Construction of a Convenient Screening Method for P- Hydroxybenzoate Hydroxylase To Enable E cient Gallic Acid and Pyrogallol Biosynthesis


 BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis of GA and pyrogallol has been emerged as an ecofriendly method to replace the traditional chemical synthesis. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high active PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM-1s-1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the mechanism explanation of the high activity. Expression of this mutant in E. coli BW25113 (F’) can generate 830 ± 33 mg/L GA from 1000 mg/L 3,4-DHBA. After that, we utilized this mutant to assemble a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (F’)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high active PobA mutant. Assembling this mutant into GA and pyrogallol biosynthetic pathways achieved the de novo production of these two compounds. Besides, this mutant has great potential for GA or pyrogallol derivatives production. The screening method could be used for other GA biosynthesis-related enzymes.

Though PobA mutants with the ability of hydroxylating 3,4-DHBA have been obtained, the hydroxylation activity was still not satis ed the demand of high-level GA and pyrogallol production. Speci cally, the low activity of PobA towards 3,4-DHBA can lead carbon source to ow into the byproduct (catechol) biosynthetic pathway. Therefore, PobA mutant with higher activity urgently needs to be investigated. Rational design of PobA mutants generally required to deeply understand the catalytic mechanism of PobA. In many cases, the mutants generated from rational design did not have the expected high activity [32]. Compared to rational design, random mutagenesis is a method with higher probability to obtain high active PobA mutants. Effective screening method can ensure the acquirement of ideal mutants. In this study, we established an e cient and simple method for screening high active PobA mutants. This method depended on the instability of GA in alkaline conditions and the generated degradation products could react with each other to form a phenolic mixture [33,34]. Speci cally, the mixture has a green color visible to naked eyes and the maximum absorption wavelength was at 640 nm. Based on that, we adopted this method to screen out a PobA mutant (Y385F/T294A/V349A PobA) from a PobA random mutagenesis library. The k cat and k cat /K m of this mutant towards 3,4-DHBA were 1.78 ± 0.16 s −1 and 0.059 µM −1 s −1 , respectively, which were 1.12-and 4.92-fold higher than that of the reported mutant (Y385F/T294A PobA), respectively. Subsequently, the in vivo conversion ability of this mutant was represented through feeding 3,4-DHBA experiments. E. coli BW25113 (F') with Y385F/T294A/V349A PobA expression could convert 1000 mg/L 3,4-DHBA into 830 ± 33 mg/L GA, representing a 75% molar conversion ratio. Meanwhile, E. coli BW25113 (F') with Y385F/T294A/V349A PobA and PDC expression could generate 323 ± 23 mg/L pyrogallol from 1000 mg/L 3,4-DHBA. After that, we employed Y385F/T294A/V349A PobA to assemble an arti cial pathway for GA production from simple carbon sources and then introduced this pathway into a 3,4-DHBA-producing strain (E. coli BW25113 (F')ΔaroE). The de novo production of GA could reach 301 ± 15 mg/L. Correspondingly, assembling Y385F/T294A/V349A PobA into a de novo pyrogallol biosynthetic pathway could enable pyrogallol production to 129 ± 15 mg/L. This work constructed an e cient screening method to screen out a high active PobA mutant, and this mutant has great potential for industrial GA, pyrogallol and their derived compounds production.

Results And Discussion
Con rmation of GA performance in alkaline conditions This study aimed to acquire the PobA mutants with high hydroxylation activity towards 3,4-DHBA. As a product GA is unstable in alkaline conditions and the degradation products can react with each other to form a phenolic mixture [33][34][35][36][37]. In this work, we rstly mixed GA and alkali NaHCO 3 . After 2 h, we found the mixture with a pH of 9.3 displayed a green color visible to naked eyes (Fig. 1A). Moreover, the green color deepened with the increase of GA concentration.
Besides, UHPLC and MS were used to analyze the mixture. Fig. S1 shows 200 mg/L GA can be completely degraded in 2 hours. MS results in Fig. S2A show several new compounds were formed in the mixture. According to MS results, we speculated one of the new compounds might be ellagic acid (Fig. S2B), whose amount was highest in the mixture. Subsequently, the mixture was scanned at full wavelength (340-820 nm). Results in Fig. 1B show the mixture has a maximum absorption wavelength of 640 nm. We then con rmed the relationship between OD 640 value and GA concentration of the mixture. Fig. 1C shows GA concentration exhibited linear relationship with OD 640 value. These results demonstrate through adding NaHCO 3 , the change of GA concentration can be observed by naked eyes, and GA concentration can be con rmed through detection of OD 640 value. These suggest addition of NaHCO 3 in the nished reaction of PobA hydroxylating 3,4-DHBA can be used as an e cient strategy for screening high active PobA mutants.
Screening and in vitro enzyme assay of PobA mutants According to the performance of GA in NaHCO 3 , we designed a complete process for screening high active PobA mutants (Fig. 1D). Firstly, error-prone PCR was conducted on gene pobA, generating a PobA mutagenesis library. The single colonies of the library were pre-incubated into 96-deep-well plates containing LB medium, and the pre-inoculum was then transferred into another 96-deep-well plates containing M9Y medium with 1000 mg/L substrate 3,4-DHBA.
After 12 h, the culture samples were taken into 96-well plates which contained 0.1 M NaHCO 3 . After reaction for 2 hours, the samples with deepest green color were picked out, and were then re-screened through detection of OD 640 value. Based on that, a high active PobA mutant (Y385F/T294A/V349A) was screened out from PobA mutagenesis library. Subsequently, this mutant was expressed and puri ed. SDS-PAGE in Fig. S3 shows the purity of puri ed Y385F/T294A/V349A PobA was greater than 95%. Enzyme assay of the puri ed mutant was then performed. Subsequently, molecular docking simulation was conducted to provide mechanism explanation for the high activity of Y385F/T294A/V349A PobA. Wild-type PobA (PDB code: 1IUV) was used as template for simulation of Y385F/T294A PobA and Y385F/T294A/V349A PobA. After that, molecular docking of the mutants with substrate 3,4-DHBA and cofactor FAD were conducted. As shown in Fig. 2A and B, Y385F/T294A PobA and Y385F/T294A/V349A PobA possess similar catalytic pocket. In the pocket, amino acid residues Y201, P293 and T294A of PobA mutants, and 4-OH of 3,4-DHBA composed a hydrogen-bond loop, which was same as the complex of wild-type PobA with native substrate 4-HBA [28]. Besides, 3-OH of 3,4-DHBA formed hydrogen bonds with P293 of PobA mutants and cofactor FAD. The hydrogen-bond loop is a stable binding. Based on that, we speculated the catalytic mechanism of PobA mutants towards 3,4-DHBA was similar to that of wild-type PobA towards 4-HBA [38,39]. First, FAD cofactor in the complex is reduced by NADPH, which is responded to the binding of 3,4-DHBA to PobA mutants. Subsequently, the oxygen in environment oxidizes the reduced FAD to produce a hydroperoxide. The hydroperoxide then attacks the C-H bond at 5 th position of 3,4-DHBA to generate a new hydroxyl group, forming product GA.

Bioconversion of 3,4-DHBA into GA
To test the in vivo conversion ability of PobA mutants towards 3,4-DHBA, Y385F/T294A PobA and Y385F/T294A/V349A PobA were individually expressed in E. coli BW25113 (F'), generating strains CTT1 and CTT2, respectively. 1000 mg/L 3,4-DHBA was added to the culture at 5.5 h. As shown in Fig. 3A, CTT1 accumulated 104 ± 18 mg/L GA at 6.5 h, representing an initial in vivo conversion rate of 27.9 ± 4.9 mg/L/h/OD. The OD 600 value of CTT1 raised rapidly from 5.5 h to 24 h and reached 7.58 ± 0.04 at 24 h. After this time point, the growth of CTT1 stopped. Within 24 h, about half of 1000 mg/L 3,4-DHBA was consumed and 651 ± 5 mg/L GA was generated in the culture. In the next 24 hours, the conversion ability of CTT1 decreased and no more GA was produced. At 48 h, GA titer reduced to 575 ± 7 mg/L because of the degradation induced by oxidation. Meanwhile, 3,4-DHBA with a titer of 502 ± 32 mg/L was detected, indicating that about half of 1000 mg/L 3,4-DHBA cannot be converted into GA by CTT1.
Similar to CTT1, 1000 mg/L 3,4-DHBA was also fed to CTT2 at 5.5 h. The results in Fig. 3B show OD 600 value of CTT2 increased steadily throughout the 48-h feeding experiment and reached 9.07 ± 0.14 at 48 h. At 6.5 h, CTT2 produced 149 ± 5 mg/L GA in the culture and displayed an initial in vivo conversion rate of 35.4 ± 1.2 mg/L/h/OD, which was 1.27-fold higher than that of CTT1. Within 36 h, 1000 mg/L 3,4-DHBA was completely consumed by CTT2 and 830 ± 33 mg/L GA was generated, representing a 75% molar conversion ratio. Signi cantly, the titer of GA was 1.27-fold higher than that of CTT1 at 24 h. These results suggest that E. coli BW25113 (F') with Y385F/T294A/V349A PobA expression exhibits higher in vivo conversion ability towards 3,4-DHBA than E. coli BW25113 (F') with Y385F/T294A PobA expression.
As a comparison, the results in Fig. 3D show CTT4 grew rapidly in the rst 12 hours and has an OD 600 value of 6.66 ± 0.11 at 12 h. Meanwhile, pyrogallol with a titer of 237 ± 21 mg/L was detected in the culture, which was 1.39-fold higher than that of CTT3 at the same time point. In addition, the byproduct catechol has a titer of 367 ± 14 mg/L, a 1.37-fold lower value when compared with that of CTT3. After 12 h, OD 600 value has no signi cant increase, which was similar to CTT3. Signi cantly, the titer of pyrogallol gradually increased to 323 ± 23 mg/L at 48 h, which was 1.45fold higher than that of CTT3. These results indicate expressing PobA mutant and PDC in E. coli BW25113 (F') could achieve the in vivo conversion of 3,4-DHBA into pyrogallol. Moreover, Y385F/T294A/V349A PobA coupling with PDC represents higher in vivo ability of converting 3,4-DHBA into pyrogallol when compared with Y385F/T294A PobA coupling with PDC.

Establishment of the biosynthetic pathway for 3,4-DHBA production
Construction of an e cient 3,4-DHBA biosynthetic pathway was signi cant for achieving the de novo production of GA and pyrogallol. In previous study, 3,4-DHBA was produced from 4-HBA through expression of heterogenous PobA in E. coli [28]. For GA and pyrogallol production, heterogenous PobA required to catalyze two reactions, hydroxylating 4-HBA into 3,4-DHBA and hydroxylating 3,4-DHBA into GA (Fig. 4). Generally, the e ciency of two reactions induced by one kind of enzyme was lower than that of one reaction induced by one kind of enzyme. In this work, to avoid the issue of PobA-catalyzing two reactions and achieve e cient GA and pyrogallol production, E. coli BW25113 (F') was engineered to produce 3,4-DHBA from 3-dehydro-shikimate (DHS) (Fig. 4). Firstly, 4-HBA biosynthetic pathway in E. coli BW25113 (F') was blocked through knockout of gene aroE (strain CTT5) or knockout of genes aroE and ydiB (strain CTT6). AroE and YdiB are isoenzymes that can catalyze DHS to produce shikimate. Fig. S5 shows CTT5 can grow in M9 medium, while CTT6 cannot grow in M9 medium because it cannot synthesize the essential amino acids phenylalanine, tyrosine and tryptophan. These results were consistent with the theoretical expectation. Subsequently, the growth curves of CTT5 and CTT6 were measured in LB medium. As shown in To achieve the de novo production of 3,4-DHBA, 3-dehydroshikimate dehydratase (AroZ) which can catalyze 3-dehydroshikimate to produce 3,4-DHBA, was individually introduced into E. coli BW25113 (F'), CTT5 and CTT6, resulting in strains CTT7, CTT8 and CTT9, respectively. Results in Fig. 5B show 3,4-DHBA titers of CTT8 and CTT9 continued to increase during the 48-h fermentation. The growth curves of CTT8 and CTT9 were similar. The OD 600 values of CTT8 and CTT9 raised rapidly in rst 12 hours and have no signi cant improvement during the next 36 hours. At 48 h, CTT8 produced 752 ± 17 mg/L 3,4-DHBA. Meanwhile, the OD 600 value was 2.03 ± 0.06. For CTT9, 420 ± 26 mg/L 3,4-DHBA accumulated in the culture at 48 h, which was 1.79-fold lower than that of CTT8. These indicate the ability of strain CTT8 to produce 3,4-DHBA was higher than that of CTT9. As a comparison, CTT7 has negligible 3,4-DHBA accumulation throughout the 48-h fermentation, suggesting without knockout of aroE or ydiB E. coli BW25113 (F') could not synthesize 3,4-DHBA in large amount. These results suggest the engineered E. coli BW25113 (F') (CTT8 or CTT9) has ability to de novo produce 3,4-DHBA and can be used as host for de novo GA and pyrogallol production.
De novo production of GA To achieve the de novo production of GA, plasmid pZE-AroZ-PobA ** was individually introduced into E. coli BW25113 (F'), CTT5 and CTT6, generating strains CTT10, CTT11 and CTT12, respectively. The fermentation results are displayed in Fig. 6. For strain CTT10, the production of GA lasted up to 36 h. At 36 h, only 14.1 ± 1.0 mg/L GA accumulated in the culture, meanwhile, the OD 600 value was 3.08 ± 0.42 (Fig. 6A). Besides, negligible 3,4-DHBA was observed in the culture, suggesting the generated 3,4-DHBA could be immediately converted into GA by strain CTT10. For strain CTT11, 3,4-DHBA and GA titers, as well as the cell growth, kept increasing throughout the 48-h fermentation (Fig. 6B).
Signi cantly, within 48 h, 3,4-DHBA with a titer of 400 ± 17 mg/L and GA with a titer of 180 ± 31 mg/L were detected in the culture. At the same time point, CTT11 has an OD 600 value of 9.10 ± 0.51. Notably, CTT11 produced 12.8-fold higher amount of GA when compared with CTT10, indicating that knockout of aroE signi cantly increased the ability of E. coli BW25113 (F') to de novo produce GA. For strain CTT12, GA titer and OD 600 value continued to increase during the 48-h fermentation (Fig. 6C). CTT12 has a GA titer of 46.5 ± 8.0 mg/L and an OD 600 value of 4.35 ± 0.84 at 48 h.
Signi cantly, GA titer of CTT12 was 3.87-fold lower than that of CTT11, suggesting that E. coli BW25113 (F')ΔaroEΔydiB has lower ability to de novo synthesize GA when compared with E. coli BW25113 (F')ΔaroE.
Overall, introducing the designed arti cial pathway into E. coli could achieve GA biosynthesis from simple carbon sources. E. coli BW25113 (F')ΔaroE demonstrates stronger ability for de novo producing GA when compared with E. coli BW25113 (F') or E. coli BW25113 (F')ΔaroEΔydiB. Assembling mutant Y385F/T294A/V349A PobA into GA biosynthetic pathway enabled more GA production than that of assembling Y385F/T294A PobA into GA biosynthetic pathway, which were consistent with the results of in vitro enzyme assay and in vivo conversion experiments.
Subsequently, plasmids pZE-AroZ-PobA *** and pCS-PDC were co-transferred into E. coli BW25113 (F'), CTT5 and CTT6, resulting in strains CTT19, CTT20 and CTT21, respectively. As shown Fig. 7D, CTT19 hardly produced 3,4-DHBA, GA, pyrogallol and catechol as CTT16. In Fig. 7E, CTT20 continued to grow in the rst 24 hours and stopped growing in the subsequent 24 hours. CTT20 yielded 67.4 ± 9.7 mg/L pyrogallol at 48 h, which was 1.39-fold higher than that of CTT17. Meanwhile, 99.7 ± 20.3 mg/L catechol accumulated in the culture, which was 1.21-fold lower than that of CTT17. These indicate the e ciency of mutant Y385F/T294A/V349A PobA was higher than that of mutant Y385F/T294A PobA for de novo biosynthesis of pyrogallol. For CTT21, pyrogallol was continuously synthesized in the rst 36 hours (Fig. 7F) and has a titer of 129 ± 15 mg/L at 36 h, a 1.91-fold higher value when compared with that of CTT20. Meanwhile, only 6.12 ± 0.46 mg/L catechol were detected, which was 12.0-fold lower than that of CTT20 at 36 h. Within 48 h, pyrogallol titer decreased to 68.5 ± 5.0 mg/L and catechol increased to 15.8 ± 2.6 mg/L. These suggest CTT21 could achieve e cient de novo pyrogallol production, meanwhile, the accumulation of byproduct catechol was trace. In all, E. coli containing the designed arti cial pathway could achieve the de novo biosynthesis of pyrogallol. Among the engineered strains, E. coli BW25113 (F')ΔaroEΔydiB with overexpression of Y385F/T294A/V349A PobA and PDC demonstrates strongest ability for de novo production of pyrogallol.

Conclusion
The low hydroxylation activity of native PobA towards 3,4-DHBA limited the high-level production of GA and pyrogallol.
Random mutagenesis was an e cient method to generate high active PobA mutants. This work rst established a simple screening method which based on the instability of GA under alkaline conditions. Using this screening method a PobA mutant (Y385F/T294A/V349A PobA) with high activity towards 3,4-DHBA was screen out from a PobA random mutagenesis library. In vitro enzyme assay demonstrates Y385F/T294A/V349A PobA possesses higher catalytic e ciency towards 4-HBA or 3,4-DHBA than Y385F/T294A PobA towards 4-HBA or 3,4-DHBA. Moreover, Y385F/T294A/V349A PobA represents higher in vivo ability of converting 3,4-DHBA into GA or pyrogallol when compared with Y385F/T294A PobA. Assembling Y385F/T294A/V349A PobA into the de novo GA or pyrogallol biosynthetic pathway achieved GA or pyrogallol production from simple carbon sources. In all, this work constructed an e cient method for screening high active hydroxylase PobA, and this method could be applied for screening other GA biosynthesis-related enzymes. The generated high active PobA has great potential for GA or pyrogallol derivatives production.

Media, strains and plasmids
Luria-Bertani (LB) medium containing 10 g NaCl, 10 g tryptone and 5 g yeast extract per liter, was used for cell inoculation and propagation. For solid medium, 20 g/L agar was added. Modi ed M9 (M9Y) medium which contains 11.28 g/L 5×M9 Minimal Salt, 10 g/L glycerol, 2.5 g/L glucose, 1 mM MgSO 4 , 0.05 mM CaCl 2 , 2 g/L MOPS and 5 g/L yeast extract, was used for feeding experiments and de novo production of GA and pyrogallol. Terri c Broth (TB) medium which contains 12 g/L tryptone, 24 g/L yeast extract, 4 g/L glycerol, 12.5 g/L K 2 HPO 4 and 2.31 g/L KH 2 PO 4 , was used for protein expression. If needed, 100 μg/mL ampicillin, 50 μg/mL kanamycin or 25 μg/mL chloramphenicol was added to the culture. E. coli XL10-Gold and E. coli BL21(DE3) were used for plasmid construction and protein expression, respectively, while E. coli BW25113 (F') was used for feeding experiments and de novo biosynthesis of GA and pyrogallol. Plasmids pZE12-luc and pCS27 were used for pathway construction. Plasmid pETDuet-1 was used for protein expression. Plasmids pKD46 and pCP20 were used for knockout of genes. Strains and plasmids used in this study are depicted in Table 1.

High-throughput screening of PobA mutants
Single colonies of PobA mutagenesis library were pre-inoculated into 96-deep-well plates which contained 1 mL LB and100 μg/mL ampicillin, and were then aerobically cultured at 37 °C for 12 h to acquire the seed cultures. After that, 10 μL seed cultures were transferred into 990 μL M9Y medium which was supplemented with 100 μg/mL ampicillin ,0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 1 g/L 3,4-DHBA in 96-deep-well plates. The cultures were left at 30 °C for incubation. After 12 h, samples were taken. The samples were rstly centrifuged at 12,000 rpm for 10 min to remove the cells and sediments in medium. After that, 50 μL supernatant was taken into 96-well plates which contained 0.1 M NaHCO 3 . After reaction for 2 hours, the samples which with deepest green color among all the samples were screened out. The screened samples were then re-screened through detecting their optical densities at 640 nm with microplate reader (BioTek Cytation 3). The screened pobAs were sequenced to con rm the mutations.
Expression and puri cation of PobA mutants E. coli BL21 (DE3) containing pETDuet-PobA ** or pETDuet-PobA *** was pre-inoculated in 5 mL LB medium which contained 100 μg/mL ampicillin, and was then cultured overnight at 37 °C. Then, 1 mL of pre-inoculum was transferred into 100 mL of fresh TB containing 100 μg/mL ampicillin and cultured at 37 °C until OD 600 reached around 0.6. After that, 0.5 mM IPTG was added to the culture to induce protein expression at 16 °C. After 12 h, the cells were harvested via centrifugation (4000 rpm for 30 min at 4 °C) and then resuspended in 20 mL lysis buffer (50 mM Tris-HCl, pH 7.5).
The cells were disrupted by ultrasonic processor. The lysed mixture was centrifuged (10000 rpm for 60 min at 4 °С).
The supernatant was collected. The proteins were puri ed by nickel column. Quick Start TM Bradford Protein Assay (BIO-RED) was used for measuring protein concentrations.
In vitro enzyme assay and modeling of PobA mutants Mutant PobAs activity was assayed by measuring the oxidation of NADPH at 340 nm using a microplate reader. A 500- Wild-type PobA (PDB code: 1IUV) was used as template to model the tertiary structure of PobA mutants by Swissmodel https://swissmodel.expasy.org/). The complexes of PobA mutants with 3,4-DHBA and FAD were modeled by AutoDock (version 4.2.6). Hydrogen bonds in the complexes were analyzed by PyMOL (version 2.4).

Knockout of genes aroE or ydiB
To acquire 3,4-DHBA-producing strains, gene aroE or ydiB of E. coli BW25113 (F') was knocked out. First, donor fragments for pre-knockout genes needed to be constructed. For knockout of gene aroE, 530 bp at the 5'-terminus of aroE, FRT from pRecA-FRT [40], kan, FRT and 340 bp at the 3'-terminus of aroE were sequentially assembled through OE-PCR, generating aroE-donor. For knockout of gene ydiB, ydiB-donor was constructed like aroE-donor. To construct E. coli BW25113 (F')ΔaroE, the aroE-donor fragment was transferred into E. coli BW25113 (F') containing pKD46, and then cultured at 30 °C overnight. To eliminate pKD46, the overnight cultures were then spread on LB solid medium with 50 μg/mL kanamycin and then cultured at 37 °C overnight. After that, plasmid pCP20 was transferred into the generated strain and cultured at 30 °C overnight. To induce FLP recombinase and eliminate plasmid pCP20, the single colonies were picked and individually incubated on LB solid medium with 100 μg/mL ampicillin, LB solid medium with

UHPLC analysis
The standards (3,4-DHBA, GA, catechol and pyrogallol) were purchased from J&K Chemicals. UHPLC (Agilent Technologies 1290 In nity II), equipped with a reverse phase column (Agilent ZORBAX SB-C18, 5 μm, 4.6×250 mm), was used for analyzing and quantifying standards and samples. Firstly, the samples were centrifuged at 12,000 rpm for 10 min to remove the cells and sediments in medium. Then, the supernatants were ltered by 0.22 μm membrane and loaded. The column temperature was set at 30 °C. Flowing phase containing solvent A (water with 0.1% formic acid) and solvent B (100% methanol) were used, with a ow rate of 1 mL/min. The gradients were used as follows: 5% to 40% solvent B for 20 min, 100% solvent B for 2 min, 100% to 5% solvent B for 2 min and 5% solvent B for an additional 6 min. 3,4-DHBA, GA, catechol and pyrogallol were quanti ed based on their peak areas at 268 nm.     The de novo biosynthetic pathway of GA and pyrogallol.

Supplementary Files
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