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Article

Involvement of Cytochrome P450 in Organic-Solvent Tolerant Bacillus subtilis GRSW1-B1 in Vanillin Production via Ferulic Acid Metabolism

by
Panaya Kotchaplai
1,2,
Jedsadakorn Ninrat
3,
Gumpanat Mahipant
4 and
Alisa S. Vangnai
4,5,*
1
Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand
2
Water Science and Technology for Sustainable Environment Research Group, Chulalongkorn University, Bangkok 10330, Thailand
3
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
4
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
5
Center of Excellence in Biocatalyst and Sustainable Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*
Author to whom correspondence should be addressed.
Fermentation 2022, 8(10), 508; https://doi.org/10.3390/fermentation8100508
Submission received: 7 September 2022 / Revised: 22 September 2022 / Accepted: 26 September 2022 / Published: 2 October 2022
(This article belongs to the Special Issue Bacillus Species and Enzymes)
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Abstract

:
The detection of vanillin during the metabolism of ferulic acid by several Bacillus strains has been reported; however, its occurrence is not yet understood. Herein, the potential enzymes involved in vanillin production during ferulic acid metabolism in the previously reported butanol-tolerant Bacillus subtilis strain GRSW1-B1 were explored. The recombinant E. coli cells that overexpressed phenolic acid decarboxylase (PadC) rapidly converted ferulic acid to 4-vinylguaiacol. The detection of vanillin was concurrent with a decrease in 4-vinylguaiacol. In addition, the reversible abiotic conversion of 4-vinylguaiacol and apocynol was observed. The overexpression of CypD, a Bacillus P450, resulted in notable production of vanillin. The two-step conversion of ferulic acid yielded 145 μM over 72 h at pH 9. Vanillin yields of approximately 258 μM and 212 μM were obtained from ferulic acid metabolism by recombinant E. coli coexpressing PadC and CypD after conversion for 72 h, at pH 9 and 10, respectively. Several possibilities that underlie the production of vanillin were discussed. This information is useful for understanding ferulic acid metabolism by Bacillus strains and for further improving this strain as a host for the production of valuable compounds from biomass.

Graphical Abstract

1. Introduction

Biomass has emerged as an important resource for a sustainable biobased economy. Lignocellulosic biomass, which is abundant, consists of three types, cellulose, hemicellulose, and lignin. While the potential application of cellulose and hemicellulose has been reported, there are still limited applications for lignin and further development remains challenging. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is one of the most abundant lignin derivatives. Using natural microorganisms and engineered strains, many studies have reported the biotransformation of ferulic acid to other value-added compounds, including vanillin.
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a globally important compound for the food, fragrance, and pharmaceutical industries. The natural vanillin extracted from vanilla pods is in high demand, with prices reaching USD 1200–USD 1400 per kg [1]; However, there is a limited supply of natural vanillin [2]. Bioproduction of vanillin, especially from the lignin derivatives, is a promising alternative source of vanillin. The well-studied and most-reported pathway for ferulic acid conversion to vanillin is via the CoA-dependent pathway, which is reported in Amycolatopsis sp. HR167, Streptomyces sp. Strain V-1, Pseudomonas strains and engineered E. coli [3,4,5,6,7,8]. In this coenzyme A-dependent, non-β-oxidative pathway, the two critical enzymes, i.e., ferulyl-CoA synthetase and enoyl-CoA hydratase/aldolase, are encoded by fcs and ech, respectively. First, the activation of ferulic acid to ferulyl-CoA is catalyzed by Fcs. Then, ferulyl-CoA is hydrated and cleaved to produce vanillin and acetyl-CoA. This pathway has been well-studied, and the CoA-dependent pathway is highly employed in microbial metabolic engineering for vanillin production [6,7,9,10]. In addition to these well-known vanillin production mechanisms, several studies have reported the metabolism of ferulic acid by Bacillus strains, which results in vanillin production [11,12,13]. However, the mechanism of vanillin production during ferulic acid metabolism has not yet been reported.
B. subtilis is a widely used industrial strain, owing to its exceptional fermentation properties. This microbe has generally been regarded as safe by the Food and Drug Administration (FDA) [14,15]. B. subtilis GRSW1-B1 was previously isolated from seawater and has been reported as a butanol-tolerant strain [16] and the genetic manipulation of GRSW1-B1 has been successfully demonstrated [17]. Accordingly, B. subtilis GRSW1-B1 appears to be a good candidate for various solvent-involving industrial applications. As several studies have reported higher vanillin production with the use of solvent-based biphasic systems [18,19], this strain was explored for its potential as a bioproduction host. The whole-genome sequencing data in this study allowed the exploration of the ferulic acid metabolism by B. subtilis GRSW1-B1, and further examination of the potential enzymes involved in vanillin production.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

2.1.1. Bacterial Strains

The bacterial strains and plasmids are listed in Table 1. The metabolism of ferulic acid was studied using B. subtilis GRSW1-B1. This strain is an organic solvent-tolerant bacteria previously isolated from seawater [16]. The strain was deposited at the BIOTEC Culture Collection, Thailand (BCC. 56597) and the NITE Biological Resource Center, Japan (NBRC. 109691). The GRSW1-B1 genome was sequenced by a shotgun sequencing approach using 454 GS FLX Titanium (Roche Basel, Switzerland) and paired-end sequencing with HiSeq 1000 (Illumina, San Diego, CA, USA) (77-fold coverage). The data were assembled by Newbler version 2.6. The NCBI Prokaryotic Genomes Automatic Annotation Pipeline was used for genome annotation with the annotation support tool GenomeMatcher [20,21]. The gene names and functions were mainly reassigned against the reviewed protein of B. subtilis 168 from the Uniprot database. This whole-genome shotgun project has been deposited at the DDBJ/EMBL/GenBank under the accession code JRXD00000000.
Escherichia coli DH5α was used for plasmid propagation, whereas E. coli BL21(DE3) and E. coli JM109(DE3) were used to overexpress Bacillus proteins.

2.1.2. Growth Conditions

Luria Bertani (LB) medium, consisting of 1% tryptone, 1% NaCl, and 0.5% yeast extract, was used for bacterial starter preparation. To cultivate the recombinant E. coli strains, LB medium was supplemented with either 50 μg/mL spectinomycin (for cells containing pCDFDuet), 100 μg/mL ampicillin (for cells containing pETDuet), or 50 μg/mL kanamycin (for cells containing pET28b). The bioconversion medium used this study was M9 minimal medium. Unless stated otherwise, bacterial cells were cultivated at 37 °C, 200 rpm. For bacterial starter preparation, a single bacterial colony was transferred to 5 mL LB and cultivated for 14–16 h.

2.1.3. Plasmid Construction

The padC gene was amplified with two primers, padC-NdeI-F and padC-AatII-R, using the genomic DNA of B. subtilis GRSW1-B1 as the template. The purified PCR product was ligated into the linearized pGEM T Easy vector and transformed into E. coli DH5α. The obtained pGEMT-padC was digested with NdeI and AatII and ligated between the NdeI and AatII sites in MCS-2 of pETDuet and transformed into E. coli DH5α. Genes encoding P450s in B. subtilis GRSW1-B1 were amplified with the primers indicated in Table 2, using the genomic DNA of B. subtilis GRSW1-B1 as the template. The PCR product was then digested and ligated into pET28b and transformed into E. coli DH5α. For pCDFD-Ado construction, the synthetic Ado fragment was inserted into pETDuet between SacI and NotI, generating pETD-Ado. pETDuet-Ado was then digested with SacI and KpnI and cloned into pCDFDuet, generating pCDFD-Ado.

2.2. Bioconversion of Ferulic Acid

2.2.1. Bioconversion of Ferulic Acid by B. subtilis GRSW1-B1

Unless stated otherwise, 1% of the overnight bacterial starter was transferred into 50 mL of M9 conversion medium supplemented with 0.01% yeast extract. The stock ferulic acid was added to obtain the initial ferulic acid concentration of 5 mM and incubated at 37 °C, with shaking at 200 rpm.

2.2.2. Potential Involvement of Bacillus P450s in the Production of Vanillin

A small-scale two-step conversion was used in this part. The growth-independent two-step conversion consists of (1) conversion of ferulic acid to 4-vinylguaiacol and (2) the conversion of a spent solution, containing 4-vinylguaiacol, to vanillin by recombinant E. coli harboring Bacillus P450s. Two Bacillus P450s, CypC (CYP152A1, P450 peroxygenase) and CypD (CYP102A2, P450 monooxygenase), were the main focus in this study. For cell preparation, 1% of the overnight bacterial starter was transferred to 300 mL of LB medium supplemented with the appropriate antibiotic in a 1 L baffle flask and cultivated at 37 °C, with shaking at 200 rpm, until the OD600 reached 0.8–1.0. Protein expression was induced by the addition of IPTG (final concentration 0.1 mM) and further cultured at 25 °C, 125 rpm for 16–18 h. δ-Aminolevulinic acid (final concentration 0.5 mM) was also added as a precursor for heme synthesis for P450 overexpression. The cells were harvested by centrifugation at 6000 rpm for 10 min 4 °C and then washed twice with 0.85% NaCl solution.
In the first step, i.e., the conversion of ferulic acid to 4-vinylguaiacol, recombinant E. coli harboring pETD-padC was resuspended in either 100 mM citrate buffer (pH 6), Tris–HCl buffer (pH 8) or glycine–NaOH buffer (pH 10) to obtain a final cell wet weight concentration of 20 mg/mL. A stock solution of ferulic acid was added to obtain a final concentration of 1 mM. The small-scale bioconversion of ferulic acid to 4-vinylguaiacol was performed using a 13 mL test tube (2 mL reaction) at 37 °C, with shaking at 200 rpm, for 1 h. For the second step, i.e., the conversion of 4-vinylguaiacol to vanillin, the reaction mixture containing 4-vinylguaiacol from the prior step was centrifuged and filtered through a 0.22 μm filter to remove bacterial cells. E. coli cells that overexpressed either CypC or CypD were resuspended in the obtained spent medium to obtain a final cell wet weight concentration of 20 mg/mL. The bioconversion of 4-vinylguaiacol to vanillin was performed in a 24-well plate (0.5 mL reaction) at 37 °C, with shaking at 200 rpm, for 24 h.

2.3. Flask-Scale Two-Step Conversion of Ferulic Acid Using Resting Cells of Recombinant E. coli

Recombinant E. coli cells harboring either pETD-PadC or pET28-CypD were prepared similarly to those previously mentioned in Section 2.2.2. This experiment was performed in M9 minimal medium (pH 7), 100 mM Tris–HCl buffer (pH 8) or 100 mM glycine–NaOH buffer (pH 9 and 10). The conversion of ferulic acid to 4-vinylguaiacol in the first step was performed for a reaction volume of 100 mL. After centrifugation and filtration, the obtained supernatants were used for further conversion of 4-vinylguaiacol by the recombinant E. coli overexpressing CypD (cell wet weight approximately of 20 mg/mL), with a reaction volume of 10 mL.

2.4. One-Pot Conversion of Ferulic Acid Using Resting Cells of Recombinant E. coli Coexpressing PadC and CypD

The recombinant E. coli cells harboring pETD-PadC and pCDFD-cypD were grown in LB medium supplemented with 100 µg/mL ampicillin and 50 µg/mL spectinomycin at 37 °C, with shaking at 200 rpm, until the OD600 reached 0.8. Then, 0.2 mM IPTG and 0.5 mM δ-aminolaevulinic acid were added to induce protein expression. The culture was further shaken at 25 °C, with shaking at 125 rpm, for 16 h before the cells were harvested and washed twice with 0.85% NaCl solution. The recombinant E. coli cells, i.e., E. coli BL21(DE3) coharboring pETDuet-padC and pCDFDuet-cypD, were resuspended in 10 mL of 100 mM potassium phosphate buffer (pH 7), 100 mM Tris–HCl (pH 8), or 100 mM glycine–NaOH (pH 9). The ferulic acid stock was added to the bioconversion medium to obtain an initial ferulic acid concentration of 1 mM.

2.5. Conversion of 4-Vinylguaiacol Using Resting Cells of Recombinant E. coli

The recombinant E. coli cells overexpressing CypD were prepared similarly to those previously mentioned for small-scale ferulic acid conversion. For E. coli cells overexpressing Ado, 1% of overnight starter (LB medium supplemented with 50 µg/mL spectinomycin) was transferred to 250 mL LB supplemented with antibiotic in a 1 L baffle flask and cultivated at 37 °C, with shaking at 200 rpm. When the OD600 reached 0.8, 0.1 mM IPTG was added together with 1 mM of Fe(II) to induce protein expression. The culture was further incubated at 25 °C, with shaking at 125 rpm, for 16 h before the cells were harvested and washed twice with 0.85% NaCl solution. This experiment was conducted in 100 mM glycine–NaOH buffer (pH 9), containing 5 mM DMSO and either 0.5 or 1.0 mM 4VG.

2.6. Analytical Methods

2.6.1. High-Performance Liquid Chromatography

For the HPLC analysis, 100 µL of cell supernatant was collected and 5 µL of 37% hydrochloric acid was added to stop the conversion reaction and then mixed with 400 µL of the mobile phase (75% water/25% acetonitrile). The sample mixture was vortexed and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.45 µM nylon filter and subjected to HPLC analysis. Chromatographic separation was achieved on a Shimadzu high-performance liquid chromatography system (Shimadzu, Kyoto, Japan), using a Synergi 4 µm Hydro-RP 80A 250 × 4.6 nm column (Phenomenex, Torrance, CA, USA) and a flow rate of 0.6 mL/min. The compounds were eluted using a gradient elution profile of solvent A (0.1% H3PO4 in water) and solvent B (acetonitrile). The A:B ratio was maintained at 75:25 for 15 min, then increased from 75:25 to 5:95 over 5 min, and held at 5:95 for 5 min, followed by a rapid decrease to 75:25 over 5 min, and then held at 75:25 for 10 min. The obtained retention times were compared with those of standard compounds.

2.6.2. Gas Chromatography-Mass Spectrometry (GC-MS)

First, 50 mL of the sample was acidified using HCl to obtain a solution pH of approximately 2. The acidified sample was then extracted with three volumes of ethyl acetate. The organic phase was then dewatered using Na2SO4 and evaporated to dryness using a rotary evaporator. The residue was resuspended in 500 μL of dioxane and 50 μL of pyridine and then derivatized using 200 μL of BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide) + 1% TCMS at 60 °C for 30 min. The derivatized samples were then filtered and submitted for GC-MS analysis. The GC-MS system in this study was an Agilent 7890B GC System equipped with an Agilent 7000C GC/MS Triple Quad (GC-QQQ) (Agilent Technologies, Santa Clara, CA, USA). Sample separation was conducted using a HP-5 ms column (30 m × 0.25 mm × 0.25 μm) with a 0.1 μL sample injection and inlet temperature of 250 °C. Helium was used as a carrier gas with a flow rate of 1 mL/min, average velocity of 25.485 cm/s, and a constant flow of 1 mL/min. The initial temperature was held at 50 °C for 5 min, then increased to 300 °C with 10 °C/min and held for 5 min (total 35 min). Post-run conditions were 310 °C (2 mL/min) for 3 min. The solvent delay time was 5 min, the m/z scan range was 33–550, the transfer line temperature was 300 °C, the EI source temperature was 250 °C, and the EI voltage was 70 eV. Compounds were identified by matching the MS spectra with those in the NIST 2011 mass spectral library.

3. Results

3.1. Whole-Genome Sequencing of B. subtilis GRSW1-B1

The draft genome of B. subtilis GRSW1-B1’s total length is 4,078,877 bp, containing 11 contigs with a GC content of 43.7%. The gene prediction was 4246 genes and 37 frame-shifted genes. The annotated genes included 4087 coding DNA sequences (CDSs), 69 pseudo genes, 5 rRNAs, 84 tRNAs, and 1 ncRNA. The average nucleotide identity of the GRSW1-B1 genome was measured against six B. subtilis, one B. pumilus, and one B. amyloliquefaciens genomes via two-way ANI with default settings (ANI calculator. Available online: http://enve-omics.ce.gatech.edu/ani/index (accessed on 3 June 2017)). The ANI value showed a similarity of more than 90% to the B. subtilis group, but only 80.46–82.10% similarity to B. pumilus SAFR-032 and B. amyloliquefaciens DSM 7. The ANI value indicated the close phylogenetic relationship between GRSW1-B1 and B. subtilis subsp. subtilis, with more than 98% identity (99.01%, 6051-HGW; 98.97%, BSn5; and 98.96%, 168), but less than 94% identity with subsp. spizizenii (93.36%, TU-B-10; and 93.11%, W23).
Based on the MegaBLAST results of GRSW1-B1 CDSs against the six B. subtilis strains above, there were 3717 and 3540 shared CDSs within the 4 strains of B. subtilis subsp. subtilis and the 2 strains of subsp. spizizenii, respectively. MegaBLAST searching against the well-known model strain 168 revealed shared 3904 CDSs and 182 unique CDSs. The unique CDSs of GRSW1-B1 compared 168 included phage-related genes, specific antibiotic-resistance genes, and phospholipid-involving genes, which indicated some exclusive characteristics, leading to different phenotype responses to solvent stress.
Several Bacillus enzymes involved in ferulic acid and vanillin metabolism, for example PadC (encoding phenolic acid decarboxylase) and YfmT (encoding vanillin/benzaldehyde dehydrogenase), have already been reported [22,23,24]. These enzymes were also found in the genome of the B. subtilis GRSW1-B1 strain. On the contrary to these well-studied enzymes, the Bacillus enzyme(s) involved in the conversion of 4-vinylguaiacol to vanillin is yet to be reported. So far, only coenzyme-dependent carotenoid oxygenases and aromatic phenol monooxygenase (Ado) were reported for their ability to convert 4-vinylguaiacol to vanillin [19,25,26,27,28]. In 2014, by using a genome mining approach with isoeugenol monooxygenase (Iso) as a query, 9-cis-epoxycerotenoid dioxygenase (Cso2) from Caulobacter segnis with 42% similarity with isoeugenol monooxygenase was reported, and the production of vanillin from 4-vinylguaicol was successfully demonstrated [25]. Several B. subtilis strains can transform isoeugenol to vanillin [29,30]. The sequences of either iso, cso2 or ado were used to explore B. subtilis GRSW1-B1; however, no similar enzyme was found.
Besides cleaving the C=C bond at the alkene side chain, the oxidation at this position also results in the production of aromatic aldehyde. In 2006, Zhang and colleagues reported the conversion of isoeugenol to vanillin via isoeugenol-diol in B. subtilis HS8, and proposed that oxidases, for example cytochrome P450 or peroxidase, might be involved [29]. Cytochrome P450s, which are heme-containing enzymes, together with metalloporphyrin (biomimetic model of cytochrome P450), have been reported to catalyze the oxidation of aryl alkene at its side chain, yielding several products, including aldehydes [31,32,33].
CypD or CYP102A2 is a self-sufficient NADPH-dependent cytochrome P450. This enzyme is a homologue to the well-studied versatile CYP102A1 or cytochrome 450-BM3 from Bacillus megaterium [34]. CypD in B. subtilis GRSW1-B1 showed 97% amino acid similarity to CypD in B. subtilis 168. The cytochrome 450-BM3 and its recombinant could catalyze the oxidation of styrene, resulting in styrene oxide [35,36]. CypC (encoded by cypC or ybdT in B. subtilis 168), a unique P450 peroxygenase, of which H2O2 can be used to catalyze the oxidation of compounds without another co-factor or redox partner required, was also found in B. subtilis GRSW1-B1. The recombinant peroxygenase from Agrocybe aegerita could catalyze the oxidation of styrene to styrene oxide and benzaldehyde [37]. Taken together, the cytochrome P450s, cypD and cypC, in B. subtilis GRSW1-B1 were selected for subsequent investigation.

3.2. Ferulic Acid Metabolism by B. subtilis GRSW1-B1

The growth rate of B. subtilis GRSW1-B1 in minimal medium with 5 mM ferulic acid as the sole carbon source was very low (Figure 1A). Upon the addition of ferulic acid, ferulic acid was rapidly converted to 4-vinylguiacol. This decarboxylation was catalyzed by the enzyme phenolic acid decarboxylase, which has been reported in several microorganisms [22,23,38,39,40]. 4-Vinylguaiacol accumulated in the conversion medium before gradually decreasing, concurrently with a slight increase in vanillin over time (Figure 1B). The vanillin yields were approximately 7.37 and 13 mg/L after 48 and 246 h (or 0.013 mg vanillin/mg ferulic acid). The metabolites of ferulic acid were identified using GC-MS and the deduced ferulic acid metabolism pathway by B. subtilis GRSW1-B1 is presented in Figure 2.

3.3. Potential Involvement of Bacillus CypD in Vanillin Production during Ferulic Acid Metabolism

In this experiment, 1 mM of ferulic acid was converted to 4-vinylguaiacol by the recombinant E. coli overexpressing PadC. The cell-free spent medium containing 4-vinylguaiacol was then used to test the potential involvement of Bacillus P450 in vanillin production. Interestingly, of the two Bacillus P450s tested, the recombinant E. coli overexpressing CypD or CYP102A2 resulted in the pH-dependent production of vanillin, yielding 26–45 μM vanillin from 1 mM ferulic acid within 24 h of conversion (Figure 3). The background vanillin concentrations in control experiments (without cells) were 13–18 μM, regardless of pH. The recombinant E. coli harboring either pET28b or pET28-cypC resulted in 7–17 μM vanillin. According to the substantially higher vanillin (2–3 fold) concentration detected in the conversion by the recombinant E. coli overexpressing CypD, CypD may be involved in vanillin production during ferulic acid metabolism. However, the in vitro enzymatic reaction of CypD with the commercial 4VG as substrate resulted in no vanillin production (data not shown). The mechanism underlying the production of vanillin in either the ferulic acid metabolism or the two-step conversion by the recombinant E. coli is still unclear. The production of vanillin is likely to be due to the oxidation of 4-vinylguaiacol by H2O2 from the uncoupled reaction of P450 enzyme. A similar observation was previously reported for the production of benzaldehyde from styrene [32]. Another possibility is that more intermediates or enzymes are involved in vanillin production; thus, no vanillin was detected in the in vitro reaction.
Apocynol, a notable byproduct of 4-vinylguaicol, was also detected in most conditions, especially in the control conditions, which were cell-free spent medium containing 4-vinylguaiacol. This compound is a product of the abiotic oxidation/hydration of 4-vinylguaiacol [41]. Similar to vanillin, apocynol is a vanilla-like aromatic compound. In this study, the pH-dependent occurrence of apocynol was observed. The detected apocynol in control conditions was 30 μM at pH 10, whereas 78 and 128 μM of apocynol were detected at pH 6 and pH 8. No apocynol was detected at pH 10 in the presence of cells. Accordingly, the pH of conversion medium/buffer may be used to manipulate the formation of apocynol during vanillin production from 4-vinylguaiacol.

3.4. pH-Dependence of the Two-Step Vanillin Production Using Recombinant E. coli Harboring CypD

The pH-dependent bioconversion of spent medium containing 4-vinylguaiacol by the recombinant E. coli harboring pET28-CypD is shown in Figure 4. After the first step, the initial concentrations of 4-vinylguaiacol were approximately 515–568 μM with no apocynol detected. After the filtration step to remove the recombinant E. coli harboring pETD-PadC, the initial 4-vinylguaiacol decreased to 66–120 μM, while the detected apocynol concentration was 157–172 μM (Figure 4, time 0). At the fourth hour of the second-step conversion, the increase in 4-vinylguaiacol was concurrent with a decrease in apocynol, suggesting a reversible conversion between 4-vinylguaiacol and apocynol. The highest yield (at 24 h) of vanillin production by the recombinant E. coli harboring pET28-CypD was obtained at pH 8 (69 μM), compared to the rates of 41 μM at pH 9 and 25 μM at pH 10. The concentration of vanillin increased to 84, 145, and 35 μM at 72 h at pH 8, 9, and 10, respectively. Only trace amounts of vanillin were detected when the two-step conversion was performed at pH 7, possibly due to further metabolism of vanillin to vanillic acid and vanillyl alcohol.

3.5. Conversion of Ferulic Acid to Vanillin via 4-Vinylguaiacol by Recombinant E. coli BL21(DE3) Coexpressing PadC and CypD

Recombinant E. coli BL21(DE3) coharboring pETD-padC and pCDFD-cypD was used to test the one-step conversion of ferulic acid. Similar to the two-step conversion, the pH-dependent metabolism of ferulic acid was observed (Figure 5). Interestingly, compared to the two-step conversion, the coexpression of PadC and CypD yielded a much higher vanillin concentration (258 and 212 μM at 72 h at pH 9 and 10, respectively). At pH 7 and 8, the vanillin concentration was increased to 185 and 321 μM at 24 h, respectively. However, in these pH environments, vanillin was rapidly converted to vanillic acid.

3.6. Vanillin Production from 4-Vinylguaiacol by Recombinant E. coli BL21(DE3) Expressing Ado or CypD

In 2018, vanillin production from 4-vinylguaiacol by aromatic dioxygenase or Ado from a thermophilic fungus, Thermothelomyces thermophila, was reported [27]. In this experiment, vanillin production from 4-vinylguaiacol using two recombinant E. coli cells, i.e., E. coli BL21(DE3) harboring pCDFD-cypD and E. coli BL21(DE3) harboring pCDFD-ado, were investigated. Vanillin production from commercial 4-vinylguaiacol using E. coli-based recombinant cells was as shown in Figure 6. The recombinant E. coli BL21(DE3) overexpressing the ado gene rapidly converted 4-vinylguaiacol to vanillin, within 6 h (Figure 6C,F,I). Vanillin was then partially converted to vanillyl alcohol, as the major byproduct, and vanillic acid, as the minor byproduct. The vanillin yield after 24 h of conversion was 240, 374, and 773 μM (36.5, 56.9, and 117.6 mg/L), when the initial concentration of 4-vinylguaiacol was 0.5, 1, and 2 mM, respectively (Figure 6C,F,I). The background concentration of vanillin generated from the abiotic control (without recombinant cells) after 120 h of conversion was 6, 9, and 19 μM (0.91, 1.4, and 2.8 mg/L), when the initial concentration of 4-vinylguaiacol was 0.5, 1, and 2 mM, respectively (Figure 6A,D,G). In the presence of the recombinant E. coli BL21(DE3) overexpressing the cypD gene, after 120 h of conversion, yields of 16, 45, and 34 μM (2.4, 6.8, and 5.1 mg/L) vanillin were produced, when the initial concentration of 4-vinylguaiacol was 0.5, 1, and 2 mM, respectively (Figure 6B,E,H). In contrast to recombinant E. coli BL21(DE3) overexpressing the ado gene, the major vanillin metabolism product produced by the recombinant E. coli BL21(DE3) overexpressing the cypD was vanillic acid, with only a trace amount of vanillyl alcohol detected. Apocynol, a product of 4-vinylguaiacol metabolism, was detected in the abiotic control and in the presence of recombinant E. coli BL21(DE3) overexpressing the cypD gene, possibly owing to the slow conversion rate, allowing 4-vinylguaiacol to be abiotically converted to apocynol. These results suggest that CypD is a potent enzyme involved in vanillin production; however, its slow conversion rate, especially in comparison with Ado, is a major challenge, and further vanillin metabolism should be limited.

4. Discussion

Several Bacillus strains have been reported for their capability to valorize lignocellulosic biomass, including lignin derivatives [11,24]. Phenolic acid decarboxylase is the enzyme that catalyzes the decarboxylation of phenolic compounds to their corresponding vinylphenols. In addition to Bacillus, this enzyme is also reported in Enterobacter [38,42], Aspergillus niger [43], and several yeast strains [29,30,31]. 4-Vinylguaiacol, a product from the decarboxylation of ferulic acid, has attracted interest as it can be used as fragrance and as a flavoring agent, as well as a precursor for other valuable compounds, including vanillin, acetovanillone and ethyl guaiacol [44]. Acetovanillone or apocynin has been reported as a major product of ferulic acid metabolism via 4-vinylguaiacol in Rhizopus oryzae [45]. While apocynol is notable product, no acetovanillone was detected in this study. Recently, many studies have reported the synthetic pathway for vanillin production from 4-vinylguaiacol [19,25,27]. These studies used carotenoid dioxygenases (Cso) or aromatic dioxygenase (Ado), which are enzymes that catalyze the coenzyme-independent C=C cleavage at the terminal alkene side chain of 4-vinylguaiacol, thereby generating vanillin and formaldehyde [19,25,27]. The vanillin yield from this synthetic pathway can be further improved by manipulating the bioconversion conditions, i.e., pH and temperature [27,46]. Typically, vanillin can be further converted to its derivatives, for example, vanillic acid and vanillyl alcohol by vanillin (aldehyde) dehydrogenase and alcohol dehydrogenase, respectively. The alcohol dehydrogenase(s) detected in E. coli-based hosts resulted in a decreased vanillin yield during extended bioconversion. In 2018, Ni and colleagues constructed a synthetic Fcs-Ech vanillin production pathway using genes from thermophilic actinomycetes and controlled the further metabolism of vanillin by applying high-temperature conditions [46]. Similarly, the vanillin production caused by the Pad-Ado pathway conducted at high temperature (50 °C) and basic pH (pH 9.5) could improve the vanillin yield by 2.35 fold compared to conversion at 30 °C and pH 7.5 [27]. Considering the toxicity of aromatic compounds, including ferulic acid, 4-vinylguaiacol and vanillin, biphasic fermentation has been adopted for vanillin production [18,26,27]. A high vanillin yield of 100 mM was obtained when the recombinant E. coli expressing PadC-Ado was used for vanillin production in a biphasic (chloroform:water) bioreactor [27].
According to the aforementioned studies, the solvent-tolerant B. subtilis GRSW1-B1 that possesses PadC appears to be a good host strain for the valorization of lignin derivatives. The vanillin yield from this strain is comparable to that reported in Enterobacter sp. Px6-4; however, it is still lower than those reported for the Bacillus strains [12,38]. 4-Vinylguaiacol and vanillin are the only two notable products of ferulic acid metabolism in B. aryabbhattai BA03 [12]. Under the optimized medium composition and biotransformation conditions, the vanillin titer increased to approximately 46 mg/L (or 0.047 mg vanillin/mg ferulic acid); the fed-batch fermentation resulted in 147 mg/L vanillin (0.082 mg vanillin/mg ferulic acid) after 216 h [12]. The optimization of fermentation medium and conditions, as well as the initial concentration of ferulic acid, increased vanillin production by the isolated B. megaterium from 48 mg/L to 218 mg/L [13].
Besides process optimization and downstream processing, metabolic engineering is also a promising approach to improve vanillin production [47]. While several studies have reported the production of vanillin from ferulic acid by Bacillus strains from the flask scale to the bioreactor scale, the vanillin production mechanism has not yet been reported [1,11,12,13,48]. To improve vanillin yield, the understanding of the enzyme(s) involved in vanillin production and metabolism is necessary. B. subtilis BS-7 (later reidentified as B. cereus BS-7), a Bacillus strain with high vanillin production, was investigated for its production efficiency in both small-scale reactions, including a 5 L bioreactor, and packed-bed stirred fermenters [48]. A high vanillin molar yield of 58.70–60.43% was observed in the immobilized cell bioreactor [11,48]. No other metabolites, for example 4-vinylguaiacol and vanillic acid, were detected in vanillin production by the strain BS-7. Whole-genome sequencing of B. cereus BS-7 revealed the presence of Ech, which is involved in the coenzyme A-dependent production of vanillin, whereas the vdh (vanillin dehydrogenase) was not detected [49]. Even though, in this study, the overexpressed CypD, a Bacillus P450 enzyme, was potentially involved in the generation of vanillin from 4-vinylguaicol, the mechanism is still unclear. Klaus and colleagues (2019) reported a synthetic vanillin production pathway that consisted of the engineered CYP102A1 or BM3-P450, which is a homolog of CypD, and vanillyl alcohol oxidase [50]. In their study, the engineered CYP102A1 catalyzed the hydroxylation of the aromatic ring and the alkyl side chain of 3-methylanisole, generating vanillyl alcohol that can be further converted to vanillin [50]. However, no product of the hydroxylation of 4-vinylguaiacol at the aromatic ring was detected in this study. Other than the CypD-catalyzed vanillin production, the generated vanillin may be the byproduct of CypD activity. Frutel and colleagues reported that H2O2 generated from the uncoupling reaction of P450 could directly oxidize the terminal alkene side chain of styrene (vinylphenol) to benzaldehyde (aromatic aldehyde) [32] and that no benzaldehyde was detected when catalase was added [51]. Other than C=C cleavage, the production of benzaldehyde from styrene by multiple enzymes was also reported [52]. The oxidation of styrene was catalyzed by the cascade containing styrene monooxygenase and epoxide hydrolase to generate the diol. This compound was then converted to benzaldehyde via a multienzyme step. Vanylglycol or 1-(4-hydroxy-3-methoxyphenyl)ethane-1,2-diol was also detected by GC-MS during ferulic acid metabolism by B. subtilis GRSW1-B1 (Table S1). Apocynol, the 4-vinylguaiacol derivative with a hydroxyl group at its vinyl group, has also been proposed as an intermediate for vanillin production from 4-vinylguaiacol [38].
Several attempts have been made to further eliminate vanillin metabolism, i.e., to enhance the vanillin yield. The deletion of vanillin dehydrogenase, an enzyme that metabolizes vanillin, resulted in a 28-fold increase in the vanillin titer from lignin degradation by the extremophile B. ligniniphilus L1 [53]. YfmT has been identified as the only vanillin dehydrogenase in B. subtilis 3NA [24]. In addition to PadC, YfmT and BsdBCD (which can convert vanillic acid to guaiacol), no other enzyme has been identified and no vanillin was detected in the study [24]. Besides padC, yfmT and bsdBCD were also found in B. subtilis GRSW1-B1. Accordingly, vanillin can be further metabolized to guaiacol via vanillic acid (Table S1).
In conclusion, this study explored ferulic acid metabolism and the potential Bacillus enzyme(s) involved in vanillin production by the B. subtilis strain GRSW1-B1. Owing to its solvent tolerant ability, this strain is a promising host strain, especially for the conversion or production of toxic compounds, which may require a bi-phasic fermentation system. While 4-vinylguaiacol is the major product of ferulic acid metabolism, this compound can be reversibly converted to apocynol. The recombinant E. coli cells that overexpressed cypD resulted in substantially higher vanillin production compared to the control experiments; however, the production mechanism is still unclear. Accordingly, it is important to further explore whether other intermediate(s) may be involved in the generation of vanillin during ferulic acid/4-vinylguaiacol metabolism. A better understanding of ferulic acid metabolism and vanillin production in the Bacillus strain will be useful for further development of the host strain.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8100508/s1, Table S1: List of intermediates identified by GC-MS.

Author Contributions

Conceptualization, P.K. and A.S.V.; methodology, P.K. and A.S.V.; investigation, P.K., J.N. and G.M.; writing—original draft preparation, P.K., J.N. and G.M.; writing—review and editing, P.K. and A.S.V.; visualization, P.K. and J.N.; supervision, A.S.V.; funding acquisition, P.K. and A.S.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Chulalongkorn University (CU_GR_63_33_61_01) and the Ratchadapisek Sompot Fund for Postdoctoral Fellowship, Chulalongkorn University. The funds by the National Research Council of Thailand (N42A640329) are also acknowledged.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors gratefully acknowledge Atsushi Yamazoe from the National Institute of Technology and Evaluation, Masao Fukuda and Michiro Tabata from Nagaoka University of Technology, and Junichi Kato from Hiroshima University for their help in whole genome sequencing and analysis. The graphical abstract was created with BioRender.com (accessed on 2 September 2022).

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 08 00508 g001 550
Figure 1. (A) Growth and (B) ferulic acid conversion by B. subtilis GRSW1-B1. (FA: Ferulic acid, 4VG: 4-vinylguaiacol, VN: vanillin).
Figure 1. (A) Growth and (B) ferulic acid conversion by B. subtilis GRSW1-B1. (FA: Ferulic acid, 4VG: 4-vinylguaiacol, VN: vanillin).
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Figure 2. Ferulic acid and vanillin metabolism by B. subtilis GRSW1-B1 deduced from HPLC and GC-MS. (a: Products detected by HPLC, B: products detected by GC-MS, and dashed bracket: product deduced from the literature).
Figure 2. Ferulic acid and vanillin metabolism by B. subtilis GRSW1-B1 deduced from HPLC and GC-MS. (a: Products detected by HPLC, B: products detected by GC-MS, and dashed bracket: product deduced from the literature).
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Figure 3. Major metabolites in the two-step ferulic acid metabolism by recombinant E. coli expressing either Bacillus CypD or CypC at (A) pH 6, (B) pH 8, and (C) pH 10, respectively. (Apo: Apocynol, VN: vanillin).
Figure 3. Major metabolites in the two-step ferulic acid metabolism by recombinant E. coli expressing either Bacillus CypD or CypC at (A) pH 6, (B) pH 8, and (C) pH 10, respectively. (Apo: Apocynol, VN: vanillin).
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Figure 4. The two-step ferulic acid conversion by the recombinant E. coli JM109(DE3) overexpressing CypD at (A) pH 7, (B) pH 8, (C) pH 9, and (D) pH 10. (4VG: 4-Vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
Figure 4. The two-step ferulic acid conversion by the recombinant E. coli JM109(DE3) overexpressing CypD at (A) pH 7, (B) pH 8, (C) pH 9, and (D) pH 10. (4VG: 4-Vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
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Figure 5. Ferulic acid metabolism by recombinant E. coli BL21(DE3) coexpressing PadC and CypD at (A) pH 7, (B) pH 8, (C) pH 9, and (D) pH 10. (FA: Ferulic acid, 4VG: 4-vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
Figure 5. Ferulic acid metabolism by recombinant E. coli BL21(DE3) coexpressing PadC and CypD at (A) pH 7, (B) pH 8, (C) pH 9, and (D) pH 10. (FA: Ferulic acid, 4VG: 4-vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
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Figure 6. Vanillin production from 4-vinylguaiacol using the recombinant E. coli harboring (B,E,H) pCDFD-cypD, (C,F,I) pCDFD-Ado. The initial concentrations of 4-vinylguaiacol were (A–C) 0.5 mM, (D–F) 1 mM, and 2 mM of 4-vinylguaiacol. (A,D,G) are the abiotic controls. (4VG: 4-Vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
Figure 6. Vanillin production from 4-vinylguaiacol using the recombinant E. coli harboring (B,E,H) pCDFD-cypD, (C,F,I) pCDFD-Ado. The initial concentrations of 4-vinylguaiacol were (A–C) 0.5 mM, (D–F) 1 mM, and 2 mM of 4-vinylguaiacol. (A,D,G) are the abiotic controls. (4VG: 4-Vinylguaiacol, VN: vanillin, Apo: apocynol, VA: vanillic acid, Valc: vanillyl alcohol).
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Table
Table 1. Bacterial strains and plasmids used in this study.
Table 1. Bacterial strains and plasmids used in this study.
Bacterial StrainsDetailsSource
B. subtilis GRSW1-B1Solvent-tolerant bacterium[16]
E. coli DH5α
E. coli BL21(DE3)
E. coli JM109(DE3)
PlasmidsDetailsSource
pET28bExpression vector pBR322 ori, kanamycin rNovagen
pETDuetExpression vector pBR322 ori, ampicillin rNovagen
pCDFDuetExpression vector CloDF13 ori; streptomycin rNovagen
pETD-PadCpETDuet harboring padC
from B. subtilis GRSW1-B1 at MCS-2		This study
pET28-CypCpET28b harboring cypC from B. subtilis GRSW1-B1This study
pET28-CypDpET28b harboring CYP102A2 (cypD)
from B. subtilis GRSW1-B1		This study
pCDFD-CypDpCDFDuet harboring CYP102A2 (cypD)
from B. subtilis GRSW1-B1		This study
pCDFD-AdopCDFDuet harboring synthetic adoThis study
Kanamycin r: kanamycin resistance, ampicillin r: ampicillin resistance, streptomycin r: streptomycin resistance.
Table
Table 2. Primers used in this study.
Table 2. Primers used in this study.
cypC-pET28 BamHI FatgcGGATCCaATGAATGAGCAGATTCCACATG
cypC-pET28 XhoI RatgcCTCGAGTTAACTTTTTCGTCTGATTCCG
cypD-pET28 BamHI F atgcGGATCCaATGAAGGAAACAAGCCCGATTC
cypD-pET28 XhoI RatgcCTCGAGCTATATCCCTGCCCAGACATC
padC_NdeI-FatcgCATATGGAAAACTTTATCGGAAGC
padC-AatII-RatcgGACGTCTTATAATCTTCCCGCGCGAATA
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Kotchaplai, P.; Ninrat, J.; Mahipant, G.; Vangnai, A.S. Involvement of Cytochrome P450 in Organic-Solvent Tolerant Bacillus subtilis GRSW1-B1 in Vanillin Production via Ferulic Acid Metabolism. Fermentation 2022, 8, 508. https://doi.org/10.3390/fermentation8100508

AMA Style

Kotchaplai P, Ninrat J, Mahipant G, Vangnai AS. Involvement of Cytochrome P450 in Organic-Solvent Tolerant Bacillus subtilis GRSW1-B1 in Vanillin Production via Ferulic Acid Metabolism. Fermentation. 2022; 8(10):508. https://doi.org/10.3390/fermentation8100508

Chicago/Turabian Style

Kotchaplai, Panaya, Jedsadakorn Ninrat, Gumpanat Mahipant, and Alisa S. Vangnai. 2022. "Involvement of Cytochrome P450 in Organic-Solvent Tolerant Bacillus subtilis GRSW1-B1 in Vanillin Production via Ferulic Acid Metabolism" Fermentation 8, no. 10: 508. https://doi.org/10.3390/fermentation8100508

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