Improving growth properties of Corynebacterium glutamicum by implementing an iron‐responsive protocatechuate biosynthesis

Abstract Corynebacterium glutamicum experiences a transient iron limitation during growth in minimal medium, which can be compensated by the external supplementation of protocatechuic acid (PCA). Although C. glutamicum is genetically equipped to form PCA from the intermediate 3‐dehydroshikimate catalysed by 3‐dehydroshikimate dehydratase (encoded by qsuB), PCA synthesis is not part of the native iron‐responsive regulon. To obtain a strain with improved iron availability even in the absence of the expensive supplement PCA, we re‐wired the transcriptional regulation of the qsuB gene and modified PCA biosynthesis and degradation. Therefore, we ushered qsuB expression into the iron‐responsive DtxR regulon by replacing the native promoter of the qsuB gene by the promoter PripA and introduced a second copy of the PripA‐qsuB cassette into the genome of C. glutamicum. Reduction of the degradation was achieved by mitigating expression of the pcaG and pcaH genes through a start codon exchange. The final strain C. glutamicum IRON+ showed in the absence of PCA a significantly increased intracellular Fe2+ availability, exhibited improved growth properties on glucose and acetate, retained a wild type‐like biomass yield but did not accumulate PCA in the supernatant. For the cultivation in minimal medium C. glutamicum IRON+ represents a useful platform strain that reveals beneficial growth properties on different carbon sources without affecting the biomass yield and overcomes the need of PCA supplementation.


INTRODUCTION
Corynebacterium glutamicum is a non-pathogenic and facultatively anaerobic soil bacterium, which was originally isolated in 1957 as a natural l-glutamate producer (Kinoshita et al., 1957). Nowadays it is an established workhorse in industrial biotechnology for the largescale production of several amino acids such as l-lysine and l-glutamate in million ton scale per year (Becker et al., 2018;Ikeda & Takeno, 2013;Wendisch, 2020Wendisch, ). et al., 2013. The wild type (WT) of C. glutamicum is not prototrophic and growth in minimal medium essentially relies on supplementation of the vitamin biotin (Peters-Wendisch et al., 2014). Although not of vital importance, the addition of small amounts of iron chelators such as protocatechuic acid (PCA) or catechol haven been shown to significantly improve the growth properties of C. glutamicum in minimal medium, that is reduction of the lag phase and constant exponential growth (Liebl et al., 1989). Consequently, PCA became a standard ingredient of the widely used CgXII minimal medium (Keilhauer et al., 1993;Unthan et al., 2014).
PCA (3,4-dihydroxybenzoic acid) is a constitutional isomer of the more frequently employed siderophore precursor 2,3-dihydroxybenzoic acid (2,3-DHBA). Likewise, it can provide the functional moiety of such iron binding molecules (e.g. petrobactin) (Barbeau et al., 2002). A common response of many bacteria to iron limited conditions is the secretion of high affinity siderophores in order to make poorly soluble ferric iron (Fe 3+ ) available. But also the catecholate compounds 2,3-DHBA and PCA themselves were detected under iron limitation in the culture supernatants of Bacillus subtilis, Paracoccus denitrificans and Bacillus anthracis, respectively (Neilands, 1981;Peters & Warren, 1968;Tait, 1975), where they can increase the iron availability by chelation of Fe 3+ and the chemical reduction to Fe 2+ (Müller et al., 2020).
The iron homeostasis of C. glutamicum was extensively studied with the focus on the transcriptional regulation, iron storage and mobilization, as well as on the utilization of alternative iron sources Brune et al., 2006;Follmann et al., 2009;Frunzke et al., 2011;Keppel et al., 2019;Küberl et al., 2016Küberl et al., , 2020Müller et al., 2020;Wennerhold et al., 2005;Wennerhold & Bott, 2006). The master regulator of iron homeostasis in C. glutamicum is DtxR which in response to the intracellular Fe 2+ concentration controls the transcription of genes encoding proteins for iron acquisition, storage and mobilization as well as proteins responsible for iron-sulphur cluster assembly. Moreover, DtxR represses under iron excess the transcription of the ripA gene coding for the transcriptional regulator of iron proteins A (RipA). Under iron limitation, RipA represses the transcription of genes encoding enzymes such as aconitase or succinate dehydrogenase. So far, it is not clear, how iron is initially transported into the cells when C. glutamicum grows as monoculture, because siderophore biosynthetic genes were not identified in the genome although a large repertoire of siderophore uptake systems is available (Brune et al., 2006;Frunzke & Bott, 2008;Wennerhold & Bott, 2006). Interestingly, C. glutamicum is genetically equipped to form PCA, but unlike in other bacteria, PCA biosynthesis nor its degradation is regulated in response to the iron availability (Brune et al., 2006;Kalinowski et al., 2003;Shen et al., 2012;Wennerhold & Bott, 2006). During growth on glucose PCA is synthesized from 3-dehydroshikimate, an intermediate of the shikimate pathway, catalysed by the 3-dehydroshikimate dehydratase (QsuB) encoded by qsuB ( Figure 1A). C. glutamicum also features the βketoadipate pathway to utilize several aromatic compounds such as PCA as sole carbon and energy source (Kubota et al., 2014;Merkens et al., 2005;Shen & Liu, 2005;Teramoto et al., 2009;Zhao et al., 2010). PCA degradation is initiated by the PCA 3,4-dioxygenase (PcaGH), which consists of two subunits encoded by pcaG and pcaH located in the pcaHGBC operon ( Figure 1) (Zhao et al., 2010). Recently, we investigated biomass formation of the C. glutamicum WT in PCA-deficient CgXII medium and observed a biphasic growth behaviour caused by a transient iron limitation (Müller et al., 2020). This growth retardation could be compensated by either the supplementation of PCA to the CgXII medium or by aeration of the bioreactor with an increased proportion of CO 2 in the inlet air. It turned out that the presence of CO 2 /HCO 3 − accelerates the chemical reduction of poorly soluble ferric iron (Fe 3+ ) to biologically active ferrous iron (Fe 2+ ) through phenolic acids and catechols including PCA (Müller et al., 2020).
To improve the growth properties of C. glutamicum in minimal medium without PCA supplementation, we installed an iron-responsive PCA biosynthesis by exchange of the native qsuB promoter with the ripA promoter (P ripA ), which is repressed by DtxR at Fe 2+ excess, integrated of a second copy of P ripA -qsuB into the genome and increased the PCA pool by mitigating expression of the pcaG and pcaH genes ( Figure 1B).

Bacterial strains, plasmids, primers and cultivation conditions
Escherichia coli DH5α was used as host for cloning applications (Hanahan, 1983). All C. glutamicum strains generated in this study were derivatives of the wild type ATCC 13032. Table 1 provides a list of all strains and plasmids used in this study. The primers are itemized in Table S1. Cryogenic stocks of all strains were prepared in 30% (v/v) glycerol and maintained at −80°C. Cell propagation during cloning was performed in complex medium (2x YT) containing 16 g Bacto-tryptone, 10 g yeast extract and 10 g sodium chloride per L (Sambrook & Russell, 2001). Plates contained additionally 18 g agar L −1 . When required a final concentration of 50 μg kanamycin mL −1 was added to the hand-warm agar prior to pouring or to liquid media immediately before inoculation. Cultivation of C. glutamicum was performed in CgXII minimal medium (Table 2) with a starting of pH 7.4 (glucose) or 6.5 (acetate).

Genetic modifications
DNA fragments for cloning were PCR amplified with the Q5 polymerase (New England Biolabs, Frankfurt, Germany) according to the manufacturers specifications. Agarose gel electrophoresis and standard techniques of molecular biology were performed as described by Sambrook & Russell, 2001. Isolation of chromosomal DNA of C. glutamicum WT, plasmid DNA and the purification of PCR products was carried out with the NucleoSpin Microbial DNA, NucleoSpin Plasmid and NucleoSpin Gel and PCR Clean-up commercial kits (all purchased from MACHEREY-NAGEL, Düren, Germany) in accordance with the manufacturer's instructions. Enzymes were obtained from New England Biolabs (Frankfurt, Germany) and oligonucleotides were supplied by Sigma Aldrich (Steinheim, Germany).
Plasmid pK19mobsacB was used for the chromosomal integrations in C. glutamicum based on selection/counter-selection as described before (Schäfer et al., 1994). Briefly, since pK19mobsacB cannot be replicated in C. glutamicum WT, kanamycin resistance can only be acquired by integration of the entire plasmid through homologous recombination. In order to excise the vector backbone and retain the markerless integration, the counter-selection is performed on sucrose containing agar plates. Only cells that eliminated the vector backbone with the sacB gene successfully can survive in this step. Cells carrying the desired genetic modification are finally verified by colony PCR (ColPCR) and discriminated from (re-established) wild type cells and cells with mutations in the sacB promoter region. Derivatives of the pK19mobsacB carried typically 500 bp regions flanking the target locus (1500 bp for targeting the CgLP4 locus).
In order to place the native qsuB gene (cg0502) of C. glutamicum WT under control of the iron-responsive transcriptional regulator DtxR, the promoter of the ripA gene was PCR amplified from the chromosomal DNA of C. glutamicum WT using the primer pair ripA1/ ripA2. Since the intergenic region between cg0501 and cg0502 is only 23 bp long we integrated the P ripA promoter upstream of the qsuB gene without removing the native genetic sequence in order to leave the coding regions intact. The 500 bp regions were PCR amplified from the chromosomal DNA with the primer pairs F1/F2 and qsuB1/qsuB2. All primers contained 20 bp overlaps with the anticipated neighbouring fragment required for homologous recombination. HindIII and BamHI linearized pK19mobsacB and all fragments were simultaneously joined by the isothermal Gibson assembly (Gibson, 2011;Gibson et al., 2009). All other plasmids were constructed accordingly. The 500 bp flanks for integration in the CgLP4 locus were F I G U R E 1 The central carbon metabolism of C. glutamicum including PCA biosynthesis via the shikimate pathway and its degradation via the β-ketoadipate pathway (A). Metabolic engineering strategies to place the PCA synthesis under control of the master regulator of the iron homeostasis, DtxR (B): (i) replacement of the native qsuB expression control with the ripA promoter (P ripA ), which is repressed by DtxR at Fe 2+ excess, (ii) integration of an additional P ripA -qsuB copy in the CgLP4 locus (Lange et al., 2017), (iii) increase of the PCA pool by mitigating expression of the pcaG and pcaH genes through the start codon exchange ATG → GTG. Abbreviations: 3-DHS, 3-dehydroshikimate; 6PG, 6-phosphogluconate; AC-CoA, acetyl-CoA; CHO, chorismate; CMA, β-carboxy-cis, cis-muconate; DAHP, 3-desoxyarabinoheptulosanat-7-phosphate; E4P, erythrose-4-phosphate; GLC, glucose; GLC-6P, glucose-6-phosphate; PCA, protocatechuic acid; PcaGH, PCA 3,4-dioxygenase; PEP, phosphoenolpyruvate; PYR, pyruvate; QsuB, 3-dehydroshikimate dehydratase; SUCC-CoA, succinyl-CoA.
amplified from the chromosomal DNA of C. glutamicum WT using the primer pairs CgLP4_1/CgLP4_2 and CgLP4_3/CgLP4_4, respectively. The integration cassette consisting of P ripA , qsuB and T rrnB was amplified with the primer pairs ripA3/ripA2 and qsuB1/qsuB3 from the chromosomal DNA, and with TrrnB1/TrrnB2 using pFEM06 as templates. The native ATG start codons of pcaG and pcaH were simultaneously replaced by the weaker GTG through homologous recombination. Three fragments were amplified from the chromosomal DNA of C. glutamicum WT using the primer pairs F3/F4, pcaG1/pcaG2 and pcaH1/pcaH2, respectively, that encoded the desired GTG start codon (Table S1). Competent cells of E. coli DH5α were transformed with the Gibson assembly mixes by electroporation (Dower et al., 1988). Positive transformants were screened by colony PCR and plasmids were finally verified by sequencing (Microsynth Seqlab, Göttingen, Germany). Competent cells of C. glutamicum were prepared according to Kirchner and Tauch (2003) and transformed with 500 ng of the purified plasmid by electroporation (van der Rest et al., 1999). Correct clones were identified by ColPCR or sequencing of the corresponding region to verify the start codon exchange.

Shaking flask cultivations
Main growth experiments were all started from the same seed train precisely as described before (Müller T A B L E 1 Bacterial strains and plasmids.

Final concentration
(NH 4 ) 2 SO 4 5 g L −1 Urea Added from sterile stocks prior to inoculation et al., 2020). A cryogenic culture was streaked on 2x YT agar plates and incubated for 2 days at 30°C. A reaction tube containing 5 mL liquid 2x YT medium was inoculated with a single colony, grown over night (O/N) at 30°C shaking (180 rpm, shaking diameter 25 mm) and used completely to inoculate a 500 mL baffled shaking flask containing 50 mL 2x YT on the next day. After 8 h incubation shaking at 30°C, cells were harvested by centrifugation (10 min, 4000 × g) and the cell pellet was resuspended in 1 mL 9 g NaCl L −1 . The OD 600 was adjusted to 25 in order to reach a starting OD 600 of 1 in the CgXII preculture by the addition of 2 mL inoculum.

Bioreactor cultivations
Bioreactor cultivations were operated in batch mode in four identical 2 L glass vessels (DASGIP®, Jülich, Germany) filled with 800 mL CgXII medium lacking MOPS buffer and urea. The pH of the cultivation medium was measured with a standard pH probe (405-DPAS-SC-K8S/325, Mettler Toledo, Giessen, Germany) and maintained at 7.4 by the addition of 25% (v/v) ammonium hydroxide. Four different experiments were performed in parallel in order to compare the C. glutamicum WT and IRON + strains aerated at 0.5 vvm with either CO 2 -enriched or ambient air. Since the glass reactor could not be operated at overpressure, 30% CO 2 was added to the inlet air in order to establish the same partial pressure of CO 2 as before Müller et al., 2020). The stirrer speed was automatically controlled to realize dissolved oxygen concentrations >35%, which was monitored with a polarographic probe (Mettler Toledo, Giessen, Germany). The bioreactors were inoculated as described above and cultivated with 20 g glucose L −1 for 14 h at 30°C.

BioLector cultivations
Microscale cultivations were performed in the BioLector device (m2p labs, Baesweiler, Germany) in 48-well flower plates. Each well was filled with a total of 1 mL CgXII medium that was supplemented as indicated above. The inoculum was pre-diluted in order to keep the total volume identical. The cultivation plate was sealed with an air-permeable membrane and incubated at 30°C at a shaking frequency of 1000 rpm. In order to monitor the cell density, the backscatter value at 620 nm was automatically measured every 10 min.

Monitoring growth and determination of the biomass concentration
The cell density was monitored during shaking flask and bioreactor cultivations by measuring the optical density at 600 nm (OD 600 ). Biomass concentrations (as cell dry weight (CDW) per L) were calculated by the correlation factor, which was specific for the spectrophotometer (Ultrospec 10 cell density meter, Harvard Biochrom, Holliston, MA, USA): c CDW = OD 600 × 0.26 g L −1 . For an accurate determination of the biomass at the end of the bioreactor cultivations 50 mL culture broth was harvested (10 min, 4000 × g, room temperature (RT)), washed three times with 20 mL 0.9 g NaCl L −1 , resuspended in 10 mL fully demineralized H 2 O and transferred to a pre-weighed 50 mL glass beaker. The beakers were dried for 48 h in a static incubator at 105°C until all humidity was evaporated. Finally, the biomass was weighed out on an analytical balance.

Quantification of sugars, alcohols and organic acids.
The consumption of glucose during bioreactor cultivations and the potential accumulation of acetate, lactate, succinate, formate and ethanol was monitored by HPLC-RID analysis as described previously (Siebert et al., 2021). Metabolites were denoted as 'not detectable', when peak areas were smaller than the quantification limit of 1 mM. The presence of PCA in culture supernatants was monitored by HPLC-DAD (Siebert et al., 2021) with a quantification limit of 1 μM.

Fluorescence experiments
In order to monitor differences in the intracellular Fe 2+ concentration, fluorescence experiments were performed with C. glutamicum FEM3-derived reporter strains (Müller et al., 2020) after 25 h of cultivation. Suspensions were diluted to an OD 600 of approximately 11 with NaCl solution (9 g L −1 ). 100 μL of the dilution was transferred into one cavity of a 96-well microtiter plate and the fluorescence was measured using a plate reader (Tecan Spark Multimode Microplate reader, Tecan, Zürich, Switzerland) as technical duplicates at the following conditions: excitation wavelength: 485 ± 20 nm, emission wavelength: 535 ± 20 nm, gain: 87. Background fluorescence of culture supernatants was subtracted from the fluorescence of the cell suspension due to the autofluorescence of the CgXII medium and the data was normalized to the biomass concentration as before (Müller et al., 2020).

Calculation of μ, Y X/S and time shift of exponential growth phases
Growth rates (μ) were determined by linear regression and least square fitting of the logarithmized cell density during the exponential growth phase. Biomass yields per substrate (Y X/S ) were calculated from the experimental data of final biomass concentration and initial glucose concentration of bioreactor cultivations once all glucose was exhausted. Different growth properties of the engineered strains were quantified by calculating the time shift of the exponential growth phases (∆t exp ). In BioLector experiments the cultivation time for reaching a backscatter value in the mid exponential growth phase (800) was extracted from the data. The ∆t exp was calculated for each cultivation by subtracting the cultivation time of the WT + PCA. In shaking flask experiments, the cultivation time until reaching a biomass concentration of 6 g CDW L −1 was calculated from the exponential growth curve. The differences between the strains and cultivation conditions were calculated as before. Standard deviations of all ∆t exp values were calculated by Gaussian error propagation.

Statistics
All experiments were performed at least as three independent biological replicates with individual seed trains starting from different colonies on the agar plates. If not stated otherwise, data represent mean values with error bars representing the standard deviation of n ≥ 3. When necessary, standard deviations were calculated by Gaussian error propagation. Significant differences of the normalized fluorescence data were analysed by a two sample Student's t-test assuming equal variances and the significance levels indicated as *, p < 0.05, **, p < 0.01, ***, p < 0.001.

R ESULTS Engineering C. glutamicum to improve the intracellular iron availability
In PCA-deficient CgXII medium with glucose we previously observed a transient iron limitation of C. glutamicum WT, which results in a growth retardation compared to medium with externally supplied PCA (Müller et al., 2020). C. glutamicum ATCC 13032 is genetically equipped to synthesize PCA on its own, but unlike in other bacteria, the endogenous pathway is not regulated in response to the iron availability (Brune et al., 2006;Teramoto et al., 2009;Wennerhold & Bott, 2006). We hypothesized that the intracellular Fe 2+ availability and thus the growth properties in non-PCA-supplemented cultures could be increased, if the qsuB gene, which is responsible for PCA formation was part of the DtxR regulon, the master regulator of iron homeostasis. Consequently, we placed the expression of the native qsuB gene under control of the ripA promoter, which is in turn only regulated by DtxR. The resulting strain should reveal a higher qsuB expression and thus PCA synthesis than C. glutamicum WT when the intracellular Fe 2+ availability is low. High Fe 2+ levels would induce a DtxR mediated repression of the qsuB expression to avoid prodigal carbon flux.
In CgXII medium supplemented with 2% (w/v) glucose without PCA, C. glutamicum WT showed biphasic growth with a growth rate of 0.38 ± 0.02 h −1 in the second of the two growth phases, whereas the supplementation of PCA resulted in constant exponential growth throughout with a rate of 0.41 ± 0.02 h −1 (Figure 2A). Compared to PCA-supplemented medium the onset of exponential growth (∆t exp ) was delayed by about 1.86 ± 1.40 h when PCA was absent ( Figure 2B). In contrast, C. glutamicum harbouring the synthetic P ripA -qsuB element recovered quicker from the transient iron limitation as the WT and showed an improved μ of 0.41 ± 0.02 h −1 in the second growth phase (Figure 2A). However, WT-like growth with PCA could not be fully restored and C. glutamicum P ripA -qsuB still exhibited a ∆t exp of 0.67 ± 0.68 h ( Figure 2B). Therefore, we integrated a second copy of the P ripA -qsuB cassette into the CgLP4 locus (Lange et al., 2017) of C. glutamicum. In the absence of PCA, the resulting strain C. glutamicum 2x P ripA -qsuB showed the same μ and a slightly reduced ∆t exp compared to the parental strain with one copy of the P ripA -qsuB cassette (Figure 2A,B). We reasoned that the PCA pool size is not solely determined by the expression strength of qsuB, but also by PCA degradation (Figure 1). To slow down the first step of the β-ketoadipate pathway, we additionally replaced the ATG start codon of the pcaG and pcaH genes, which encode two subunits of the protocatechuate 3,4-dioxygenase with the weaker GTG start codon in C. glutamicum 2x P ripA -qsuB. The resulting strain C. glutamicum IRON+ could grow on PCA as sole carbon and energy source. However, its growth rate was reduced by 37% (μ = 0.26 ± 0.01 h −1 compared to μ = 0.41 ± 0.06 h −1 of the WT strain; data not shown). During the cultivation with 2% (w/v) glucose, this strain still grew at μ = 0.41 ± 0.02 h −1 during the exponential phase without PCA and showed an almost identical growth pattern compared to C. glutamicum WT cultivated in the presence of PCA with an average ∆t exp of only 0.25 ± 0.83 h (Figure 2A,B). In the applied shaking flask system all engineered strains reached the same biomass concentration at the end of the cultivation and neither showed an improved growth property compared to the WT in the presence of PCA nor secreted PCA into the culture supernatant in concentrations higher than 1 μM (quantification limit; data not shown).
In order to investigate the impact of the introduced genetic modifications on the intracellular Fe 2+ availability, we re-engineered the genetic modifications of C. glutamicum IRON+ in the reporter strain background of C. glutamicum FEM3. In this strain high Fe 2+ concentrations induce the binding of DtxR to P ripA , thus provoking repression of lacI, which eventually results in egfp expression under the control of the strong tac promoter (P tac ) (Müller et al., 2020). The biomass-specific fluorescence of C. glutamicum FEM3 IRON+ after 25 h of cultivation in CgXII medium without PCA was more than twice as high as that of the basic reporter strain C. glutamicum FEM3 (Figure 3), indicating significantly increased intracellular Fe 2+ levels (p-value = 1.17 × 10 −4 ) as a result of the introduced genetic modifications. However, the metabolic engineering approach to increase the carbon flux towards PCA could not completely replace the external addition of PCA, which provoked highest biomass-specific fluorescence in both strains (Figure 3).

Bioreactor cultivations with C. glutamicum IRON+
Next, we analysed the growth performance of C. glutamicum IRON+ in bioreactor cultivations in CgXII medium with 2% (w/v) glucose in the absence of PCA. As observed before (Müller et al., 2020), when aerated with pressurized air with 0.04% CO 2 the C. glutamicum WT showed biphasic growth with a μ of 0.34 ± 0.02 h −1 in phase two and exhibited improved and constant growth with a μ of 0.41 ± 0.02 h −1 when the inlet air was enriched with 30% CO 2 (Figure 4). When the newly constructed strain C. glutamicum IRON+ was cultivated, we could not observe such a growth retardation under both conditions. C. glutamicum IRON+ grew under standard conditions (pressurized air) and with CO 2 enriched air exponentially throughout the cultivation with a μ of 0. 41 ± 0.01 h −1 , which is identical to the cultivation of the WT with 30% CO 2 (Figure 4).

Growth properties of C. glutamicum IRON+ at small inoculum sizes
To achieve reliable and reproducible growth with C. glutamicum in PCA-free CgXII medium, a relatively high F I G U R E 2 Shaking flask cultivation of C. glutamicum WT and engineered derivatives in CgXII minimal medium containing 2% (w/v) glucose and PCA as indicated. Growth curve over time (A) and time shifts of the exponential growth curves with regard to the C. glutamicum WT cultivation with PCA as reference (B). All data points represent mean values with error bars indicating the standard deviation of n ≥ 3. Standard deviation in (B) was calculated by error propagation.

F I G U R E 3
Biomass-specific fluorescence after 25 h cultivation in shaking flasks containing CgXII minimal medium, 2% (w/v) glucose and PCA as indicated. Genetic modifications of C. glutamicum IRON+ were re-engineered in the reporter strain background C. glutamicum FEM3 (Müller et al., 2020). Bars represent mean values with error bars indicating the standard deviation of n = 5. Significance levels indicated as *, p < 0.05, **, p < 0.01, ***, p < 0.001. *** *** starting biomass concentration of at least 0.3 g CDW L −1 is commonly applied. Unthan et al. (2014) showed that PCA supplementation also ensured constant growth with smaller inoculum size. Consequently, we analysed the effect of a decreasing initial cell density on the course of the cultivation. Microscale cultivations were performed in the BioLector system that allows to continuously monitor the cell density via the backscatter signal. In this system we compared the effect of different starting ODs (OD start = 1, which corresponds to 0.3 g CDW L −1 , OD start = 0.2 and 0.02) on growth of C. glutamicum WT and C. glutamicum IRON+. When PCA was supplemented in cultivations with a OD start = 1, C. glutamicum WT grew at μ = 0.52 ± 0.01 h −1 , and the biomass concentration peaked between 8 and 9 h ( Figure S1). We compared the shift of exponential growth phases at lower OD start as before. Since backscatter values are not accurately proportional at low OD 600 we calculated the theoretical shift of exponential growth phases if cultivations did not experience a lag phase. Assuming a constant μ = 0.52 h −1 independent of the inoculum density, exponential growth phases should be shifted theoretically by 3.1 h when the cultivation is started from OD start = 0.2 instead of 1. A further reduction of the OD start by the factor of 10 would result in a shift by 7.5 h compared to OD start = 1. The actual time shifts of 3.4 ± 0.2 and 7.6 ± 0.2 h for cultivations of C. glutamicum WT in the presence of PCA with an OD start = 0.2 and 0.02 are thus in good agreement with the expectations (Figure 5A-C), indicating that growth is not limited by the lower inoculum sizes as long as PCA is supplemented. As before, the exponential growth phase of C. glutamicum WT in a non-PCA-supplemented culture was significantly retarded in comparison with PCA-supplemented cultivations ( Figure S2). ∆t exp increased from 2.8 ± 0.4 h at an OD start = 1 to 8.1 ± 0.9 and 37.1 ± 15.7 h at an OD start of 0.2 and 0.02, respectively ( Figure 5A-C). In contrast, C. glutamicum IRON+ showed an improved performance at low OD start . Compared to the reference condition (C. glutamicum WT in PCA-supplemented medium), the ∆t exp was shifted by only 1.2 ± 0.2 h at OD start = 0.2 and 5.2 ± 0.5 h at OD start = 0.02, which is a reduction of the growth retardation by 85% and 86%, respectively. At starting OD 600 = 1 a delay of C. glutamicum IRON+ compared to the PCA-supplemented WT cultivation was completely vanished.

Growth properties of C. glutamicum IRON+ on acetate
Then, we characterized the growth of C. glutamicum IRON+ on acetate as an example of a gluconeogenic substrate. Similar to the cultivations on glucose, growth of C. glutamicum WT on acetate ceased about 2 h later, when PCA was not supplemented in the cultivation medium ( Figure 6). Interestingly, the exponential growth rate (0.26 ± 0.01 h −1 ) was reduced by about one third compared to the PCA-supplemented condition (μ = 0.39 ± 0.01 h −1 ). Such prominent differences of the exponential growth rate were not noted when using glucose as the carbon source. The engineered strain IRON+ did not reveal a growth retardation in any cultivation condition and grew at a similar rate as the PCA-supplemented WT (μ = 0.37 ± 0.00 h −1 (without PCA) and 0.38 ± 0.00 h −1 ). It was interesting to note, that IRON+ initiated growth on acetate quicker than in any other conditions when PCA was supplemented.

DISCUSSION
Although genetically equipped for the biosynthesis of PCA, C. glutamicum does not regulate the expression of the responsible genes in response to the intracellular iron availability (Brune et al., 2006;Wennerhold & Bott, 2006). We have recently shown, that C. glutamicum WT experiences a transient iron limitation in CgXII medium lacking PCA, which results in a growth delay during the initial phase of the cultivation (Müller et al., 2020). The external supplementation of PCA in CgXII minimal medium is a commonly employed strategy (Keilhauer et al., 1993), but might be undesired because of being expensive and providing an additional carbon source (Graf et al., 2019;Shen et al., 2012). In this study we artificially ushered the control of qsuB expression into the iron-responsive DtxR regulon. This approach was inspired by nature, reflecting that other organisms regulate their biosynthesis of PCA or the structural analogue 2,3-DHBA in response to the intracellular iron availability in turn (Garner et al., 2004;Neilands, 1981;Peters & Warren, 1968;Tait, 1975). We replaced the native promoter of the qsuB gene by P ripA and introduced a second copy of the P ripA -qsuB F I G U R E 4 Bioreactor fermentation of C. glutamicum WT and C. glutamicum IRON+ with ambient air and 30% (v/v) CO 2 in the inlet air, respectively. Growth curves represent mean values with error bars indicating the standard deviation of n = 3. C. glutamicum WT std CO 2 C. glutamicum WT high CO 2 C. glutamicum IRON+ std CO 2 C. glutamicum IRON+ high CO 2 cassette into the genome of C. glutamicum, which indeed improved the growth properties but not to the level of PCA-supplemented cultures. It was necessary to additionally reduce the degradation of PCA in order to enhance the growth phenotype further. This step is plausible since C. glutamicum possesses a functional β-ketoadipate pathway and can efficiently utilize several aromatic compounds such as PCA (Shen et al., 2012). Accordingly, Okai et al. (2017) and Kallscheuer and Marienhagen (2018) engineered C. glutamicum for the production of hydroxybenzoic acids and showed the necessity of an inactive degradation of PCA for efficient overproduction of this aromatic compound. With our previously designed iron reporter strain C. glutamicum FEM3 (Müller et al., 2020) we show, that the improved growth properties in minimal medium caused by the introduced genetic modifications correlates with an increased intracellular Fe 2+ availability. Interestingly, the beneficial growth properties of C. glutamicum IRON+ could also be exploited when acetate was used as sole carbon and energy source. Recently, Graf et al. (2019) evolved a fast-growing variant of C. glutamicum WT (designated as EVO5), which proliferates independently of PCA. Genome resequencing of C. glutamicum EVO5 identified overall 10 mutations with three mutations located in the genes F I G U R E 5 Microscale cultivations (BioLector) of C. glutamicum WT and C. glutamicum IRON+ in CgXII minimal medium containing 2% (w/v) glucose and PCA as indicated. Bars represent the time shifts (∆t exp ) of the exponential growth curves with regard to the C. glutamicum WT cultivation with PCA as reference, starting the cultivation at an initial optical density of OD = 1 (A), OD = 0.2 (B) and OD = 0.02 (C). All data points represent mean values with error bars indicating the standard deviation of n ≥ 5. The standard deviation was calculated by error propagation. coding for the transcriptional regulators DtxR, RipA and RamA. Re-engineering of the ramA mutation in C. glutamicum WT improved the growth rate in PCA-deficient minimal medium on glucose, however, led to significantly impaired growth on acetate (Graf et al., 2019). In contrast, C. glutamicum IRON+ showed also in CgXII medium with acetate improved growth properties. We could not simply compensate the transient iron limitation during the initial growth phase on acetate with C. glutamicum IRON+ but the growth rate was 42% higher than that of the WT during the entire cultivation. A reduction of the growth rate has been reported previously (Wendisch et al., 2000) and might be due to an uncoupled membrane potential during growth on weak acids (Axe & Bailey, 1995;Baronofsky et al., 1984;Kiefer et al., 2020). In this context it is not clear, whether PCA can alleviate the growth limiting stress conditions through its inherent redox chemistry (i.e. acting as an electron shuttle, Perron & Brumaghim, 2009) or whether the enzymatic stress-response benefits from the increased Fe 2+ availability of the strain. Given the fact, that acetate gains increasing importance as an alternative carbon and energy source for microbial production processes (Kiefer et al., 2020;Merkel et al., 2022;Schmollack et al., 2023), future research needs to address this effect as well as the performance of C. glutamicum IRON+ when exposed to different environmental stresses.

C. glutamicum
Although the qsuB gene is monocistronically transcribed in ATCC 13032 (Pfeifer-Sancar et al., 2013) it is located in a gene cluster with qsuC and qsuD encoding dehydroquinate dehydratase and quinate/shikimate dehydrogenase, respectively. Consequently, the replacement of the native promoter by P ripA might have increased qsuCD expression, too, which could additionally increase carbon flux towards the QsuB substrate 3-dehydroshikimate (Kubota et al., 2014;Teramoto et al., 2009). Notably, not only PCA has a growth promoting effect. Also (di-)phenolic compounds, such as catechol (Liebl et al., 1989), ferulic acid and vanillin facilitate growth of C. glutamicum (Siebert et al., 2021). We showed that functionalized aromatic compounds, with a mix of amino and hydroxyl groups or two adjacent hydroxyl groups chelate iron and/or reduce Fe 3+ , which improves the overall intracellular iron availability (Müller et al., 2020). And even indole was found to reduce extracellular Fe 3+ (Walter et al., 2020). Therefore, when feedstocks such as lignocellulosic hydrolysates, which contain diphenolic compounds, are supplemented to the minimal medium or strains, which overproduce such molecules are utilized (Kallscheuer & Marienhagen, 2018;Kim et al., 2022;Okai et al., 2016Okai et al., , 2017, the application of C. glutamicum IRON+ might not be required. The fact that the soil bacterium C. glutamicum naturally encounters this substrate mixture might also explain the greater iron availability in its natural habitat, and why qsuB expression is not evolutionarily placed under control of the iron homeostasis regulators. However, the iron acquisition mode of C. glutamicum is not known, yet. An interesting future research objective is to differentiate whether the entire Fe 3+ -chelates are internalized by C. glutamicum prior to the release of Fe 2+ (i.e. via one of the siderophorespecific transport systems) or whether the chemical reduction takes place spontaneously in the extracellular environment and iron is then taken up via Fe 2+ -specific transport proteins that are also annotated in the genome (Frunzke & Bott, 2008).
The final strain C. glutamicum IRON+ performs well at different cultivation scales, does not accumulate quantifiable levels of PCA in the culture supernatant and maintains an equal Y X/S as the WT. By that, C. glutamicum IRON+ features an interesting genetic basis that could be further engineered for production purposes and represents a neat host strain for laborious screening approaches, as well as large-scale fermentations. Moreover, the introduced genetic modifications might be combined with the engineered biotin prototrophic C. glutamicum strains Peters-Wendisch et al., 2014) to obtain a prototrophic platform strain, which provides reliable growth even at low initial biomass concentrations.

CONCLUSION
This study demonstrates the successful reorganization of transcriptional control by applying a naturally inspired approach. C. glutamicum might not encounter iron restricted conditions in its natural habitat, but does so in monoseptical cultivations. Consequently, by controlling the expression of the endogenous PCA synthesis gene qsuB in response to the iron availability and by mitigating F I G U R E 6 Shaking flask cultivation of C. glutamicum WT and C. glutamicum IRON+ in CgXII minimal medium containing 1% (w/v) acetate and PCA as indicated. Data points of the growth curve over time represent mean values with error bars indicating the standard deviation of n = 3. C. glutamicum WT with PCA C. glutamicum WT without PCA C. glutamicum IRON+ with PCA C. glutamicum IRON+ without PCA its degradation, we ended up with a strain that exhibits a significantly higher intracellular Fe 2+ concentration, superior growth properties on different substrates and retains an identically high biomass yield as the WT. Hence, C. glutamicum IRON+ represents an interesting platform for further engineering approaches in an academic as well as industrial environment, because it overcomes the need of undesirable PCA supplementation, which is expensive on the one hand and provides another carbon source on the other.

A C K N O W L E D G E M E N T S
This study was funded by the German Federal Ministry of Education and Research (BMBF) as part of the project association ForceYield (grant 031B0825D). Open Access funding enabled and organized by Projekt DEAL.

F U N D I N G I N F O R M AT I O N
No funding information provided.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that there are no competing interests associated with this work.