEM images before and after the catalytic reaction in the supplementary material, as shown in Figure S4.

2.3. Fed-Batch Strategy for Improved Biosynthesis of 4-HBA

According to Figure 3B, when 480 mM of 4-HBD was added at one time, the production curve of 4-HBA tended to be flat after 36 h of reaction, and the final 4-HBA concentration was comparable to that of when 4-HBD concentration was 360 mM. This result indicated that 480 mM of 4-HBD had a strong inhibitory effect on the enzyme. In order to avoid this phenomenon, 360 mM of 4-HBD was used as the initial substrate concentration, and the concentrations of 4-HBD and 4-HBA were continuously monitored during the reaction. The results are depicted in Figure 4. After 24 h of reaction, the concentration of 4-HBD dropped to 88.5 mM, at which point 120 mM of 4-HBD was added. When the reaction time was 48 h, 343.6 mM of 4-HBA was obtained with a yield of 71.5%, which was 21.9% higher than when 480 mM of 4-HBD was added at one time. The 4-HBA concentration reached the maximum value of 376.4 mM after 84 h of reaction, corresponding to a yield of 78.4%. However, when the 4-HBD concentration dropped to about 120 mM for the second time, adding 120 mM of 4-HBD to continue the reaction did not cause the 4-HBA concentration to rise again (data not shown), which indicated that long-term use of free cells is limited.

2.4. Biosynthesis of 4-HBA by Immobilized Cells

Immobilized E. coli BL21 (DE3)/pRSF-Deoc in K-carrageenan was used in order to realize the reuse of cells. Each batch of reaction was carried out at 120 mM 4-HBD and 60 °C for 24 h, and the results are shown in Figure 5. The obtained immobilized cells were used for multiple batches of catalysis. Each batch was run at 120 mM 4-HBD and 60 °C for 36 h. In the first 10 batches of reaction, the product concentration fluctuated between 50 and 90 mM. Among them, the conversion rate gradually decreased from batch 1 to batch 3, indicating that the immobilized cells were in the mass transfer stage, and that the conversion rate stabilized after the third batch and decreased after 10 batches. The residual cell activity remained at 90% after the 10 cycles (360 h), and after 13 batches of biosynthesis, the molar conversion rate dropped to 34%, indicating a deactivation of immobilized cells.

3. Materials and Methods

3.1. Strains, Plasmids, Primers, and Chemicals

The strains, plasmids, sequence of Deoc, and primers used in this work were listed in Table S1–S3. 4-HBD and acetone were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Isopropyl β-D-thiogalactoside (IPTG), genome kit, antibiotics, and protein markers were obtained from Sangon (Shanghai, China). Prime STAR MAX DNA polymerase, DNA marker, DNA ligation kit, and restriction enzymes were provided by Takara (Shanghai, China). FastPure Plasmid Mini Kit and FastPure Gel Extraction DNA Mini Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). SDS-PAGE gel quick preparation kit was obtained from Beyotime Biotechnology, Shanghai, China. K-carrageenan was obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade.

3.2. Construction of Gene Overexpression Plasmid

E. coli JM109 (Novagen, Germany) was used for plasmid construction, and E. coli BL21 (DE3) (Novagen, Germany) was used for plasmid cloning and recombinant molecule production. The expression vector pRSFDuet-1 was supplied by Novagen (Darmstadt, Germany). Plasmids construction and DNA manipulation were performed following the standard molecular cloning protocols. The whole genome of E. coli BL21 (DE3) was extracted according to the instructions of the genome kit, and the target gene was obtained by PCR amplification with the designed primers of Deoc-F and Deoc-R. Firstly, the pRSF-duet-1 and the Deoc gene were cut by the restriction enzymes of Bam HI and Hind III, respectively. Secondly, Bam HI- and Hind III-digested PCR products of Deoc were ligated to the Bam HI- and Hind III-digested plasmids to construct pRSF-Deoc. All plasmids were constructed in E. coli JM109 and transferred into the expression host E. coli BL21 (DE3).

3.3. Preparation of Whole-Cell Biocatalysts

3.3.1. Medium and Culture Conditions

The strains E. coli BL21/ pRSF-Deoc stored in glycerol were inoculated on solid LB medium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl, and 20 g/L of agar) and cultured at 37 °C for 12 h. A single colony was picked and inoculated into 30 mL of LB medium before being cultured for 12 h at 37 °C and 220 r/min. Subsequently, the culture with an amount of 2% was inoculated into the Terrific broth (TB) medium (12 g/L of tryptone, 24 g/L of yeast extract, 2.2 g/L of KH₂PO₄, 12.4 g/L of K₂HPO₄, and 1% of glycerol) and cultured at 37 °C and 220 r/min. A total of 50 mg/L of kanamycin was added to the culture medium during the whole culture process. In addition, 0.8 mM of IPTG was added when the optical density (OD) at 600 nm reached 0.6−0.8, and the medium was cultured at 20 °C and 220 r/min for 20−24 h.

3.3.2. Preparation of Whole-Cell Biocatalysts

Wet cells were obtained by centrifuging the cells cultured in the TB medium at 8000× g for 10 min at 4 °C. Next, 10 mL of phosphate buffer at pH 7.5 was added to the wet cells in order to resuspend the cells. Afterwards, the mixture was centrifuged at 8000× g for 10 min at 4 °C, and the obtained pellets were placed at 4 °C before use.

3.4. Biosynthesis of 4-HBA

In a 5 mL reaction system, 1 g of wet cells was added into the mixture solution of acetone and phosphate buffer solution (pH 7.5) in order to form a total volume of up to 5 mL. The concentration of acetone in the mixture solution varied from 3% to 80% (v/v). The biosynthesis was carried out in a water bath shaker at 40 °C and 150 r/min for 12 h in 10 mL stoppered glass vials, with optimization of reaction temperature by varying the temperature from 30 °C to 70 °C (all reactions were performed in triplicate).

Fed-Batch Biosynthesis of 4-HBA

At the reaction temperature of 60 °C and the rotation speed of 150 r/min, first, 4-HBD with a final concentration of 360 mM was added to the mixture consisting of 2 g of wet cells and 8 mL of acetone. The concentration of 4-HBD remaining in the reaction system and the generation of 4-HBA were detected every 12 h. After 24 h of reaction, 120 mM of 4-HBD was added to the reaction system in order to continue the reaction until the concentration of 4-HBA no longer increased.

3.5. Biosynthesis of 4-HBA by the Immobilized Cells

In order to form a concentrated 50 wt.% bacterial suspension, 8 g of wet cells were suspended in normal saline. Next, the suspension was mixed with aqueous 4.0 wt.% K-carrageenan solution at 50 °C. The obtained homogeneous mixture was placed at 10 °C for 30 min, and the obtained colloid was placed into 0.3 M of KCl solution at 10 °C for 4 h. Afterwards, the hardened colloid was cut into 3 × 3 × 3 mm pieces, and the immobilized cells were obtained.

Once 8.5 g of immobilized cells were added to 5 mL of reaction mixture composed of acetone and 120 mM 4-HBD, the biosynthesis was carried out at 60 °C for 36 h. Repeated batch reactions were undertaken to test the stability of the immobilized cells.

3.6. Analysis Method

The whole-cell biocatalytic activity was assayed by measuring 4-HBA concentration in the reaction system. After reaction, the system was centrifuged at 12,000 r/min in order to remove cells, and the supernatant was diluted 10× with methanol. Qualitative and quantitative analysis methods of 4-HBA were reported in previous studies [23]. The products were detected by absorbance measurements at 222 nm using a high-performance liquid chromatography (HPLC) (Waters Co., Ltd., Milford, MA, USA) and a reverse-phase Amethyst C18-H column (4.6 × 250 nm²) (Beijing Dicoma Technology Co., Ltd, Beijing, China) for 20 min. The flow rate was 1 mL/min, and the mobile phase A was water (80%) with 0.1% (v/v) phosphoric acid. Mobile phase B was acetonitrile (35%). The retention times for 4-HBD and 4-HBA were 9.50 min and 20.68 min, respectively. Standard curve of 4-HBA and 4-HBD and HPLC of our product and 4-HBA standard were shown as Figures S2 and S3.

For SDS-PAGE analysis, the cells were harvested by centrifugation at 5500× g for 10 min at 4 °C, and then were resuspended in PBS (pH 7.5) with ultrasonic disruption. The suspension was centrifugated at 8000 r/min for 10 min. Standard SDS-PAGE (15% gel) was applied for the target protein assay. The images of the gels were scanned using a Tanon 3500 R gel imaging system (Tanon, Shanghai, China).

The molar conversion and yield of 4-HBA were calculated according to the following equation:

$$\mathrm{Molar}\mathrm{conversion}=\frac{mol of consumed 4HBD}{mol ofinitial4HBD}\times 100\%$$

$$Yield=\frac{molofproduced4HBA}{molofinitial4HBD}\times 100\%$$

4. Conclusions

In our study, E. coli BL21 (DE3) overexpressing DERA_Ec was used as a whole-cell catalyst to synthesize 4-HBA in a pure acetone system with 4-HBD as the substrate. We obtained 111.3 mM of 4-HBA from 120 mM of 4-HBD with a yield of 92.8%, which was greatly improved compared to the reported value of 0.9 mM [13]. The highest cumulative amount of 4-HBA was 376.4 mM using the fed-batch strategy, and the reusability of K-carrageenan immobilized cells was confirmed. However, in the acetone reaction system with a high substrate concentration, the catalytic activity of E. coli BL21 (DE3)/pRSF-Deoc whole cells still needs to be improved.

5. Patents

There are no patents resulting from the work reported in this manuscript.