Synthesis of 3,4-Dihydroxybenzoic Acid in E. coli and C. glutamicum Using Dehydroshikimate Dehydratase of Different Types

Abstract

Dehydroshikimate (DHS) dehydratase (DSD) catalyzes the conversion of DHS into 3,4-dihydroxybenzoic acid (3,4-DHBA), a compound with promising applications across various industries. The DSD from Podospora anserina (DSD_Pa) was characterized and its catalytic properties were compared with those of previously investigated enzymes, AsbF (Bacillus thuringiensis), Qa-4 (Neurospora crassa), and QsuB (Corynebacterium glutamicum), both in vitro and in vivo using tube fermentation. Escherichia coli and C. glutamicum were used as platforms to construct model 3,4-DHBA producers. To increase DHS availability in both hosts, shikimate dehydrogenase AroE was inactivated, and the plasmid pVS7-aroG4, encoding 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (E. coli), was introduced. In E. coli, heterologous 3,4-DHBA synthesis was achieved through chromosomal integration of dsd genes. The fungal genes were codon-optimized for this bacterium. The same genes were cloned into the pVK9 vector and introduced into C. glutamicum, where 3,4-DHBA degradation was disrupted (ΔpcaHG). AsbF (k_cat ~ 1 s⁻¹) showed poor 3,4-DHBA accumulation in both hosts (1–1.5 g/L). The enzymes with better catalytic characteristics, QsuB (k_cat ~ 60 s⁻¹), DSD_Pa (k_cat ~ 125 s⁻¹), and Qa-4 (k_cat ~ 220 s⁻¹), provided 5 g/L 3,4-DHBA in E. coli and 3 g/L 3,4-DHBA in C. glutamicum, except for Qa-4. The low production (~1.5 g/L) observed for Qa-4 in C. glutamicum might be attributed to a non-optimal nucleotide sequence rich in codons rare for C. glutamicum.

1. Introduction

3,4-Dihydroxybenzoic acid (3,4-DHBA), also known as protocatechuic acid, is a natural metabolite. In soil microorganisms, 3,4-DHBA serves as an intermediate in quinate/shikimate catabolism. 3,4-DHBA is a phenolic acid with functional –COOH and –OH groups, which can form complexes with divalent and trivalent cations. Consequently, in some Bacillus spp., 3,4-DHBA acts as an iron-chelating component of the siderophore [1,2,3].

In recent decades, 3,4-DHBA has been found to possess antioxidant, neuroprotective, and anti-inflammatory properties [4,5]. This makes it possible to use 3,4-DHBA in cosmetic and pharmaceutical industries [6]. Moreover, 3,4-DHBA was shown to be used as an intermediate for the synthesis of other valuable compounds such as vanillin, catechol, adipic acid, muconic acid, and biopolymers [7,8,9,10,11,12]. Current methods for 3,4-DHBA production have several disadvantages. Extraction from plants gives a low yield and, subsequently, high cost of the target compound [13,14]. Chemical synthesis implies an energy-consuming process and environmentally harmful reagents [15]. Therefore, microbial fermentation of renewable raw materials can be applied for green and sustainable 3,4-DHBA production.

The construction of 3,4-DHBA-producing strains has been described previously [16,17,18,19,20,21,22]. Such strains were created based on C. glutamicum, Escherichia coli, Bacillus licheniformis, and Pseudomonas putida. The 3,4-DHBA synthesis from D-xylose using C. glutamicum as a host has been described [21]. However, other 3,4-DHBA-producing strains utilized glucose as a carbon source.

Two biosynthetic routes for microbial 3,4-DHBA production have been established: dehydration of 3-dehydroshikimate (DHS), an intermediate of the common aromatic pathway, or a two-step conversion of chorismate (CHA), the end product of this pathway (Figure 1). The latter route may be less favorable due to additional requirements for ATP, PEP, and NADPH. Interestingly, 3,4-DHBA synthesis via CHA appeared to be effective when combined with DHS-based synthesis in a C. glutamicum producing strain. This approach achieved the highest reported titer to date (~ 82 g/L 3,4-DHBA) under aerobic growth-arrested fermentation conditions [22].

Here, only 3,4-DHBA synthesis by DHS dehydratase (EC: 4.2.1.118) (DSD) was considered, as we aimed to compare DSDs from different sources. Based on their structural properties, DHS dehydratases have been divided into five types: bacterial one-domain, bacterial two-domain, fungal one-domain, bacterial membrane-associated, and QuiC2-like enzymes [23,24]. Previously, we compared 3,4-DHBA production in vitro and in vivo using enzymes of the first three types and employing E. coli as a platform strain [17,25]. Two-domain QsuB from C. glutamicum and one-domain Qa-4 from the filamentous fungus Neurospora crassa showed higher specific activity at physiological pH 7.5 and provided better 3,4-DHBA accumulation than the one-domain AsbF from Bacillus thuringiensis. In this work, we biochemically characterized DSD from Podospora pauciseta/anserina (DSD_Pa) for the first time. DSD_Pa possessed 72% identity with Qa-4 and demonstrated catalytic properties similar to those of Qa-4 and QsuB.

C. glutamicum has been described as a host for 3,4-DHBA synthesis, primarily using its native enzyme QsuB [21,22]. In this work, we compared DHS dehydratases of different types in a C. glutamicum platform strain. Unlike E. coli, C. glutamicum both synthesizes and degrades 3,4-DHBA as an intermediate of the quinate/shikimate utilization pathway (Figure 1B). Therefore, 3,4-DHBA degradation was disrupted in the C. glutamicum model producer. To reduce DHS flux into the common aromatic pathways, the aroE gene, encoding the main biosynthetic shikimate dehydrogenase, was inactivated in both host strains. The investigated bacteria possess additional minor enzymes with this catalytic function (Figure 1). YdiB exhibits low activity in E. coli, and its inactivation does not affect the growth in M9 minimal medium [26]. Quinate/shikimate dehydrogenase QsuD is essential for utilizing these compounds as carbon sources in C. glutamicum [27]. To enhance the carbon flux into the aromatic pathways and subsequent precursor DHS accumulation, we introduced a plasmid encoding E. coli 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase (pVS7-aroG4). Thus, we aimed to reveal more promising enzymes for 3,4-DHBA production.

2. Materials and Methods

2.1. Strains, Plasmids and Growth Conditions

E. coli and C. glutamicum strains and plasmids are listed in

Table 1. Bacterial strains and plasmids used in this work.

Strain or Plasmid	Relevant Characteristics	Source
E. coli strains
BL21(DE3)	F–ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K−12(λS)	Novagen (Merck Millipore, Darmstadt, Germany)
MG1655∆aroE	F− λ− ilvG− rfb-50 rph-1ΔaroE	[25]
MG1655∆aroE PlacUV5-qsuBattφ80	3,4-DHBA producing strains	[17]
MG1655∆aroE PlacUV5-asbFattφ80
MG1655∆aroE PlacUV5-qa-4attφ80
MG1655∆aroE PlacUV5-dsdPaattφ80		This work
C. glutamicum strains
AJ1511	ATCC13869 lacking cryptic plasmid pAM330	[28]
K118	AJ1511ΔaroEΔpcaHG	This work
K118ΔqsuB	−	This work
Plasmids
pAH162-λattL-TcR-λattR- PlacUV5-qsuB	TcR, oriRγ, phi80attP; used for the creation of integrative vector with the dsdPa gene	[17]
pAH162-λattL-TcR-λattR-PlacUV5-dsdPa	TcR, oriRγ, phi80attP; used for PlacUV5-dsdPa integration into E. coli chromosome	This work
pAH123	ApR, the helper for the PlacUV5-dsdPa integration	[29]
pMW-int-xis	ApR, oriR101, repA101ts, λcIts857, λPR → λxis-int; the helper for marker removal in E. coli.	[30]
pMW119-lox66-cat-lox71	CmR; contained excisable Cm-marker. lox71/lox66—mutant lox sites that exclude chromosomal deletions occurring after further introduction of identical markers.	A. Krylov (JSC “AGRI”)
pVC-AprR-lacI-Ptrc-id2-recE564T	AprR, ori pMB1, ori pAM330; the helper for RecET-mediated recombination in C. glutamicum	[31]
p06-PdapA-cre	GmR, ori pMB1, ori pCG1; the helper for Cre-mediated marker removal in C. glutamicum	[32]
pET22b-dsdPa-His6	ApR; production of DSDPa protein with and without His-tag	This work
pET22b-dsdPa
pVK9	KmR; ori pCG1, ori ColE1	[33]
pVK9-lacI-Ptrc-id2-gfp	KmR; oripCG1, oriColE1, lacI, IPTG inducible Ptrc-id2	A. Krylov (JSC “AGRI”)
pVK9-lacI-Ptrc-id2-asbF	KmR; plasmids containing dsd genes	This work
pVK9-lacI-Ptrc-id2-qa-4
pVK9-lacI-Ptrc-id2-qsuB
pVK9-lacI-Ptrc-id2-dsdPa
pVS7	SpeR; ori pMB1, repBI1	[34]
pVS7-aroG4	SpeR; ori pMB1, repBI1, aroG4	This work

. E. coli and C. glutamicum cells were cultivated with aeration (240 rpm) in LB (Tryptone, 10; NaCl, 10; Yeast extract, 5 (g/L)) and BHI (Thermo Scientific™, Detroit, MI, USA) at 37 °C and 30 °C, respectively. To prepare plates, agar (20 g/L) was added. Antibiotics were used in the following concentrations (mg/L): ampicillin (Ap), 200, tetracycline (Tc), 12.5, spectinomycin (Spe), 50 (E. coli); chloramphenicol (Cm), 6, kanamycin (Km), 25, spectinomycin (Spe), 50, gentamicin (Gm), 1.5 (C. glutamicum).

C. glutamicum cells were grown (~2 h) in BHI supplemented with glycine (2%) and Tween 80 (0.1%) before electroporation.

2.2. Fermentation

Fermentation of E. coli and C. glutamicum strains was performed in tubes (18 mm × 200 mm), containing 2 mL of fermentation medium. Tubes were inoculated with 0.2 mL of seed culture. To prepare seed culture, one loop (∅ 3 mm) of cells taken from a fresh Petri dish was introduced into tubes (13 mm × 150 mm) containing 3 mL of medium and incubated as described below.

The E. coli fermentation medium contained (g/L) glucose, 40; CaCO₃, 60; tryptone, 10; NaCl, 10; yeast extract, 5; (NH₄)₂SO₄, 0.5; KH₂PO₄, 0.5; MgSO₄∙7H₂O, 0.3; FeSO₄∙7H₂O, 0.005; thiamine, 0.01; and 4-hydroxybenzoic acid, para-aminobenzoic acid, and 2,3-dihydroxybenzoic acid, 0.005 (pH 7.0). Fermentation was performed at 34 °C and 240 rpm for 44 h. The seed culture was grown in LB at 34 °C with aeration (240 rpm) for 3 h.

The C. glutamicum fermentation medium contained (g/L): glucose, 50; MgSO₄·7H₂O, 0.4; CaCO₃, 60; (NH₄)₂SO₄, 5; KH₂PO₄, 2; FeSO₄∙7H₂O, 0.01; MnSO₄∙4H₂O, 0.01; ZnSO₄∙7H₂O, 0.0072; soybean hydrolysate, 0.75 (Ajinomoto Co., Inc., Kawasaki, Japan); biotin, 0.00012; thiamine, 0.01; para-aminobenzoic acid, 0.005; L-Phe, 1; L-Tyr, 1; L-Trp, 1. Fermentation was performed at 30 °C and 240 rpm until complete glucose consumption. The seed medium (pH 7.5) contained (g/L) glucose, 5; tryptone, 10; yeast extract, 10; KH₂PO₄, 1; MgSO₄·7H₂O, 0.4; FeSO₄·7H₂O, 0.01; MnSO₄·5H₂O, 0.01; urea, 3; and biotin 0.0001. The seed culture was grown for 18 h at 30 °C, 240 rpm.

Residual glucose was tested by GlukoPHAN test strips (Erba Lachema, Brno, Czech Republic) and concentrations were determined with BIOSEN C_line (EKF-diagnostic GmbH, Barleben, Germany). To make samples transparent for optical density (OD) measurements, 2 N HCl was added to convert CaCO₃ present in the fermentation medium into CaCl₂. DHS and 3,4-DHBA concentrations were analyzed by HPLC as described previously [17].

2.3. Strains Construction

The integration of the dsd_Pa gene into the E. coli chromosome was performed as described previously for genes asbF, qa-4, and qsuB [17]. For this purpose, the integrative vector pAH162-λattL-Tc^R-λattR-P_lacUV5-dsd_Pa was created. Helper plasmids pAH123 and pMW-int-xis were used for integration into the native φ80attB site of MG1655ΔaroE chromosome and for subsequent elimination of the Tc resistance marker, respectively (Figure 2A).

Gene deletions in the chromosome of C. glutamicum were performed using RecET-mediated recombineering [31]. Helper plasmids pVC-Apr^R-lacI-P_trc-id2-recE⁵⁶⁴T and p06-P_dap-cre were used for replacement of the target gene by the marker gene and marker elimination, respectively (Figure 2B).

2.4. DNA Manupulations

All DNA procedures, including PCR and Circular Polymerase Extension Cloning (CPEC), were performed as described previously [17,25]. The DNA sequence of dsd_Pa was optimized for E. coli based on amino acid sequence CAP65279.1 and chemically synthesized (Twist Bioscience, South San Francisco, CA, USA). The obtained gene was used for expression in both E. coli and C. glutamicum. Primers for PCR used in this study are listed in Table S1.

DNA fragments used for the gene replacement (aroE (cgl1629), pcaHG, and qsuB) were obtained by overlap PCR of three fragments. These included the excisable marker lox71-cat-lox66 and two flanking homologous arms (~800 bp) targeting chromosomal regions upstream and downstream the insertion site. Plasmid pMW119-lox66-cat-lox71 served as the template for marker amplification.

All plasmids were obtained by CPEC [35] using pre-amplified DNA fragments with overlapping ends. The DNA of chemically synthesized asbF, qa-4, and dsd_Pa genes was used as templates. Chromosomal DNA from AJ1511 [28] and VKPM B-10747 [36] strains served for amplifying the qsuB and aroG4 genes, respectively. To obtain the vector parts, DNA of pVK9-lacI-P_trc-id2-gfp, pAH162-λattL-Tc^R-λattR-P_lacUV5-qsuB, pET22b, and pVS7 plasmids was used as the templates after digestion with PstI, PvuII, SalI, and BamHI, respectively. Translation initiation regions (included in primers) for all genes inserted into the pVK9 vector were designed to ensure similar expression levels using UTR Designer [37]. The expression indices of dsd genes were similar in both E. coli and C. glutamicum, as determined by GenScript (https://www.genscript.com/tools/rare-codon-analysis, accessed on 17 January 2025). The resulting plasmids were verified by sequencing (Evrogen, Moscow, Russia).

2.5. Enzyme Activity Assays

The activity was measured as previously described [25,38] by monitoring the change in absorbance of 3,4-DHBA at 290 nm (ε₂₉₀ = 3.89 × 10³ M⁻¹ × sm⁻¹) for 0.5 min using Genesys10S UV-visible spectrophotometer (Thermo Scientific, Madison, WI, USA). The standard reaction was performed at 25 °C in a quartz cuvette in 1 mL of reaction mixture with the following composition: 0.1 M Tris/HCl buffer (pH 7.5), 10 mM metal salt (CoCl₂, MgCl₂, and MnCl₂), 0.1–5 mM DHS and purified enzyme (150 nM AsbF, 10 nM Qa-4, 20 nM QsuB, 45 nM DSD_Pa).

To exclude the potential influence of the His-tag on enzyme activity, DHS dehydratase activity was analyzed in crude extracts of BL21(DE3)/pET22b-dsd_Pa and BL21(DE3)/pET22b-dsd_Pa-His₆ strains (Figure S1). Crude protein extracts were prepared using xTractor-Buffer (Takara Bio, San Jose, CA, USA). Since the activities were identical (mean statistical error < 0.5%), the purified C-terminally His₆-tagged DSD_Pa protein was used for subsequent kinetic analyses. The His₆-tagged protein was purified using the Capturem™ His-Tagged Purification Miniprep Kit (Takara Bio, San Jose, CA, USA). To remove residual divalent cations, the enzyme was incubated with 1 mM EDTA on ice for 1 h prior to activity assays. For metal cofactor testing, 1 mM DHS was used. The K_m and V_max values of DSD_Pa were determined by non-linear regression analysis [39]. The inhibition type was investigated by the “quotient velocity plot” method [40]. For this analysis, DHS was used at concentrations 0.2, 0.4, and 1 mM, while 3,4-DHBA was evaluated as a potential inhibitor at concentrations ranging from 0 to 0.6 mM.

2.6. Sequence Alignment

The amino acid sequences of DHS dehydratases, QuiC from Acinetobacter baylyi (AAC37159.1), QuiC1 from P. putida (AAN68163.1), QuiC2 from Listeria monocytogenes (CAD00312.1), DSD_Pa (CAP65279.1), Qa-4 (CAA32750.1), QsuB (BAB97815.1), and AsbF (AOM10649.1), were obtained from the protein database (https://www.ncbi.nlm.nih.gov/protein, accessed on 15 April 2024). Analysis of homologous sequences was performed using the blastp algorithm (https://www.uniprot.org/blast, accessed on 15 April 2024), and phylogenetic tree rendering (https://www.uniprot.org/align, accessed on 15 April 2024) was conducted using UniProt services.

2.7. Statistical Analysis

All values in tables represent arithmetic means of at least three independent experiments, with errors given as confidence interval. Microsoft Excel 2010 was used for calculations. To assess the significance of differences between the investigated enzymes, we performed Analysis of Variance (ANOVA) followed by Tukey’s HSD (honestly significant difference) test. This statistical analysis was applied to both catalytic properties (Table S2) and 3,4-DHBA accumulation in test tube fermentations (Tables S3–S5).

3. Results

3.1. DHS Dehydratase from P. anserina and Its Expression in T7 System

The DHS dehydratase from P. anserina (DSD_Pa) and Qa-4 both belong to type IV DHS dehydratases (Figure 3). DSD_Pa was applied for vanillin production in Schizosacharomyces pombe, though it was not characterized biochemically [7]. The precursor of vanillin producer with the dsd_Pa gene accumulated ~ 0.3 g/L 3,4-DHBA, while E. coli producer expressing the qa-4 gene produced ~ 3 g/L 3,4-DHBA [17]. These results were obtained in different host organisms in separate studies and did not provide an insight into the diversity of enzymes of the same type.

To enable direct comparison between DSD_Pa, Qa-4, and the enzymes of other types, we characterized DSD_Pa using the same experimental approach previously applied to QsuB, AsbF, and Qa-4 [17,25].

3.2. Catalytic Properties of DSD_Pa and Their Comparison with Those of AsbF, Qa-4, QsuB

DSD is a metal-dependent enzyme, and we measured DSD_Pa activity in the presence of various divalent metal ions (Mg²⁺, Ca²⁺, Zn²⁺, Co²⁺ and Mn²⁺). Unlike previously tested enzymes [25], DSD_Pa retained its activity after incubation with EDTA, which was used to remove residual metal ions from the active site (Figure 4A). DSD_Pa was slightly inhibited in the presence of Ca²⁺, Zn²⁺ and Mg²⁺, while Mn²⁺ and Co²⁺, especially, activated the enzyme. Therefore, Co²⁺ was a preferable cofactor. All further experiments were conducted with 10 mM CoCl₂.

The optimal pH for DSD_Pa was in the range of 8.1–8.5, similar to QsuB (pH 8.0–8.4) (Figure 4B). Since we intended to use the enzyme in bacterial cells, we studied the enzyme activities at physiological pH 7.5.

The kinetic parameters of DSD_Pa (

Table 2. Kinetic properties of DHS dehydratases.

Enzyme	Km, μM	kcat, s−1	Keff, 10−3/μM/s	Source
DSDPa	236 ± 29	125 ± 7	527 ± 167	This work
QsuB	960 ± 80	61 ± 1	60 ± 12	[25]
Qa-4	600 ± 20	219 ± 1	370 ± 70	[17]
AsbF	36 ± 7	1.1 ± 0.1	29 ± 7

) were derived from the corresponding kinetic curve (Figure 5).

DSD_Pa had higher substrate specificity, intermediate meaning of k_cat and the best K_eff, when compared to other catabolic enzymes, QsuB and Qa-4 (Table 2). According to ANOVA and Tukey’s HSD tests, K_eff of DSD_Pa and Qa-4 were statistically similar, while that of QsuB was closer to the less active AsbF (Table S2). k_cat and K_eff characterize the enzyme activity at high and low substrate concentrations, respectively. The fungal enzymes DSD_Pa and Qa-4, being phylogenetically related, exhibited the most favorable kinetic properties, suggesting they would be particularly effective for 3,4-DHBA production.

The enzyme’s susceptibility to inhibition by the target compound is a crucial parameter for biotechnological applications. Previous studies [17] demonstrated that AsbF showed the highest sensitivity to 3,4-DHBA inhibition compared to QsuB and Qa-4. Our analysis revealed that DSD_Pa undergoes mixed-type inhibition by 3,4-DHBA (Figure 6), like it was determined previously for QsuB (Figure 6). 3,4-DHBA could bind to both the free enzyme (K_i) and the enzyme-substrate complex (K’_i). The inhibition constants were similar in magnitude to those reported for QsuB (

Table 3. Inhibition constants of DSD_Pa and QsuB.

Enzyme	Ki, mM	K’i, mM	Source
DSDPa	0.33 ± 0.04	0.61 ± 0.07	This work
QsuB	~0.38	~0.96	[17]

).

3.3. 3,4-DHBA Production in E. coli Strains

The E. coli MG1655ΔaroE strain naturally accumulated DHS. In our previous work [17], we have already used derivatives of this strain with integrated DHS dehydratase genes to compare 3,4-DHBA production.

The isogenic MG1655∆aroE P_lacUV5-dsd_Pa strain was additionally constructed in this work. All strains were evaluated in test tube fermentation (

Table 4. Accumulation of 3,4-DHBA and DHS in the culture broth of the E. coli MG1655∆aroE-based strains containing different genes for DSD in test tube fermentation.

N	DSD	pVS7-aroG4	1 mM IPTG	OD540	DHS	3,4-DHBA	Res. Glc *
					g/L
1	−	−	−	38 ± 1	1.5 ± 0.1	<0.1	3.0 ± 0.1
2	AsbF			33 ± 1	<0.1	<0.1	4.0 ± 0.1
3	Qa-4			32 ± 1	<0.1	1.1 ± 0.1
4	QsuB			33 ± 1	0.4 ± 0.1	0.7 ± 0.1
5	DSDPa			33 ± 1	<0.1	0.9 ± 0.1
6	−	−	+	33 ± 1	1.3 ± 0.1	<0.1	2.5 ± 0.1
7	AsbF			33 ± 1	<0.1	0.2 ± 0.1	5.5 ± 0.1
8	Qa-4			32 ± 1	<0.1	1.1 ± 0.1
9	QsuB			31 ± 1	<0.1	1.2 ± 0.1
10	DSDPa			32 ± 1	<0.1	1.2 ± 0.1
11	−	+	+	37 ± 1	7.2 ± 0.1	<0.1
12	AsbF			43 ± 1	7.3 ± 0.7	0.5 ± 0.1
13	Qa-4			37 ± 1	<0.1	4.9 ± 0.1
14	QsuB			38 ± 1	<0.1	4.8 ± 0.1
15	DSDPa			38 ± 2	<0.1	5.0 ± 0.5

* Res. Glc—residual glucose determined after cultivation time (CT) = 44 h.

).

All investigated genes were under the control of the P_lacUV5 promoter, which normally requires IPTG induction for transcription. However, strains expressing the more active enzymes, Qa-4, QsuB, and DSD_Pa, accumulated 3,4-DHBA even without IPTG (Table 4, lines 3–5), likely due to promoter leakage. Notably, IPTG addition did not significantly increase 3,4-DHBA accumulation (Table 4, lines 8–10). As previously reported, the MG1655∆aroE P_lacUV5-asbF strain showed limited 3,4-DHBA production, resulting from both low enzyme activity and strong product inhibition by 3,4-DHBA [17]. To increase DHS supply, we introduced plasmid pVS7-aroG4, which carries the mutant variant of E. coli 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase that is resistant to L-Phe feedback inhibition [36]. Strain with the asbF gene and the parent MG1655ΔaroE accumulated significant amounts of DHS (~ 7 g/L) after introduction of pVS7-aroG4. The plasmid strains expressing Qa-4, QsuB, and DSD_Pa accumulated about 5 g/L 3,4-DHBA. These results demonstrate that Qa-4, QsuB, and DSD_Pa show comparable performance in the engineered E. coli platform strain MG1655ΔaroE/pVS7-aroG4 (Table S3).

3.4. 3,4-DHBA Production in C. glutamicum Strains

To assess DHS dehydratase activity in C. glutamicum, we constructed plasmids pVK9-lacI-P_trc-id2-dsd (Figure S2), designed to maintain similar expression levels of dsd genes. These plasmids, along with the empty pVK9 vector, were transformed into the K118 strain. The resulting strains were evaluated in test tube fermentation (

Table 5. Accumulation of 3,4-DHBA and DHS in the culture broth of the K118/pVK9-lacI-P_trc-id2-dsd strains in test tube fermentation.

N	DSD *	pVS7-aroG4	1 mM IPTG	OD540	DHS	3,4-DHBA	CT **, h
					g/L
1	−	−	−	85 ± 1	0.6 ± 0.1	0.7 ± 0.1	24
2	AsbF			84 ± 1	0.6 ± 0.1	0.7 ± 0.1
3	Qa-4			81 ± 1	0.5 ± 0.1	0.6 ± 0.1
4	QsuB			90 ± 1	<0.1	2.7 ± 0.1
5	DSDPa			88 ± 1	<0.1	2.4 ± 0.1
6	−	−	+	85 ± 1	0.5 ± 0.1	0.7 ± 0.1	24
7	AsbF			84 ± 2	0.2 ± 0.1	1.5 ± 0.3
8	Qa-4			83 ± 3	0.2 ± 0.1	1.2 ± 0.1
9	QsuB			62 ± 2	<0.1	2.2 ± 0.2	29
10	DSDPa			72 ± 1	<0.1	2.1 ± 0.1
11	−	+	+	87 ± 1	0.8 ± 0.1	0.7 ± 0.1	24
12	AsbF			83 ± 2	0.3 ± 0.1	1.3 ± 0.1
13	Qa-4			86 ± 1	0.3 ± 0.1	1.4 ± 0.2
14	QsuB			82 ± 6	<0.1	2.7 ± 0.1	27
15	DSDPa			86 ± 1	<0.1	3.4 ± 0.1	24

* The control strain “−” contained pVK9 vector. K118/pVK9 and K118/pVK9/pVS7 strains had the similar performance. ** CT—cultivation time. Fermentation was terminated when glucose (40 g/L) was consumed.

).

The K118 strain harboring the empty pVK9 vector accumulated ~ 0.7 g/L 3,4-DHBA due to the expression of the native chromosomal qsuB gene (Table 5, lines 1, 6, 11). Strains carrying plasmids with qsuB and dsd_Pa genes produced 3,4-DHBA even without IPTG addition, likely due to promoter leakage and the high activity of these enzymes (Table 5, lines 4, 5). This conclusion was supported by the prolonged cultivation time (CT), extended from 24 to 29 hours until complete glucose consumption, and by reduced biomass accumulation (OD) under IPTG addition (Table 5, lines 9, 10). Strains expressing AsbF and Qa-4 demonstrated lower 3,4-DHBA accumulation, exceeding the basal production of the K118 strain only in the presence of IPTG (Table 5, lines 6–8).

Following the approach used for E. coli 3,4-DHBA producers, C. glutamicum strains were transformed with pVS7-aroG4. Both 3,4-DHBA accumulation and biomass increased in strains expressing QsuB and DSD_Pa; however, DHS was still absent (Table 5, lines 14–15). This suggests that the substrate DHS might be limited in these strains. Strains expressing AsbF and Qa-4 did not show an increase in 3,4-DHBA titer and continued to accumulate DHS (Table 5, lines 12–13). Therefore, Qa-4 activity was as low as that of AsbF in C. glutamicum (Table S4). In the K118 strain, 3,4-DHBA production was also supported by native QsuB. To assess whether the native enzyme might influence the activity of heterologous ones, we analyzed 3,4-DHBA production in a ΔqsuB derivative of the K118 strain.

3.5. 3,4-DHBA Production in C. glutamicumΔqsuB Strains

Inactivation of the native qsuB gene reduced 3,4-DHBA accumulation in all plasmid strains containing dsd genes (

Table 6. Accumulation of 3,4-DHBA and DHS in the culture broth of the C. glutamicum K118ΔqsuB/pVK9-lacI-P_trc-id2-dsd strains in test tube fermentation.

N	DSD *	pVS7-aroG4	1 mM IPTG	OD540	DHS	3,4-DHBA	Res. Glc **
					g/L
1	−	−	−	70 ± 7	1.0 ± 0.1	<0.1	0
2	AsbF			75 ± 1	0.9 ± 0.1	0.1 ± 0.1
3	Qa-4			69 ± 1	0.9 ± 0.1	0.1 ± 0.1
4	QsuB			71 ± 1	<0.1	0.6 ± 0.1
5	DSDPa			65 ± 1	0.8 ± 0.1	0.3 ± 0.1
6	−	−	+	68 ± 1	0.9 ± 0.1	0.1 ± 0.1	0
7	AsbF			71 ± 4	0.3 ± 0.1	0.4 ± 0.1
8	Qa-4			70 ± 2	0.4 ± 0.1	0.6 ± 0.1
9	QsuB			38 ± 1	<0.1	0.7 ± 0.1	1.8 ± 0.1
10	DSDPa			40 ± 3	<0.1	0.6 ± 0.1	1.6 ± 0.2
11	−	+	+	67 ± 1	0.9 ± 0.1	0.1 ± 0.1	0
12	AsbF			71 ± 3	0.4 ± 0.1	0.6 ± 0.1
13	Qa-4			72 ± 1	0.5 ± 0.1	0.6 ± 0.1
14	QsuB			33 ± 1	<0.1	0.6 ± 0.1	1.6 ± 0.1
15	DSDPa			43 ± 1	<0.1	0.8 ± 0.1	1.5 ± 0.1

* The control strain “−” contained pVK9 vector. K118ΔqsuB/pVK9 and K118ΔqsuB/pVK9/pVS7 strains had the similar performance. ** CT = 24 h for strains with Res. Glc = 0 and CT = 28 h for strains with Res. Glc.

, lines 2–5; 7–10). The K118ΔqsuB expressing QsuB and DSD_Pa showed decreased biomass accumulation under IPTG induction, and introduction of the pVS7-aroG4 plasmid did not restore biomass levels (Table 6, lines 14–15). These results suggest that DHS availability was reduced in the ΔqsuB strain.

Deletion of the qsuB gene might have affected the translation of the downstream aroD (qsuC) gene, which is located in the same operon (Figure 7). Although the deletion was designed to keep the reading frame, the removed DNA region may have contained additional transcription signals, or the inserted DNA fragment may have attenuated aroD transcription.

The strains expressing Qa-4 and AsbF accumulated higher levels of DHS than the strains expressing QsuB and DSD_Pa (Table 6 and Table S5). Therefore, the activities of Qa-4 and AsbF were lower than those of QsuB and DSD_Pa in the ΔqsuB strain.

3.6. Sequence Comparison of qa-4 and dsd_Pa Genes

The observed difference in Qa-4 and DSD_Pa activities in E. coli and C. glutamicum may stem from variations in gene expression efficiency between these hosts. The presence of codons with a usage frequency less than 10 in C. glutamicum was analyzed in qa-4 and dsd_Pa genes (

Table 7. Comparative analysis of codon usage frequencies for qa-4 and dsd_Pa genes in E. coli and C. glutamicum.

AA	Codon	C. glutamicum Frequency	E. coli Frequency	Number of Codons in qa-4	Number of Codons in dsdPa
Arg (R)	CGA	6.6	4.3	3	-
	CGG	4.9	4.1	13	19
	AGA	2.2	1.4	2	5
	AGG	3.2	1.6	0	0
Cys (C)	UGU	2.3	5.9	5	5
	UGC	4.1	8.0	2	2
Gly (G)	GGG	6.7	8.6	3	2
His (H)	CAU	6.7	15.8	5	4
Ile (I)	AUA	1.8	3.7	3	1
Leu (L)	UUA	5.1	15.2	27	17
	CUA	5.8	5.3	5	2
Pro (P)	CCC	9.7	6.4	3	0
Val (V)	GUA	8.1	11.5	0	1
Ser	AGU	4.9	7.2	0	0
	UCA	8.2	7.8	0	0
	UCG	7.6	8.0	7	1
Tyr	UAU	7.4	16.8	6	4
Thr	ACA	7.6	6.4	1	0
	ACG	8.8	1.5	0	0
∑ codons rare for C. glutamicum relatively E. coli				48	33

). The usage frequencies of some of these codons differed by two times or more in C. glutamicum and E. coli (indicated in bold in Table 7). In a whole, the qa-4 gene sequence contained more codons problematic for C. glutamicum than dsd_Pa. The most noticeable difference was observed for Leu codon UUA (27 in qa-4 versus 17 in dsd_Pa) rare for C. glutamicum. Thus, poor expression of Qa-4 in C. glutamicum may be considered as a major factor underlying its reduced enzymatic activity and consequent low 3,4-DHBA production.

4. Discussion

Relatively few studies have reported comparative screening of DHS dehydratases [11,18]. The natural diversity of these enzymes enables selection of optimal candidates for microbiological synthesis, depending on specific hosts requirements. In previous work, we demonstrated that Qa-4 and QsuB enzymes, related to quinate/shikimate catabolic pathway, exhibited higher specific activity and greater resistance to 3,4-DHBA inhibition compared to AsbF, a siderophore biosynthesis enzyme [17]. In this study, we characterized another catabolic DHS dehydratase from P. anserina. Both DSD_Pa and Qa-4, which originate from closely related fungi, showed superior catalytic efficiency, k_cat and K_eff values, compared to QsuB (Table 2). Surprisingly, no significant differences were observed between the E. coli MG1655ΔaroE-based strains expressing these enzymes, even when DHS supply was enhanced through introduction of the pVS7-aroG4 plasmid (see Figure 8 for final comparison of the enzymes and host strains). All strains expressing Qa-4, DSD_Pa and QsuB reached 3,4-DHBA accumulation up to 5 g/L. Presumably, the differences and in vitro kinetic advantages of a particular enzyme may become apparent in optimized high-performance strains or under fed-batch fermentation conditions.

Significant functional differences emerged between phylogenetically related fungal enzymes when expressed in C. glutamicum. The expression of the qa-4 gene in C. glutamicum appears problematic and may explain the observed 3,4-DHBA production impairment. Both qa-4 and dsd_Pa genes were chemically synthesized with codon optimization for E. coli expression. While E. coli and C. glutamicum genes are typically used interchangeably without optimization, sequence analysis revealed the qa-4 gene contains substantially more codons that are rare in C. glutamicum compared to dsd_Pa. The presence of rare codons in native sequences is known to modulate translational kinetics, facilitating proper protein folding [41]. However, when heterologous rare codons are incompatible with the host’s tRNA pool, this can result in translational stalling, premature termination, or protein misfolding. Notably, in contrast to E. coli, DSD_Pa demonstrated marginally better performance than QsuB in the presence of pVS7-aroG4 plasmid in C. glutamicum platform strain.

Parallel experiments with 3,4-DHBA-producing strains revealed the differences in metabolic networks in E. coli and C. glutamicum. C. glutamicum platform strain had a bottleneck in DHS accumulation, even in the presence of the pVS7-aroG4 plasmid. E. coli MG1655ΔaroE/pVS7-aroG4 accumulated DHS up to 7 g/L (Table 4, line 11), while C. glutamicum K118/ pVS7-aroG4 produced less than 1 g/L DHS (Table 5, line 11). The observed DHS accumulation deficiency in C. glutamicum platform strain may result from metabolic flux diversion through downstream reactions of the common aromatic pathway. The minor shikimate dehydrogenase QsuD is more active than nearly silenced YdiB of E. coli. At the same time, the qsuC gene, which encodes the DHS-synthesizing dehydroquinate dehydratase (see Figure 1), is part of the qsu operon. This operon is known to be induced by shikimate or chorismate in C. glutamicum [27,42], explaining its suboptimal expression in our K118 strain. The DHS deficiency and consequent decrease in 3,4-DHBA production were further exacerbated in the ΔqsuB strain, likely due to the possible polar effect of this modification on qsuC transcription (Figure 8). In contrast, aroD expression is constitutive in the E. coli MG1655 strain [43]. Thus, C. glutamicum platform strain requires metabolic engineering to achieve 3,4-DHBA production levels comparable to E. coli.

Based on our findings, DSD_Pa is suitable for heterologous 3,4-DHBA biosynthetic pathways creation in both E. coli and C. glutamicum.