A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

doi:10.21203/rs.3.rs-3037160/v1

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Research Article

A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

https://doi.org/10.21203/rs.3.rs-3037160/v1

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published 29 Aug, 2023

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Background

Microbial production of aromatic chemicals is attractive as a means of obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid 4-amino-3-hydroxybenzoic acid (4,3-AHBA) is expected to be a precursor of highly functional polybenzoxazole polymers; however, its microbial production methods have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) in the metabolic pathway of the industrially relevant bacterium Corynebacterium glutamicum.

Results

Six different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains and screened by detecting the concentration of 4,3-AHBA produced following cultivation using glucose as the carbon source. The highest 4,3-AHBA concentration was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). The combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBH^M106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) of 4,3-AHBA and 0.059 g/L (0.43 mM) of the precursor 4-ABA in the fed-batch culture.

Conclusions

Identifying PHBH mutants that efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to an improved understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

4-amino-3-hydroxybenzoic acid

4-aminobenzoic acid

polybenzoxazole

artificial biosynthetic pathway

shikimate pathway

Corynebacterium glutamicum

flavoprotein monooxygenases

4-hydroxybenzoate 3-hydroxylase

Microbial production of aromatic chemicals from biomass-derived carbon sources is an attractive way to obtain renewable precursors for high-performance materials in the face of climate change and fossil fuel resource depletion [1, 2, 3]. A non-proteinogenic amino acid 4-amino-3-hydroxybenzoic acid (4,3-AHBA) retains ortho-aminophenol and carboxylic acid groups in its molecular structure; therefore, it is expected to be a precursor for polybenzoxazole (PBO), a class of high-performance materials with excellent thermal stability and mechanical strength formed by a ring-closing condensation reaction of those two functional groups [4, 5]. Recent studies demonstrated that 3-amino-4-hydroxybenzoic acid (3,4-AHBA), a structural isomer of 4,3-AHBA, can be biosynthesized from biomass-derived carbon source using recombinant Corynebacterium glutamicum possessing two-step enzymatic reactions originally identified in Streptomyces griseus [6, 7] and be utilized as a precursor for poly(2,5-benzoxazole-co-2,5-benzimidazole) plastic films [8]. In contrast, to the best of our knowledge, no study has reported the microbial production of 4,3-AHBA from biomass-derived carbon sources.

So far, there have been several reports on the formation of 4,3-AHBA, such as the chemical reduction of 4-nitro-3-hydroxybenzoic acid [9], the biological conversion of 4-hydroxylaminobenzoic acid [10], and in vitro enzymatic 3-hydroxylation of 4-aminobenzoic acid (4-ABA) [11, 12]. Notably, biochemical studies on 4-hydroxybenzoate 3-hydroxylases (PHBHs, EC 1.14.13.2) from Pseudomonas aeruginosa (PaPHBH) and Pseudomonas fluorescens (PfPHBH) using 4-ABA as a structural analogue of 4-hydroxybenzoic acid (4-HBA) have shown that these enzymes exhibit low 3-hydroxylation activity against 4-ABA [11, 12]. In nature, various microorganisms biosynthesize 4-ABA as a precursor of folate via 4-amino-4-deoxychorismate (ADC) by ADC synthase (EC 2.6.1.85) and ADC lyase (EC 4.1.3.38) from chorismic acid, the endpoint of the shikimate pathway [13]. The shikimate pathway constitutes the upstream reactions of aromatic amino acid biosynthetic pathways, such as those for L-phenylalanine, L-tyrosine, and L-tryptophan, and is preferably used to produce various aromatic chemicals from biomass-derived carbon sources [1, 2]. Indeed, efficient production of 4-ABA (43 g/L) from glucose has been achieved using genetically engineered Corynebacterium glutamicum [13]. In this context, we hypothesized that 4,3-AHBA could be obtained from glucose as a biomass-derived carbon source by developing a PHBH that efficiently catalyzes 4-ABA 3-hydroxylation and expressing them in a suitable host microorganism.

Regarding the host microorganism, C. glutamicum was deemed suitable because it is relatively resistant to several aromatic chemicals, such as 4-ABA and 4-HBA, and can produce high concentrations of these chemicals [13, 14]. In addition, C. glutamicum has been widely used for the industrial production of amino acids, such as L-glutamic acid and L-lysine; its products have been expanded to include commodity chemicals and cosmetics through metabolic engineering techniques [15, 16, 17, 18]. Furthermore, C. glutamicum does not contain endotoxins and secretes only a few endogenous proteins into its extracellular culture medium [19]. These properties are advantageous as they simplify the purification process for both protein and chemical production.

Here, we report the production of 4,3-AHBA from glucose using a C. glutamicum strain genetically engineered to express a PHBH mutant that catalyzes the 3-hydroxylation of 4-ABA (Fig. 1). After screening six different PHBHs and their mutants, we obtained a PHBH mutant that efficiently produces 4,3-AHBA in C. glutamicum. Subsequently, a recombinant C. glutamicum strain expressing the PHBH mutant with an enhanced 4-ABA biosynthetic pathway was constructed and examined for gram-scale production of 4,3-AHBA using a fed-batch culture method.

Screening of wild type PHBHs for 3-hydroxylation of 4-ABA in C. glutamicum

To obtain a wild type enzyme suitable for the 3-hydroxylation of 4-ABA, a series of C. glutamicum strains expressing heterologous PHBHs were constructed and evaluated based on 4,3-AHBA concentration produced in the culture supernatant.

An Escherichia coli/C. glutamicum shuttle vector pKCG_P_tuf_T1 was constructed to express the protein of interest in C. glutamicum, which has an expression cassette comprising a strong constitutive promoter (P_tuf) derived from upstream of the tuf gene (gene number: cg0587) encoding the elongation factor TU [20] and the rrnB T1 terminator sequence derived from pVWEx1 (GenBank ID: MF034723.1). Then, the pobA genes for expression of PHBHs from Bradyrhizobium diazoefficiens (BdPHBH, NCBI accession number: WP_011089160.1), Caulobacter vibrioides (CvPHBH, WP_010920262.1), Rhodopseudomonas palustris (RpPHBH, WP_011157287.1), Sinorhizobium meliloti (SmPHBH, WP_010976283.1), Cupriavidus metallidurans (CmPHBH, WP_011519894.1), and Rhodococcus fascians (RfPHBH, WP_027494688.1) were selected, codon-optimized for C. glutamicum, and inserted into the expression cassette on the pKCG_P_tuf_T1. The amino acid sequences of the six PHBHs were less than 67% identical to each other (Additional file 1: Table S1). The resulting plasmids were used to construct C. glutamicum transformant strains KN001–KN007 (Table 1).

Table 1

*C. glutamicum* strains used in this study
Strain	Relevant characteristics	Reference
NBRC 12168	Wild type C. glutamicum strain identical to ATCC 13032	^aNBRC
NBRC 12169	Wild type C. glutamicum strain identical to ATCC 13058	NBRC
HT23	NBRC 12168 derivate; ΔcglIM (cg1996), ΔcglIR (cg1997), ΔcglIIR (cg1998)	[42]
KC265	HT23 derivate; P_tuf_aroG (cg2391)	This study
KC282	KC265 derivate; P_tuf_aroE3 (cg1835)	This study
KC300	KC282 derivate; P_tuf_aroB (cg1827)	This study
KC314	KC300 derivate; P_tuf_aroA (cg0873)	This study
KC315	KC314 derivate; ^bΔpobB (cg1226), P_tuf_qsuC (cg0503)	This study
KC367	KC315 derivate; ^cP_tuf_aroC (cg1829)	This study
KC376	KC367 derivate; ^dP_tuf_tkt (cg1774)	This study
KC408	KC376 derivate; P_tuf_ppsA (cg0644)	This study
KC525	KC408 derivate; ΔqsuB (cg0502)	This study
KC551	KC525 derivate; ^eP_tuf_pabAB (cg1134)	This study
KC594	KC551 derivate; ^fP_tuf_aroG^D146N	This study
KC617	KC594 derivate; ^gP_tuf_aroF^P155L	This study
KN001	^hKm^R; KC551 harboring pKCG_P_tuf_T1	This study
KN002	Km^R; KC551 harboring pKCG_P_tuf_BdPHBH_T1	This study
KN003	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH_T1	This study
KN004	Km^R; KC551 harboring pKCG_P_tuf_RpPHBH_T1	This study
KN005	Km^R; KC551 harboring pKCG_P_tuf_SmPHBH_T1	This study
KN006	Km^R; KC551 harboring pKCG_P_tuf_CmPHBH_T1	This study
KN007	Km^R; KC551 harboring pKCG_P_tuf_RfPHBH_T1	This study
KN008	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^Y201F_T1	This study
KN009	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294G_T1	This study
KN010	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294A_T1	This study
KN011	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294V_T1	This study
KN012	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294L_T1	This study
KN013	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294I_T1	This study
KN014	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294S_T1	This study
KN015	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^T294L_T1	This study
KN016	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^Y201F/T294S_T1	This study
KN017	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^Y161S/D357V_T1	This study
KN018	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^Y161S_T1	This study
KN019	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^D357V_T1	This study
KN020	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106T_T1	This study
KN021	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106G_T1	This study
KN022	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106A_T1	This study
KN023	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106V_T1	This study
KN024	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106L_T1	This study
KN025	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106I_T1	This study
KN026	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106S_T1	This study
KN027	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106C_T1	This study
KN028	Km^R; KC551 harboring pKCG_P_tuf_CvPHBH^M106A/T294S_T1	This study
KN029	Km^R; KC617 harboring pKCG_P_tuf_CvPHBH^M106A/T294S_T1	This study
^a NBRC: Biological Resource Center, National Institute of Technology and Evaluation, Japan.
^b The endogenous pobB (cg1226) gene encoding PHBH was disrupted to prevent potential background 3-hydroxylation.
^c The aroK (cg1828) gene is adjacent downstream and constitutes an operon.
^d The tal (cg1776), zwf (cg1778), opcA (cg1779), and pgl (cg1780) genes are adjacent downstream and constitute an operon.
^e The pabC (cg1135) gene is adjacent downstream and constitutes an operon.
^faroG^D146N: the codon-optimized aroG gene variant encoding D146N mutant of DAHP synthase from E. coli.
^garoF^P155L: the aroF (cg1129) gene variant encoding P155L mutant of DAHP synthase.
^h Km^R: kanamycin resistance.

To evaluate the PHBHs under sufficient 4-ABA supply, the strain KC551 (Table 1), endowed with an enhanced 4-ABA biosynthetic pathway, was used as the parental strain for transformation. The strain KC551 has P_tuf promoters inserted upstream of the endogenous genes that comprise the pentose phosphate pathway, the shikimate pathway, the subsequent 4-ABA production pathway, and the ppsA gene encoding phosphoenolpyruvate synthase, and has two additional genes (qsuB and pobB) deleted to prevent undesirable reactions. To confirm the adequate supply of 4-ABA, we measured 4-ABA concentration, glucose concentration, and optical density at 600 nm (OD₆₀₀) every 24 h for 96 h in deep-well plate culture of strain KN001 (Fig. 2A), in which the pKCG_P_tuf_T1 empty vector was introduced into strain KC551. At 72 h of culture, glucose was depleted, OD₆₀₀ reached 40, while 1.2 g/L (8.8 mM) of 4-ABA was detected in the culture supernatant, indicating successful 4-ABA overproduction.

Subsequently, six strains (KN002–KN007) carrying the plasmids harboring the heterologous pobA genes were cultured, and the concentrations of 4-ABA and 4,3-AHBA in the culture supernatants at 96 h were analyzed. Analysis of cell lysates from strains KN001–KN007 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed an additional band around 40 kDa in all strains except KN001 (Additional file 2: Fig. S1), suggesting that each PHBH was successfully expressed. Analysis of 4,3-AHBA and 4-ABA concentrations in the culture supernatants of strains KN002–007 using high-performance liquid chromatography (HPLC) revealed that 4,3-AHBA was detected in the supernatants of all six strains (Fig. 2B). Since 4,3-AHBA was detected in the culture supernatant of the strains KN002–007 except for KN001, it was suggested that the intracellularly expressed heterologous PHBHs could catalyze 3-hydroxylation of 4-ABA to produce 4,3-AHBA. The highest mean 4,3-AHBA concentration (4.3 ± 0.1 mM) and mean 4,3-AHBA percentage (58%) was detected in the culture supernatant of strain KN003, which expressed CvPHBH that had 61% identity to the amino acid sequence of PaPHBH. Thus, CvPHBH was selected as the wild type enzyme for further amino acid mutagenesis studies.

Site-directed mutagenesis targeting the active site of Cv PHBH

Using CvPHBH as a template enzyme, amino acid mutations effective for increasing 4,3-AHBA productivity in C. glutamicum were investigated by site-directed mutagenesis to the residues located in the active site.

PHBH is classified as a flavoprotein monooxygenase (FPMO) and normally catalyzes 3-hydroxylation of 4-HBA to produce 3,4-dihydroxybenzoic acid using reduced nicotinamide adenine dinucleotide phosphate (NADPH), molecular oxygen (O₂), and flavin adenine dinucleotide (FAD) [12]. Based on a three-dimensional structure model of CvPHBH constructed by a homology modelling method, 11 residues (G46, V47, W185, L199, Y201, L210, S212, R214, Y222, P293, and T294) located within 4 Å from 4-ABA and a residue (Y385) involved in the formation of hydrogen-bond networks from the substrate to protein surface [12] are shown in Fig. 3A. All 12 of these amino acid residues were identical to residues in the same position in the amino acid sequence of PaPHBH (Additional file 2: Fig. S2). Among them, in light of the studies on PaPHBH and PfPHBH [12, 21, 22, 23, 24], three residues (S212, R214, and Y222) that could form non-covalent bonds with the carboxy group of 4-ABA and another three (Y201, P293, and T294) that could form a hydrogen-bond loop with the amine group of 4-ABA were considered to be largely responsible for the 3-hydroxylation activity of CvPHBH toward 4-ABA. In particular, given that deprotonation of the phenolic group of 4-HBA is an important step in the catalytic process of PaPHBH [12], Y201 was deemed a pivotal residue, as the phenol group of the side chain could form hydrogen bonds with the amino group of 4-ABA. Furthermore, T294 was considered a suitable residue for modifying CvPHBH activity by mutagenesis while maintaining the enzyme function since the carbonyl group of the main chain, whose molecular species is unaffected by mutagenesis, could be involved in hydrogen-bond formation with Y201. Indeed, mutations of T294 have been found in PaPHBH mutants exhibiting increased hydroxylation activity toward 3,4-dihydroxybenzoic acid [24, 25, 26]. On the other hand, mutations of P293, which are reportedly responsible for the movement of FAD in the catalytic cycle [27], were expected to disrupt the catalytic function of the enzyme. Therefore, we focused on Y201 and T294 as target residues for site-directed mutagenesis and examined eight mutants (CvPHBH^Y201F, CvPHBH^T294G, CvPHBH^T294A, CvPHBH^T294V, CvPHBH^T294L, CvPHBH^T294I, CvPHBH^T294S, and CvPHBH^T294C), excluding mutations that were expected to disrupt the catalytic function beforehand.

The C. glutamicum strains expressing selected CvPHBH mutants were constructed (KN008–KN015), and the concentrations of 4,3-AHBA and 4-ABA in the culture supernatants of those strains after 96 h were analyzed. The results showed improvement in mean 4,3-AHBA concentration and mean 4,3-AHBA percentage when the five single-mutants (CvPHBH^Y201F, CvPHBH^T294G, CvPHBH^T294A, CvPHBH^T294S, and CvPHBH^T294C) were expressed (Fig. 3B). Conversely, expression of three single-mutants (CvPHBH^T294V, CvPHBH^T294L, and CvPHBH^T294I) did not enhance the 4,3-AHBA productivity compared to that of the wild type CvPHBH. The highest mean 4,3-AHBA concentration was detected in the strain KN008 expressing CvPHBH^Y201F (6.1 ± 0.8 mM, 94%), while the highest mean 4,3-AHBA percentage was detected in the CvPHBH^T294S-expressing KN014 strain (5.8 ± 0.3 mM, 98%).

Following the observation of a marked increase in the 4,3-AHBA productivity in strains expressing two mutants, namely CvPHBH^Y201F and CvPHBH^T294S, we evaluated the 4,3-AHBA productivity of the strain KN016 expressing the double-mutant CvPHBH^Y201F/T294S. The results showed that strain KN016 exhibited a higher mean 4,3-AHBA concentration (4.7 ± 0.7 mM) and mean 4,3-AHBA percentage (87%) than strain KN003 expressing the wild type CvPHBH; however, the improvement was not as remarkable as that observed when the respective single-mutants were expressed.

Random mutagenesis screening with colorimetric detection of 4,3-AHBA

In parallel with site-directed mutagenesis, a random library of CvPHBH mutants was screened using a colorimetric assay method newly constructed to simply estimate the concentration of 4,3-AHBA in the culture supernatants.

For rapid screening, we first established a method to estimate 4,3-AHBA concentration by oxidizing 4,3-AHBA with a commercially available laccase and spectrophotometrically measuring the amount of dye produced (Fig. 4A). Fortunately, it has been reported that oxidative dimerization of 4,3-AHBA naturally occurs under aerobic conditions at room temperature, forming 2-aminophenoxazin-3-one-7-carboxylic acid (2,3,7-APOC), which is easily observed as orange-yellow by the naked eye [28]. In addition, studies on multi-copper oxidases such as tyrosinase and laccase have shown that these enzymes oxidize molecules retaining ortho-aminophenol group with O₂ and the subsequent non-enzymatic dimerization reaction produces phenoxazinone dyes [29, 30]. Given the commercial availability of laccases, a colorimetric assay method based on the oxidation of 4,3-AHBA with laccase was devised. As shown in Fig. 4B, the addition of 4,3-AHBA to 0.1 M citrate buffer (pH 4.5) containing the laccase immediately caused a change in the color of the solution, which turned orange-yellow. The absorption spectrum of the reaction solution showed an absorption peak at ca. 446 nm, which was consistent with previously reported results [28]. In addition, the molecular mass of the eluate with an absorption peak at ca. 446 nm in the liquid chromatography-mass spectrometry (LC-MS) measurement was m/z = 257 (Additional file 2: Fig. S3), which was consistent with that of 2,3,7-APOC ([M + H]⁺ = 257).

Then, a plasmid library harboring the gene encoding random CvPHBH mutants in the expression cassette of the pKCG_P_tuf_T1 vector was generated by error-prone PCR and used to transform the C. glutamicum strain KC551. The 1152 colonies of the resulting transformants were screened based on the absorbance at 446 nm of the mixture of the culture supernatants and the laccase solutions. Sequencing of the CvPHBH mutant expressed in the strains with the highest absorbance resulted in the identification of the CvPHBH^Y161S/D357V double-mutant. The effect of the double-mutation on the 4,3-AHBA productivity was re-evaluated using HPLC analysis, along with the effect of the two single mutations that comprise it (Fig. 4C). The analysis of the culture supernatant of strain KN017 expressing CvPHBH^Y161S/D357V after 96 h resulted in 7.7 ± 0.5 mM of mean 4,3-AHBA concentration and 96% of mean 4,3-AHBA percentage. On the other hand, when strains KN018 and KN019 expressing CvPHBH^Y161S and CvPHBH^D357V were used, 6.0 ± 0.3 mM (70%) and 5.1 ± 0.5 mM (69%) of the 4,3-AHBA productivity were observed, respectively. All three mutants had improved 4,3-AHBA productivity compared to that of the wild type CvPHBH but were inferior to the improvement of the 4,3-AHBA productivity exhibited by the single-mutant CvPHBH^T294S.

Exploration of effective mutations around FAD

Of the two mutation sites found in the random screening, we noted that Y161 was located around FAD, a cofactor required in the catalytic cycle, in the three-dimensional structure model of CvPHBH (Fig. 5A). Similarly, we were interested in introducing mutations into residue M106, which is located opposite Y161 across FAD and expected to affect the position and movement of the cofactor. Therefore, eight strains (KN020–KN027) expressing CvPHBH mutants with a seemingly non-destructive mutation introduced at residue M106 (CvPHBH^M106G, CvPHBH^M106A, CvPHBH^M106V, CvPHBH^M106L, CvPHBH^M106I, CvPHBH^M106S, CvPHBH^M106T, CvPHBH^M106C) were constructed and evaluated for 4,3-AHBA productivity (Fig. 5B). Unexpectedly, the strain KN023 expressing CvPHBH^M106A exhibited even higher 4,3-AHBA productivity (6.6 ± 0.5 mM, 99%) than the strain KN014 expressing the active site mutant CvPHBH^T294S. Furthermore, the evaluation of the strain KN028 expressing a double-mutant CvPHBH^M106A/T294S also resulted in high 4,3-AHBA productivity (5.9 ± 0.3 mM, 98%), indicating that the M106A mutation located around FAD was compatible with the T294S mutation located at the active site.

Production of 4,3-AHBA in fed-batch culture

To investigate the capability of gram-scale 4,3-AHBA production, a C. glutamicum strain expressing CvPHBH^M106A/T294S was cultivated in fed-batch culture. To further enhance the supply of 4-ABA, aroG^D146N from E. coli and aroF^P155L from C. glutamicum that encode 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase with feedback-resistance [31, 32] were introduced into the strain KC551; the resulting strain KC617 was transformed with the plasmid pKCG_P_tuf_CvPHBH^M106A/T294S_T1, obtaining the KN029 strain. The fed-batch culture of KN029 was provided glucose under constant pH, temperature, and dissolved O₂ conditions with an initial culture volume of 60 mL (Fig. 6A and Additional file 2: Fig. S4). After 75 h of culture (final volume, 84 mL), the concentration of 4,3-AHBA in the culture supernatant reached 13.5 g/L (88 mM; Fig. 6B), and that of 4-ABA was 0.059 g/L (0.43 mM). The 4,3-AHBA percentage was calculated as 99.5%.

In this study, we designed an artificial biosynthetic pathway to produce the potential PBO precursor 4,3-AHBA from glucose in C. glutamicum and investigated the applicability of PHBHs to implement the pathway (Fig. 1). Six heterologous PHBHs were first examined with a sufficient intracellular supply of 4-ABA; we found that any of the PHBHs could produce 4,3-AHBA (Fig. 2). The subsequent site-directed mutagenesis targeting the active site of CvPHBH, the best wild type PHBH, yielded mutants CvPHBH^Y201F and CvPHBH^T294S that highly improved the 4,3-AHBA productivity (> 94% of mean 4,3-AHBA percentage) of the corresponding strains (Fig. 3). In addition, the random mutation screening using the newly constructed laccase-mediated colorimetric assay method yielded the CvPHBH^Y161S/D357V double-mutant (Fig. 4). Further exploration allowed us to obtain the CvPHBH^M106A mutant with the highest mean 4,3-AHBA percentage (99%) of the corresponding strain (Fig. 5). Moreover, the fed-batch culture of the strain KN029 expressing the double-mutant CvPHBH^M106A/T294S and the feed-back resistant DAHP synthases yielded 13.5 g/L (88 mM) of 4,3-AHBA with a 4,3-AHBA percentage of 99.5% (Fig. 6). These results indicated that application of the CvPHBH mutants implemented the artificial biosynthetic pathway to produce a gram-scale 4,3-AHBA from glucose in C. glutamicum.

During CvPHBH mutagenesis, three single mutations on different residues, namely Y201F, T294S, and M106A, were identified as being responsible for markedly improving 4,3-AHBA productivity (Fig. 3B and 5B). One reason underlying the enhanced 4,3-AHBA productivity following the Y201F mutation may be the loss of the hydrogen bond between the phenolic group on the side chain and the amine group of 4-ABA. These findings were consistent with a report on the enhanced hydroxylation activity for 4-aminophenylacetic acid (4-APA) by the S146A mutation in 4-hydroxyphenylacetate 3-hydroxylase (HPAH, EC 1.14.14.9) from Acinetobacter baumannii (AbHPAH), which belongs to a different group of FPMOs [33]. Dhammaraj et al. proposed that the interaction between the hydroxy group of S146 and the phenolic group of 4-hydroxyphenylacetic acid (4-HPA) in the active site of AbHPAH contributed to substrate recognition and that the S146A mutation removes this interaction rendering the enzyme able to recognize its non-natural substrate, 4-APA, similarly to its natural substrate 4-HPA [33]. Thus, the alteration of substrate recognition by CvPHBH^Y201F due to the removal of the hydrogen bond between Y201 and 4-ABA may have contributed to the increased 4,3-AHBA productivity. However, removal of the phenolic group in position Y201 does not appear to be essential for efficient 3-hydroxylation of 4-ABA by CvPHBH, as indicated by the finding of highly active mutants, such as CvPHBH^T294S and CvPHBH^M106A, that did not contain the Y201F mutation. In particular, the T294S mutation to the T294 residue, whose carbonyl group in the main chain could form a hydrogen bond including substrates and Y201, enhanced the 4,3-AHBA productivity even though the type of molecular environment that directly interacts with 4-ABA is unlikely to change. Besides, it could be observed that mutations to sterically smaller amino acids such as Gly, Ala, Ser, and Cys in position T294 tended to increase 4,3-AHBA productivity (Fig. 3B). Previously, Chen et al. reported that introducing the T294A mutation effectively improved the reactivity of PaPHBH toward 3,4-dihydroxybenzoic acid; they proposed that the increased flexibility of the hydrogen-bond loop by the disruption of hydrogen bond between T294 and T347 contributed to this enhancement [24]. Our results do not fit with this proposal in that the 4,3-AHBA productivity was increased even for mutations such as T294S that do not appear to disrupt the hydrogen bond with T347. However, since mutation to sterically smaller amino acids is thought to generate conformational space and increase the flexibility of the residue, the effect of increasing the flexibility of the hydrogen-bond loop would be expected for the T294S mutation in CvPHBH like the T294A mutation in PaPHBH. Therefore, one possible explanation for the effectiveness of the mutation at position T294 in improving the 4,3-AHBA productivity is the increased flexibility of the hydrogen-bond loop at the active site of CvPHBH. As for the M106A mutation, it was surprising that the introduction of a single mutation at residue M106, which is located away from the active site, dramatically improved the 4,3-AHBA productivity (Fig. 5B). In addition, the strain expressing CvPHBH^M106A/T294S showed 4,3-AHBA productivity comparable to that of the strains expressing CvPHBH^M106A or CvPHBH^T294S, suggesting that these two beneficial single mutations could be combined without interfering with each other. It should be noted, however, that the comparisons made in this study regarding PHBHs were based on the concentration of 4,3-AHBA produced in the culture supernatant of C. glutamicum strains that express PHBHs intracellularly. Further detailed biochemical analysis of CvPHBH and its mutants will provide a better understanding of mutation effects on the substrate specificity of CvPHBH and the 4,3-AHBA productivity of C. glutamicum when the CvPHBH mutants are expressed.

Currently, several enzymes classified as FPMOs are known to perform ortho-hydroxylation of aniline derivatives. FPMOs are classified into eight groups (Group A–H) based on their sequence and structural similarities [34, 35]. Those responsible for the hydroxylation of aromatic chemicals are found in the one-component Group A FPMOs, which are represented by PHBH, and the two-component Group D FPMOs, which are represented by HPAH. One example is kynurenine 3-monooxygenase (Group A, EC 1.14.13.9), which catalyzes the hydroxylation of kynurenine using O₂ and NADPH as co-substrates to give 3-hydroxykynurenine in the L-tryptophan degradation pathway [36]. Another example is anthranilate 3-monooxygenase (Group D, EC 1.14.14.8) from Geobacillus thermodenitrificans NG80-2, which converts anthranilate to 3-hydroxyanthranilate in the anthranilate degradation pathway [37]. Incidentally, it should be noted that the 4-aminobenzoate hydroxylase, also known as 4-aminobenzoate 1-monooxygenase (Group A, EC 1.14.13.27), catalyzes the decarboxylative hydroxylation of 4-ABA to form 4-hydroxyaniline, but not the ortho-hydroxylation of aniline groups [38]. FPMOs are thought to have wide application as biocatalysts for synthesizing value-added chemicals because they can catalyze various regioselective monooxygenation reactions, such as hydroxylation, Baeyer-Villiger oxidation, and epoxidation [34, 35]. The findings for CvPHBH and its mutants would be new examples of FPMOs that catalyze the ortho-hydroxylation of aniline derivatives and contribute to our understanding of this group of enzymes and the expansion of their application.

From a practical point of view, there is much room to improve the productivity and purity of 4,3-AHBA using further metabolic and culture engineering technologies. It has been reported that the introduction of heterologous pabAB and pabC genes encoding ADC synthase and ADC lyase contributed to improving 4-ABA productivity [13], which can be adopted to further increase the supply of intracellular 4-ABA. In addition, since PHBHs simultaneously consume O₂ and NADPH during their reaction, controlling the dissolved O₂ content during culture and supplying sufficient NADPH by altering the metabolic redox balance in the cell could increase productivity. Furthermore, the purity and cost of downstream processing can be improved by controlling the oxidative colorization of the culture supernatant. We expect these improvements will enable the production of 4,3-AHBA from biomass resources at an industrially feasible cost for practical application.

Expression of the CvPHBH mutants in the industrially relevant bacterium C. glutamicum enabled efficient 3-hydroxylation of 4-ABA derived from the shikimate pathway, leading to the de novo production of 4,3-AHBA from glucose. The fed-batch culture of the C. glutamicum strain KN029 expressing CvPHBH^M106A/T294S yielded 13.5 g/L of 4,3-AHBA with a 4,3-AHBA percentage of 99.5%. The productivity of 4,3-AHBA would be improved by further exploration of industrial production. In addition, the CvPHBH mutants obtained in this study would be used to explore the molecular mechanism of regioselective monooxygenation of the aniline moiety. These findings would contribute to the diversification of aromatic chemicals derived from biomass resources to address the global demands for sustainable chemical products.

Chemicals and reagents

Standard chemicals for analysis of 4-ABA and 4,3-AHBA were purchased from Tokyo Chemical Industry (Tokyo, Japan). All other chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). DNA synthesis and sequencing were performed by Eurofins Genomics (Tokyo, Japan). DNA amplification was performed using KOD One® Master Mix (TOYOBO, Tokyo, Japan), PrimeSTAR® Mutagenesis Basal Kit (Takara Bio, Shiga, Japan), or Diversify™ PCR Random Mutagenesis Kit (Takara Bio). The plasmid pHSG299, the restriction enzyme DpnI, NucleoSpin® PCR and Gel Clean-up, In-Fusion® HD Cloning Kit, and NucleoSpin® Plasmid EasyPure were purchased from Takara Bio and used for DNA manipulation. The plasmid pHM1519 was extracted from C. glutamicum NBRC 12169 [39]. ECOS™ Competent E. coli JM109 (NIPPON GENE, Tokyo, Japan) were used for plasmid preparation. Difco™ Terrific Broth and Difco™ LB Broth were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA) and appropriately supplemented with 1.5% agar and 50 mg/L kanamycin sulfate for the cultivation of E. coli strains. Bacto™ Tryptone was purchased from Life Technologies Corporation (Waltham, MA, USA). Mini-PROTEAN® TGX™ Stain-Free Gels (Any kD, 15-well; Bio-Rad Laboratories, Hercules, CA, USA), Precision Plus Protein™ Unstained Standards (Bio-Rad Laboratories), and 3X SDS-PAGE Loading Buffer (BioVision, Milpitas, CA, USA) were used for SDS-PAGE analysis.

Construction of plasmids and bacterial strains

The C. glutamicum strains used in the study are listed in Table 1. The plasmids and primers used in the study are listed in Additional file 1: Table S2 and S3, respectively. The genomic DNA sequence and gene numbers for C. glutamicum were from previous reports [40, 41]. The nucleotide sequences of the pobA genes codon-optimized for C. glutamicum are listed in the Additional file 3. Each codon-optimized pobA gene was amplified from synthetic DNA using the appropriate primer pair and assembled with the pKCG_P_tuf_T1 vector. The plasmids for expression of site-directed PHBH mutants and aroF^P155L were constructed using appropriate overlapping primer pairs. The random mutant library was generated by assembling the DNA fragment of the pobA gene for CvPHBH amplified with error-prone PCR and the linearized pKCG_P_tuf_T1 vector. The error-prone PCR was performed in buffer condition 5 (nine mutations per 1000 bp) according to the kit instructions. The genetically engineered C. glutamicum strains were constructed from C. glutamicum NBRC 12168 using a two-step homologous recombination system with a suicide vector containing the sacB gene from Bacillus subtilis [42, 43]. The C. glutamicum strains used for evaluating 4,3-AHBA productivity were constructed by incorporating the appropriate plasmids into strains KC551 or KC617. The C. glutamicum strains were transformed using an electroporation method [44] with an ELEPO21 (Nepa Gene, Chiba, Japan) under the following conditions: poring pulse (1400 V voltage, 3.5 ms pulse length, 50 ms pulse interval, 1 number of pulses, + polarity) and transfer pulse (200 V voltage, 50 ms pulse length, 50 ms pulse interval, 3 number of pulses, ± polarity).

Growth conditions of C. glutamicum strains

Single colonies of the positive transformants were aerobically cultivated in CGTG15 medium (per liter: 50 g glucose, 10 g Tryptone, 20 g (NH₄)₂SO₄, 5 g urea, 1 g KH₂PO₄, 1 g K₂HPO₄, 0.25 g MgSO₄·7H₂O, 10 mg CaCl₂, 10 mg FeSO₄·7H₂O, 10 mg MnSO₄·5H₂O, 1 mg ZnSO₄·7H₂O, 0.2 mg CuSO₄·5H₂O, 0.02 mg NiCl₂·6H₂O, 0.2 mg biotin) supplemented with kanamycin sulfate (50 mg/L). The composition of the CGTG15 medium was designed with reference to the CGXII medium [45, 46, 47]. The transformants of strain KC551 were cultured at 30°C and 800 rpm in 750 µL of the medium in deep-well plates sealed with Axygen® Breathable Sealing Film (Corning Inc., Corning, NY, USA) using M·BR-034P shaker (TAITEC Corporation, Saitama, Japan). To produce 4,3-AHBA in fed-batch culture, strain KN029 was pre-cultured for 24 h at 30°C in 6 mL of the medium in a test tube. Subsequently, 3 mL of the pre-cultured cells were used to inoculate 60 mL of the medium containing 250 mg/L of antifoam FERMOL1000 (Kao Corporation, Tokyo, Japan) in a small-scale multi-channel fermenter (Bio Jr.8; ABLE, Tokyo, Japan), which was cultured at 32°C (aeration, 60 mL/min). The agitation was automatically controlled to keep the dissolved O₂ level constant at 0.2 ppm. The pH was maintained at 7.2 with the automatic addition of 10% ammonia solution, and glucose in the culture medium was replenished as needed with 60% (w/w) glucose solution to prevent depletion. Aliquots of culture medium were taken to calculate cell density, followed by quantification of the products and glucose. Cell density was calculated by monitoring the optical density at 600 nm (OD₆₀₀) using a spectrophotometer.

SDS-PAGE analysis

Aliquots (60 µL) of culture medium were mixed with 540 µL of 0.1 M potassium phosphate buffer (pH 7.4) and 700 mg of glass beads YGB01 (0.1 mm, Yasui Kikai Corporation, Osaka, Japan). The cells in the mixture were then disrupted using Multi-beads Shocker MB901U(S) (Yasui Kikai Corporation) at 4°C with six cycles of 2500 rpm for 60 s and intervals for 60 s. Subsequently, the mixture was centrifuged at 21600 ×g for 5 min at 4°C, and 10 µL of the supernatant was mixed with 5 µL of the loading buffer, followed by incubation for 15 min at 95°C. The resulting sample (5 µL) was used for electrophoresis (200 V, 30 min).

Analytical method

The quantification of 4-ABA and 4,3-AHBA in culture supernatants was performed using an HPLC system (Chromaster; Hitachi High-Tech Science Corporation, Tokyo, Japan) equipped with a reversed-phase column (L-column® ODS, 5 µm [4.6 × 150 mm]; Chemicals Evaluation and Research Institute, Tokyo, Japan) and a photodiode array detector (detection wavelength: 280 nm). Gradient elution (eluent A: 0.1 M KH₂PO₄ in 0.1% (v/v) H₃PO₄/H₂O, eluent B: 70% (v/v) methanol/H₂O) was conducted at a flow rate of 1.0 mL/min and a column temperature of 40°C. The quantification of glucose in culture supernatants was performed using the HPLC system equipped with an organic acid analysis column (ICSep ION-300, 5 µm [7.8 × 300 mm]; Tokyo Chemical Industry) and a refractive index detector. Elution (eluent: 5 mM H₂SO₄/H₂O) was performed under isocratic conditions at a flow rate of 0.5 mL/min and a column temperature of 50°C. The culture medium was diluted with 37 mM H₂SO₄ aqueous solution, and insoluble material was removed using AcroPrep™ Advance 96-well Filter Plates (350 µL, 0.2 µm WWPTFE; Nihon Pall Ltd., Tokyo, Japan). The resulting solution was allowed to stand overnight or longer for hydrolysis of N-glucosyl byproduct [13] before analysis. The 4,3-AHBA percentage was calculated as the percentage of 4,3-AHBA concentration relative to the total concentration of 4,3-AHBA and 4-ABA.

Colorimetric detection of 4,3-AHBA

The enzyme solution was prepared by mixing 0.1 g of laccase M120 powder (Amano Enzyme, Aichi, Japan) with 20 mL of 0.1 M sodium citrate buffer (pH 4.5), followed by centrifugation. The substrate solution was prepared by dissolving 16 mg of 4,3-AHBA in 20 mL of 0.1 M sodium citrate buffer (pH 4.5). On a 96-well assay plate, 5 µL of the substrate solution was mixed with 195 µL of the enzyme solution and allowed to stand for 20 min at room temperature. The absorbance spectrum was measured using Infinite® 200 PRO (TECAN, Männedorf, Switzerland). Thereafter, 100 µL of the resulting solution was mixed with 200 µL of methanol and filtrated using AcroPrep™ Advance 96-well Filter Plates (350 µL, 0.2 µm WWPTFE). The molecular mass was analyzed using an LC-MS system (LCMS-2020; Shimadzu Co., Kyoto, Japan) equipped with a reversed-phase column (Sunrise C28 [2.0 × 150 mm]; ChromaNik Technologies, Osaka, Japan). Gradient elution (eluent A: 10 mM ammonium acetate aqueous solution, eluent B: 10 mM ammonium acetate in 99% (v/v) methanol/H₂O) was conducted at a flow rate of 0.2 mL/min and a column temperature of 40°C. Electrospray ionization was performed in the positive ion mode. The detection of 4,3-AHBA in the culture supernatant of C. glutamicum was performed by mixing 5 µL of the culture supernatant with 195 µL of the enzyme solution and measuring absorbance at 446 nm.

Structure modelling

The three-dimensional structural model was constructed using the SWISS-MODEL server [48] based on a crystal structure of PaPHBH (PDB ID: 1IUT) that contained 4-ABA and FAD in its molecule [12].

4-ABA

4-Aminobenzoic acid

ADC

4-Amino-4-deoxychorismate

3,4-AHBA

3-Amino-4-hydroxybenzoic acid

4,3-AHBA

4-Amino-3-hydroxybenzoic acid

4-APA

4-Aminophenylacetate

2,3,7-APOC

2-Aminophenoxazin-3-one-7-carboxylic acid

DAHP

3-deoxy-D-arabino-heptulosonate-7-phosphate

4-HBA

4-Hydroxybenzoic acid

4-HPA

4-Hydroxyphenyllactic acid

PBO

Polybenzoxazole

FPMO

Flavoprotein monooxygenase

PHBH

4-Hydroxybenzoate 3-hydroxylase

HPAH

4-Hydroxyphenylacetate 3-hydroxylase.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

Patent applications related to this study have been filed with Kao Corporation, to which all the authors belong, as the applicant.

Funding

This study was supported by Kao Corporation and the project P20011 (Development of bioderived product production technology that accelerates the realization of carbon recycling) for the New Energy and Industrial Technology Development Organization (NEDO) under the Ministry of Economy, Trade and Industry (METI), Japan.

Authors' contributions

KN and FT contributed to the design of this study. Material preparation, data collection, and analysis were performed by KN and TO. KN wrote the draft of the manuscript. All authors approved the final manuscript.

Acknowledgements

We would like to thank Editage (http://www.editage.com) for English language editing.

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Competing interest reported. Patent applications related to this study have been filed with Kao Corporation, to which all the authors belong, as the applicant.

Additionalfile1.pdf
Additional file 1: Table S1 The identity (%) matrix for amino acid sequences of PHBHs. Table S2 Plasmids used in this study. Table S3 Primers used in this study.
Additionalfile2.pdf
Additional file 2: Fig. S1 SDS-PAGE analysis for the cell lysate of the strains KN001–007. Fig. S2 The amino acid sequence alignment of CvPHBH and PaPHBH. Fig. S3Mass profile of the eluate with the absorption peak at ca. 446 nm in LC. Fig. S4Time variation of fermentation process parameters in the fed-batch culture of C. glutamicum strain KN029.
Additionalfile3.txt
Additional file 3: The nucleotide sequences of the pobA genes codon-optimized for C. glutamicum.

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published 29 Aug, 2023

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Editorial decision: Major revision
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A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Random mutagenesis screening with colorimetric detection of 4,3-AHBA

Exploration of effective mutations around FAD

Production of 4,3-AHBA in fed-batch culture

Discussion

Conclusions

Methods

Chemicals and reagents

Construction of plasmids and bacterial strains

SDS-PAGE analysis

Analytical method

Colorimetric detection of 4,3-AHBA

Structure modelling

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1