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Article

Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation

by
Amanda G. Godar
1,
Francesca Cristobal
2,
Luis Taquillo
2,
Xuan Wang
1 and
David R. Nielsen
3,*
1
School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
2
School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA
3
Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ 85287, USA
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(2), 101; https://doi.org/10.3390/fermentation12020101
Submission received: 23 December 2025 / Revised: 4 February 2026 / Accepted: 6 February 2026 / Published: 11 February 2026
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

While the biobased, fermentative production of succinate by Escherichia coli represents a sustainable alternative to its conventional synthesis from petroleum, this process requires substantial amounts of inorganic carbon (Ci) to support CO2-fixing reactions in the reductive branch of the tricarboxylic acid (rTCA) cycle. Accordingly, intracellular Ci availability represents a potential limiting factor during E. coli succinate fermentations. Here, we first investigate the role and importance of E. coli’s native CO2 concentrating mechanism (CCM)—comprising two carbonic anhydrases (CAs), Can and CynT—by comparing and contrasting the behaviors of wild-type E. coli and the engineered succinate-producing strain, KJ122. Deletion of can and cynT significantly impaired the aerobic growth of both strains under low CO2 atmosphere and/or low pH, outcomes that were further exacerbated under anaerobic conditions for KJ122. During bioreactor fermentations, KJ122 Δcan ΔcynT further exhibited a prolonged lag phase (~48 h) and 44% reduced succinate production relative to KJ122 by 96 h. Next, the relative functions and performance of mechanistically diverse, heterologous CCM components were investigated by characterizing their ability to restore growth and/or succinate production. While the cyanobacterial bicarbonate transporter SbtA and the Ci transporter DabAB from Halothiobacillus neapolitanus each complemented growth at 0.05% CO2 and pH 6.5–7.5, neither fully restored succinate production by KJ122 Δcan ΔcynT. Moreover, individual overexpression of sbtA, dabAB, or can in KJ122 rendered no additional improvements to succinate production. Collectively, while these results point to the critical importance of CA for supporting efficient fermentative succinate production by E. coli, they also suggest that this native CCM alone is sufficient for ensuring Ci acquisition at requisite levels under the conditions examined.

1. Introduction

As rising atmospheric CO2 levels contribute to increasingly frequent and intense weather events while accessible petroleum sources continue to decline [1,2], microbial conversion of biomass-derived sugars and/or CO2 into biochemicals remains a promising strategy for simultaneously addressing both challenges. Organic acids, many of which can be microbially produced at high titers and yields, have a broad range of applications in the food, pharmaceutical and chemical industries [3]. In particular, succinate is a four-carbon dicarboxylic acid that serves as a precursor to diverse chemicals, including 1,4-butanediol, γ-butyrolactone and tetrahydrofuran [4]. Succinate further represents a useful monomer for producing various biodegradable plastics [4], including polybutylene succinate (PBS) and PBS/polylactic acid blends [5]. The global market of succinate was ~211 million USD in 2023 and is predicted to increase to 322 million USD by 2032, representing a compound annual growth rate of 6.2% [6].
Succinate production presently occurs via microbial fermentation or, more prominently, chemical synthesis. The latter, however, involves the use of non-renewable, petroleum-derived feedstocks (e.g., paraffin, maleic anhydride, fumaric acid), energy intensive reaction conditions (e.g., 120–180 °C and up to 4.0 MPa H2) and heavy metal catalysts (e.g., Ni or Pd), all of which contribute to high environmental impacts [7]. Alternatively, various microorganisms natively or have been engineered to efficiently convert biomass-derived sugars to succinate with high selectivity and under mild process conditions [8,9]. As a result, relative to its chemical synthesis, biobased succinate production has been estimated to reduce CO2 emissions by as much as 2- to 12-fold (depending on the feedstock and purification processes used) [10,11].
One important consideration during fermentative succinate production is the need to exogenously supply significant amounts of inorganic carbon (Ci) [12,13] which serves as an essential co-substrate of the reductive branch of the tricarboxylic acid (rTCA) cycle, being fixed by phosphoenolpyruvate (PEP) carboxykinase (Ppc) and/or PEP carboxylase (Pck) to produce oxaloacetate (OAA) (Figure 1). This need stands in contrast to the fermentative growth of wild-type Escherichia coli which, despite also having a small requirement for Ci (e.g., for fatty acid biosynthesis by biotin carboxylase (accC) and for other minor reactions [14]), is typically capable of meeting such demands solely via endogenous CO2 production (e.g., by the formate hydrogenlyase complex under fermentation conditions and during fatty acid biosynthesis) and/or through diffusion of atmospherically available CO2. However, engineered succinate producing strains typically carry deletions blocking the formation of other fermentative side products (i.e., lactate, acetate, formate and ethanol), which limit endogenous CO2 evolution while making succinate production the primary means of cofactor regeneration; outcomes that render extracellular Ci acquisition as an essential requirement for growth and succinate production. Accordingly, even more so than wild-type E. coli, engineered succinate producers have the potential to serve as excellent backgrounds for investigating the behaviors of different Ci acquisition systems.
Multiple physicochemical and biological barriers exist with respect to ensuring that Ci is sufficiently abundant, first, in the extracellular medium and, second, intracellularly. For instance, in aqueous solutions, the relative abundance of dissolved Ci species (i.e., CO2 (aq), HCO3− and CO32−) depends on pH (Supplemental Figure S1A) whereas the absolute concentration of each (and, therefore, total Ci) further depends on the partial pressure of CO2 (g) (Supplemental Figure S1B). At pH 7.0, the optimum for E. coli succinate fermentations, the majority of extracellular Ci is present as HCO3− (82%) with the remainder being CO2 (aq) (18%). In contrast, at pH 5.5, for example, the opposite is true, with the majority then being CO2 (aq) (88%) and the rest HCO3− (12%). Inside the cell, E. coli’s intracellular pH is maintained constant at about 7.4, meaning that CO2 (aq) always comprises just ~10% of the total intracellular Ci [15]. However, while CO2 (aq) is capable of freely diffusing across the inner membrane, it just as easily diffuses back out. Meanwhile, as an anion, HCO3− is unable to freely diffuse across the inner membrane, either into or out of the cell [16] (note: E. coli has no known HCO3− transporter(s)). Thus, to ensure retention and intracellular accumulation, CO2 (aq) that has diffused into the cell must subsequently be hydrated. However, while CO2 (aq) spontaneously reacts with water to form H2CO3 (which then rapidly dissociates to HCO3−), the initial reaction is slow and, without a catalyst, intracellular Ci levels can become limiting [17,18]. E. coli solves this problem with the aid of two cytoplasmic β-carbonic anhydrases (CAs), Can and CynT, that reversibly catalyze the intracellular interconversion of CO2 and HCO3− (Figure 1). Both genes exhibit conditional expression, with can expression being inversely proportional to growth rate [14]. cynT, meanwhile, is not active under standard fermentation conditions, but rather is expressed primarily in the presence of cyanide to prevent HCO3− depletion [19,20]. While deletion of cynT has no impact on E. coli’s doubling time during aerobic growth [20], deletion of can prevents growth under ambient CO2 conditions (i.e., air with ~0.05% CO2) [14]. These two CAs exclusively comprise E. coli’s native CO2 concentrating mechanism (CCM) and, to our knowledge, their role in Ci acquisition in support of fermentative succinate production has not yet been comprehensively investigated.
Unlike E. coli, other microorganisms have developed alternative and complementary CCMs with which to facilitate efficient Ci acquisition. For instance, photoautotrophic cyanobacteria employ a multi-pronged CCM which importantly includes the use of membrane-bound HCO3− transporters. Among these, SbtA is a high affinity, low flux Na+/HCO3− symporter (Figure 1) [21,22]. The chemolithoautotrophic bacterium Halothiobacillus neapolitanus, meanwhile, utilizes the membrane-associated DabAB Ci transporter to facilitate proton symport across the membrane while also converting CO2 to HCO3− (Figure 1) [23,24]. This activity has been proposed to be particularly advantageous in species where CO2 diffusion is limited, such as by mucus or biofilm [25]. Finally, whereas Can and CynT are cytoplasmic CAs, other evolutionarily distinct families of CAs are localized elsewhere in bacterial cells [26,27]. For instance, α-CA from Neisseria gonorrhoeae (CAN.g.) is uniquely localized in the periplasm (Figure 1), where its exact role has not been experimentally determined, but it is predicted to ether aid in Ci concentration or support inner membrane integrity in low pH environments [28]. To our knowledge, a direct comparison of the relative efficacy of these mechanistically distinct CCMs during E. coli fermentative growth is yet to be reported.
The first goal of this study was to investigate the importance of E. coli’s native CCM with respect to its role in supporting fermentative succinate production. Next, three mechanistically distinct and heterologous CCM components—sbtA, dabAB and CAN.g.—were evaluated in terms of their ability to complement growth and/or succinate production in CA-free (Δcan ΔcynT) E. coli strains under different environmental conditions impacting extracellular Ci availability. Lastly, these same CCM components were investigated with respect to their potential for improving fermentative succinate production by engineered E. coli via further enhancement of its native CCM.

2. Materials and Methods

2.1. Media and Culture Conditions

All strains were routinely cultured at 37 °C in Luria–Bertani (LB) broth or on LB agar plates containing 100 µg/mL ampicillin, where appropriate. AM1 mineral salt medium supplemented with 100 mM KHCO3 was used for all experiments, as previously described [29]. For growth on agar plates under different CO2 atmospheres, KHCO3 was excluded, and pH was controlled via addition of 100 mM MOPS (pH 6.5 and 7.5) or 100 mM MES (pH 5.5). Agar plates were additionally supplemented with 100 mM NaCl and, where appropriate, 10 µM IPTG and 100 µg/mL ampicillin. For fermentation experiments, KHCO3 was included in AM1 media at an initial concentration of 6.1 g/L (100 mM) whereas IPTG was added at specified concentrations.
Fermentation vessels consisted of 250 mL media bottles outfitted with a pH probe, a base addition port, a venting port and a sampling port. All fermentations were performed using an initial volume of 200 mL and maintained at a constant temperature of 37 °C. Throughout the fermentation, a custom-built pH controller was used to maintain pH at the 7.0 set point via automated base addition of a base solution consisting of 2.4 M KHCO3 and 1.2 M KOH. Cell growth was measured as optical density at 600 nm (OD600) using a UV/Vis spectrophotometer (Beckman Coulter DU-730, Beckman Coulter Life Sciences, Brea, CA, USA). In all cases, overnight seed cultures grown in an incubator with a 5% CO2 headspace were used to inoculate fermentations at an initial OD600 of 0.1, after which cultures were then sampled every 24 h to monitor growth, glucose consumption and metabolite production.

2.2. Strain and Plasmid Construction

All strains and plasmids constructed and/or used in this study are summarized in Table 1. The gene cynT was deleted from E. coli strains EDCM636 and KJ122 via a modified Datsenko and Wanner method [30], with pKD3 serving as the template for the FRT-cmR-FRT fragment. Strains were unmarked using pCP20 which encodes FLP recombinase. The gene can was deleted from KJ122 ΔcynT using the sucrose counterselection method [31]. All gene deletions were confirmed via colony PCR using primer pairs as described in Supplemental Table S1.
The dabAB operon from plasmid pFE-dabAB, a gift from Dr. David Savage (Addgene #133002), was cloned into pTrc99A using the EcoRI site. All other plasmids were constructed by cloning the gene(s) of interest into pTrc99A via Gibson Assembly [32] or Golden Gate Assembly [33] utilizing either BsaI or PaqCI. The gene can was amplified from E. coli ATCC 8739. The genes sbtA from Cyanobium sp. PCC 7001 and CAN.g. gene from Neisseria gonorrhoeae were codon optimized for E. coli and synthesized as gBlocks by Integrated DNA Technologies (IDT, Coralville, IA, USA). All PCR primers used were custom designed then synthesized by IDT. The full list of primers and gene sequences used are included in Supplemental Table S1 and Supplemental Sequences, respectively.

2.3. Cultivation on Agar Plates Under Controlled Atmospheric Conditions

For growth under aerobic conditions, agar plates were incubated at 37 °C in a CO2 incubator (Caron Oasis 6400, Caron Products and Services, Inc., Marietta, OH, USA) operated at the desired CO2 concentration (0.05 to 5%). For growth under anaerobic conditions, agar plates were incubated inside of a sealed benchtop vacuum chamber (Terra Universal, Fullerton, CA, USA). The box was first flushed for 20 min with Argon gas. A pair of mass flow controllers (Alicat Scientific Products, Tucson, AZ, USA) were then used to create custom Ar-CO2 gas mixtures containing the desired CO2 concentration (0.05 to 5%), as confirmed using a Vaisala infrared CO2 gas meter with MI70 indicator and GMP70 probe. This process was repeated each time after removing plates for inspection and imaging. All plates were imaged every 24 h for a total of 96 h.

2.4. Fermentation Product Analysis

Concentrations of glucose and fermentation products of interest were determined using high-performance liquid chromatography (HPLC; 1100 series, Agilent, Santa Clara, CA, USA). Analytes were separated isocratically using an OA-1000 Organic Acid column (300 × 6.5 mm × 9 µm; Alltech, Nicholasville, KY, USA) maintained at 50 °C and a mobile phase of 5 mM H2SO4 flowing at a constant rate of 0.55 mL/min. External calibrations prepared using authentic standards were used to identify species as well as to determine their concentrations.

3. Results and Discussion

3.1. E. coli’s Native CCM Is Critical for Efficient Succinate Fermentation

While it has been shown that carbonic anhydrases are essential for aerobic growth of wild-type E. coli under atmospheric CO2 conditions [14,23], to our knowledge, their importance under anaerobic conditions has not yet been reported. To address this gap, can and cynT were first both deleted in MG1655 and the resulting strain, MG1655ΔCA (Table 1), was fermented at pH 7 in the presence of 100 mM KHCO3. Under these conditions, however, there was no difference in growth or sugar consumption between MG1655 and MG1655ΔCA (Figure 2A,B). In this case, the main fermentation product of both strains was lactate (Figure 2C) which, unlike succinate production, allows for NAD+ regeneration without the need for significant Ci input. These results were compared with fermentative succinate production by also deleting can and cynT from KJ122 (a strain previously engineered to produce succinate at high titers and yields: e.g., up to 82.6 g/L and 88% maximum theoretical yield when fermenting 100 g/L glucose) [29], creating KJ122ΔCA (Table 1). When KJ122ΔCA was similarly fermented at pH 7 in the presence of 100 mM KHCO3, the result was an extended lag phase lasting up to 48 h (Figure 2D). Furthermore, glucose consumption and succinate production by 96 h were both significantly decreased (by 36.9% and 43.7%, respectively) relative to KJ122 (Figure 2E,F). Rather, only by 168 h had KJ122ΔCA consumed the same amount of glucose and produced an equivalent amount of succinate (80.2 ± 2.6 vs. 86.1 ± 1.1 g/L). By the time all glucose was consumed in each respective culture (i.e., by 168 vs. 96 h), this difference amounted to a 46.8% reduction in average volumetric productivity (i.e., 0.48 ± 0.02 vs. 0.90 ± 0.01 g-succinate/L-h), thereby highlighting the important role of E. coli’s native CAs for enabling efficient succinate production. Finally, the fermentative performance of KJ122 vs. KJ122ΔCA was also compared at pH 5.5; a condition in which provided KHCO3 would be predominantly converted to and driven out of the medium as CO2 (g) (Supplemental Figure S1), thereby reducing the total availability of extracellular Ci relative to pH 7. Here, while growth of KJ122 was expectedly slower than at pH 7, differences between the two strains became even more pronounced, with KJ122ΔCA displaying almost no growth, succinate production, or glucose consumption by 96 h (Figure 2G–I).

3.2. E. coli’s Native CCM Enables (An)Aerobic Growth in Low Extracellular Ci Environments

Previous studies have shown that the growth deficit caused in CA-free E. coli strains can be overcome by culturing under a high CO2 atmosphere (typically 5–10%) [23,34]. Again, however, these studies were exclusively performed under aerobic conditions, and a more comprehensive investigation of other environmental factors influencing extracellular Ci availability has not yet been reported. Accordingly, a growth assay using agar plates incubated under different, controlled CO2 atmospheres and pH conditions was performed to further investigate the importance of E. coli’s native CCM. First, under ambient air conditions (0.05% CO2) and across all tested pH conditions (5.5, 6.5 and 7.5), KJ122 and MG1655 were able to grow, demonstrating that native CA activity in both strains was sufficient, even under low extracellular Ci availability (Figure 3). Unlike their respective parents, however, MG1655ΔCA and KJ122ΔCA were both incapable of growth under these same conditions (Figure 3); a deficit that also continued at 0.5% and 1% CO2 (10- and 20-times atmospheric CO2 concentrations, respectively). Growth was finally restored, albeit visibly reduced, at 2.5% CO2, with full restoration observed only at 5% CO2. At 2.5% CO2, meanwhile, overall growth of MG1655ΔCA and KJ122ΔCA was also reduced with each successive drop in pH (i.e., 5.5 < 6.5 < 7.5). Of course, as a neutrophile, E. coli has a decreased ability to grow in low pH environments. However, since this pH-dependent growth decline seemingly occurred to a greater extent for both CA-free strains relative to their respective parents (i.e., MG1655 and KJ122), this suggests that reduced availability of total aqueous Ci under increasingly acidic conditions (Supplemental Figure S1B) was an even greater contributor in this case.
A parallel experiment was also conducted to investigate and compare the importance of E. coli’s native CCM under anaerobic conditions. Previously, Flores et al. found that a related E. coli succinate producing strain was unable to grow on agar plates in an anaerobic atmosphere devoid of CO2 [35]; however, a minimum threshold of Ci availability required for growth was never determined. Here again, across all tested pH conditions, KJ122 and MG1655 both grew when supplied with 0.05% CO2 or higher (Figure 4). In contrast, no growth was observed for KJ122ΔCA below 5% CO2 whereas minimal growth was observed for MG1655ΔCA at pH 7.5 and 2.5% CO2 (with growth across all pH conditions only possible at 5%). Similar to aerobic conditions, the growth of MG1655ΔCA and KJ122ΔCA again declined at 5% CO2 with each successive drop in pH. When taken together with the results of Figure 3, these findings collectively demonstrate that: (i) E. coli’s native CCM is required for growth in low extracellular Ci environments under both aerobic and anaerobic conditions; and (ii) fermentative growth of KJ122ΔCA displays an even greater sensitivity to Ci availability.

3.3. Heterologous CCM Components Restore Growth of KJ122ΔCA

Whereas E. coli’s native CCM consists exclusively of cytoplasmic CAs, as discussed above, the CCMs of other CO2-fixing microbes are known to employ other functionally distinct mechanisms. Accordingly, we next investigated the ability of three of these—namely SbtA, DabAB and CAN.g.—with respect to their ability to restore the growth of KJ122ΔCA under conditions of reduced extracellular Ci availability. While SbtA and DabAB have been shown to restore the growth of other CA-free E. coli mutants under aerobic conditions [36,37], similar characterizations have yet to be performed anaerobically. These three genes along with E. coli can (control) were individually cloned into pTrc99A and then transformed into KJ122ΔCA. The resulting strains were first grown aerobically at 0.05% CO2 to confirm their function with respect to facilitating Ci acquisition. All except CAN.g. restored growth of KJ122ΔCA at pH 7.5 (Figure 5), with can and dabAB expression restoring growth equivalently across all pH conditions. Expression of sbtA, meanwhile, restored growth in a pH-dependent manner, being less effective at pH 6.5 and wholly ineffective at pH 5.5; an outcome that is consistent with the fact that SbtA transports HCO3−, which becomes significantly less abundant at lower pH (Supplemental Figure S1A). This pH-dependent difference in functional efficacy is also consistent with a past observation that dabAB-like genes were 4.8-times more abundant than sbtA in metagenomes obtained acidic environments (<pH 5.4), whereas this ratio shifts to just 0.43 for alkaline environments (pH 8.5–10) [36]. When the experiment was repeated at 0.5% CO2, growth for all strains was improved, notably now including the strain expressing sbtA at pH 5.5.
Anaerobically, expression of can, sbtA and, dabAB, all restored KJ122ΔCA growth at 0.05% CO2 and pH 7.5 (Figure 5). Whereas expression of can also equally restored KJ122ΔCA growth at pH 6.5 and 5.5, dabAB and sbtA expression now showed only partial growth restoration at pH 6.5 and none at pH 5.5. At 0.5% CO2, can and dabAB expression resulted in similar levels of growth restoration under all pH conditions, whereas sbtA expression again showed diminishing growth restoration with decreasing pH. These collective results suggest that DabAB and SbtA could potentially serve as functional replacements to E. coli’s native CCM under fermentative conditions, or possibly even be used to enhance it. In contrast, CAN.g. failed to restore growth of KJ122ΔCA under any conditions was accordingly excluded from the remainder of the study.

3.4. Heterologous CCM Components Partially Restore Succinate Production in KJ122ΔCA

The potential ability of SbtA and DabAB (along with Can as control) to restore the succinate fermentation performance of KJ122ΔCA was next investigated in batch bottle bioreactors maintained at pH 7. Furthermore, two induction levels (10 and 100 µM IPTG) were compared for the expression of each gene. Under these conditions, can expression resulted in 15.8- and 8.5-fold higher growth by 48 h than that of KJ122ΔCA for 10 and 100 µM IPTG induction, respectively, after which a similar maximum OD600 was finally achieved by 96 h in all cases (Figure 6A). By 96 h, final succinate titers were 38.2% higher than by KJ122ΔCA yet still 35% lower relative to KJ122 for both induction conditions (Figure 6C). Despite representing an improvement over KJ122ΔCA, these results also point to potential differences in can expression from a plasmid versus natively in KJ122. Expression of sbtA with 100 µM IPTG, meanwhile, resulted in only minimal differences relative to KJ122ΔCA (Figure 6D–F). The greatest restoration of growth and succinate production was observed for dabAB expression at 100 µM IPTG where, despite still experiencing a ~24 h lag, supported 20% greater growth than that of even KJ122 by 48 h (Figure 6G) and equivalent final succinate production by 96 h (both ~87 g/L; Figure 6I). While these findings suggest that heterologous CCM components can function in support of E. coli succinate fermentations, further studies are required to determine if the persistent limitations are due to differences in gene expression, Ci affinity and/or other factors, as well as if they can ultimately be overcome.

3.5. Functional Enhancement of E. coli’s Native CCM Does Not Improve Succinate Fermentation

Finally, with the goal of improving the succinate fermentation performance of KJ122, the effects of enhancing E. coli’s native CCM via overexpression of native and heterologous CCM components were investigated. Unfortunately, as seen in Table 2, can, sbtA and dabAB expression in KJ122 provided little to no improvement with respect to glucose consumption, final succinate titer, yield, or productivity. Overall, while only can overexpression appeared to offer a slight benefit, those differences were ultimately not statistically significant relative to an empty vector control. These results collectively suggest that E. coli’s native CCM is sufficient for supporting Ci acquisition by KJ122 under the fermentation conditions examined. If Ci acquisition is not limiting, this suggests that another, downstream bottleneck may instead exist in KJ122. This could include, for example, subsequent rates of Ci fixation by Ppc/Pck. Interestingly, others have shown that co-expressing Ppc or Pck together with a heterologous CCM component (namely, a CA similar to can with ppc, sbtA with ppc, or sbtA with pck) does result in improved succinate production by other E. coli strains [38,39,40,41]. A similar, future approach may likewise prove useful for KJ122. Meanwhile, expression of non-native CCM may still represent useful tools for improving succinate fermentation in other strains/microorganisms and/or under different fermentation conditions.

4. Conclusions

Inorganic carbon acquisition from the extracellular medium represents a key process during fermentative succinate production by E. coli. The importance of E. coli’s native CCM in this regard was first demonstrated where, in the absence of CA, cultures suffered a significant (48 h) lag along with 46.8% reduced succinate productivity. Although several heterologous CCM components were found to be capable of restoring the growth of CA-free E. coli strains under both aerobic and anaerobic conditions and across diverse environmental conditions affecting extracellular Ci availability, none were ultimately capable of fully compensating for the loss of CA activity during fermentative succinate production. Moreover, their overexpression also provided no significant benefit to succinate production when native CA activity remained intact. Taken together, the collective findings suggest that E. coli’s native CCM plays a critical and sufficient role in supporting fermentative succinate production.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation12020101/s1, Figure S1: Availability and speciation of dissolved inorganic carbon varies with pH and headspace CO2 pressure; Table S1: Primers used in this study; Supplemental Sequences.

Author Contributions

A.G.G., X.W. and D.R.N. conceived the idea. A.G.G., F.C. and L.T. performed the experiments. A.G.G., X.W. and D.R.N. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported in part by grants from the National Science Foundation (CBET-1705409, CBET-2148629). A.G.G. also received financial support in the form of Completion Fellowships from Arizona State University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 12 00101 g001
Figure 1. Overview of native and heterologous CCM components in Escherichia coli and the influence of pH. Native carbonic anhydrase is in gray whereas heterologous components are colored. Ci species abundance is indicated by percentages of total Ci. Arrows between Ci species indicate transport or conversion reactions, with dashed arrows representing unfacilitated (i.e., passive) diffusion. Arrow weight indicates relative contribution at the specified pH based on species abundance. The favored direction of equilibrium reactions is indicated by arrow length. Dotted arrows indicate that there are multiple enzymatic reactions not shown between species.
Figure 1. Overview of native and heterologous CCM components in Escherichia coli and the influence of pH. Native carbonic anhydrase is in gray whereas heterologous components are colored. Ci species abundance is indicated by percentages of total Ci. Arrows between Ci species indicate transport or conversion reactions, with dashed arrows representing unfacilitated (i.e., passive) diffusion. Arrow weight indicates relative contribution at the specified pH based on species abundance. The favored direction of equilibrium reactions is indicated by arrow length. Dotted arrows indicate that there are multiple enzymatic reactions not shown between species.
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Fermentation 12 00101 g002
Figure 2. E. coli’s native CCM is important for Ci acquisition during fermentative succinate production. (A–C). Fermentation of MG1655 (gray upwards facing triangles, solid line) and MG1655ΔCA (green downwards facing triangles, dashed line) at pH 7. (D–F). Fermentation of KJ122 (teal circles, solid line) and KJ122ΔCA (purple squares, dashed line) at pH 7. (G–I). Fermentation of KJ122 (teal circles, solid line) and KJ122ΔCA (purple squares, dashed line) at pH 5.5. All cultures were initially supplemented with 100 mM KHCO3. For the pH 5.5 fermentation, pH was adjusted following KHCO3 addition. Error bars represent standard error from triplicate cultures.
Figure 2. E. coli’s native CCM is important for Ci acquisition during fermentative succinate production. (A–C). Fermentation of MG1655 (gray upwards facing triangles, solid line) and MG1655ΔCA (green downwards facing triangles, dashed line) at pH 7. (D–F). Fermentation of KJ122 (teal circles, solid line) and KJ122ΔCA (purple squares, dashed line) at pH 7. (G–I). Fermentation of KJ122 (teal circles, solid line) and KJ122ΔCA (purple squares, dashed line) at pH 5.5. All cultures were initially supplemented with 100 mM KHCO3. For the pH 5.5 fermentation, pH was adjusted following KHCO3 addition. Error bars represent standard error from triplicate cultures.
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Fermentation 12 00101 g003
Figure 3. Carbonic anhydrase deletion impairs aerobic growth of E. coli in a CO2 and pH dependent manner. Plate based growth of CA-free strains and their respective parents under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) MG1655, (2) KJ122, (3) KJ122ΔCA, (4) MG1655ΔCA.
Figure 3. Carbonic anhydrase deletion impairs aerobic growth of E. coli in a CO2 and pH dependent manner. Plate based growth of CA-free strains and their respective parents under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) MG1655, (2) KJ122, (3) KJ122ΔCA, (4) MG1655ΔCA.
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Figure 4. Carbonic anhydrase deletion impairs anaerobic growth of E. coli in a CO2 and pH dependent manner. Plate based growth of CA-free strains and their respective parents under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) MG1655, (2) KJ122, (3) KJ122ΔCA, (4) MG1655ΔCA.
Figure 4. Carbonic anhydrase deletion impairs anaerobic growth of E. coli in a CO2 and pH dependent manner. Plate based growth of CA-free strains and their respective parents under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) MG1655, (2) KJ122, (3) KJ122ΔCA, (4) MG1655ΔCA.
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Figure 5. Heterologous CCM components provide conditional complementation of E. coli’s native CCM under both aerobic and anaerobic conditions. Plate based growth of under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) KJ122 pTrc99A, (2) KJ122ΔCA pTrc99A, (3) KJ122ΔCA pDabAB, (4) KJ122ΔCA pCan, (5) KJ122ΔCA pSbtA and (6) KJ122ΔCA pCAN.g..
Figure 5. Heterologous CCM components provide conditional complementation of E. coli’s native CCM under both aerobic and anaerobic conditions. Plate based growth of under different pH conditions and CO2 atmospheres at 96 h. All plates were streaked with the following strains as indicated by the legend provided with the plate at the top left: (1) KJ122 pTrc99A, (2) KJ122ΔCA pTrc99A, (3) KJ122ΔCA pDabAB, (4) KJ122ΔCA pCan, (5) KJ122ΔCA pSbtA and (6) KJ122ΔCA pCAN.g..
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Figure 6. Heterologous CCM components only partially restore fermentative succinate production by KJ122ΔCA. For all plots, KJ122 is indicated by teal circles and KJ122ΔCA is indicated by purple squares. For (A–C), KJ122ΔCA pCan is indicated with green triangles (downward light green: 10 µM IPTG; upward dark green: 100 µM IPTG). For (D–F), KJ122ΔCA pSbtA is indicated with blue triangles (downward light blue: 10 µM IPTG; upward dark blue: 100 µM IPTG). For (G–I), KJ122ΔCA pDabAB is indicated with red triangles (downward light red: 10 µM IPTG; upward dark red: 100 µM IPTG). Error bars represent standard error from triplicate cultures.
Figure 6. Heterologous CCM components only partially restore fermentative succinate production by KJ122ΔCA. For all plots, KJ122 is indicated by teal circles and KJ122ΔCA is indicated by purple squares. For (A–C), KJ122ΔCA pCan is indicated with green triangles (downward light green: 10 µM IPTG; upward dark green: 100 µM IPTG). For (D–F), KJ122ΔCA pSbtA is indicated with blue triangles (downward light blue: 10 µM IPTG; upward dark blue: 100 µM IPTG). For (G–I), KJ122ΔCA pDabAB is indicated with red triangles (downward light red: 10 µM IPTG; upward dark red: 100 µM IPTG). Error bars represent standard error from triplicate cultures.
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Table 1. Strains and plasmids engineered and/or used in this study.
Table 1. Strains and plasmids engineered and/or used in this study.
StrainGenotype/FeaturesSource
MG1655F-lambda-ilvG-rfb-50 rph-1Nielsen Lab
EDCM636MG1655 ΔlacZY, can< >FLK2CGSC
MG1655ΔCAMG1655 Δcan cynT::FRTThis study
KJ122ATCC 8739, pck*, ptsI*, galR*, galS*, ΔldhA, ΔadhE, ΔackA, Δ(focA-pflB) ΔmgsA, ΔpoxB, ΔtdcDE, ΔcitF, ΔaspC, ΔsfcA; pck* stands for a mutated form of pck (G to A at position−64 relative to the ATG start codon), ptsI* stands for a mutated form of ptsI (single base deletion at position 1673 causing a frameshift mutation in the carboxyl-terminal region), galR* stands for an IS1 element inserted at position 261 in the galR ORF, galS* stands for an adenine insertion at position 231 in the galS ORF.[29]
KJ122ΔCAKJ122 Δcan cynT::FRTThis study
PlasmidGenotype/FeaturesSource
pFE-dabAB2Source of dabABAddgene
pTrc99AColE1 ori, lacI, ampR, PtrcNielsen Lab
pCanpTrc99A-Trc-canThis study
pDabABpTrc99A-Trc-dabABThis study
pCAN.g.pTrc99A-Trc-CAN.g.This study
pSbtApTrc99A-Trc-sbtAThis study
Table 2. Overexpression of native and heterologous CCM components provides no significant benefit to succinate fermentation performance by E. coli KJ122. CCM components are expressed using 100 µM IPTG. All metrics were determined after 96 h. Error is reported as standard error from triplicate cultures, except in the case of the empty vector control where error is reported as represent standard error from three sets of controls (9 cultures total).
Table 2. Overexpression of native and heterologous CCM components provides no significant benefit to succinate fermentation performance by E. coli KJ122. CCM components are expressed using 100 µM IPTG. All metrics were determined after 96 h. Error is reported as standard error from triplicate cultures, except in the case of the empty vector control where error is reported as represent standard error from three sets of controls (9 cultures total).
CCM Component ExpressedGlucose Consumed
(g/L)		Succinate Produced
(g/L)		Yield
(gsuccinate/gglucose)		Succinate Production Rate
(g/L-h)
Can99.6 ± 0.690.3 ± 0.70.91 ± 0.020.94 ± 0.02
DabAB96.9 ± 0.783.5 ± 0.30.86 ± 0.000.87 ± 0.00
SbtA97.2 ± 0.873.2 ± 3.80.75 ± 0.030.76 ± 0.04
Empty Vector97.2 ± 2.984.4 ± 5.50.87 ± 0.060.87 ± 0.06
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Godar, A.G.; Cristobal, F.; Taquillo, L.; Wang, X.; Nielsen, D.R. Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation. Fermentation 2026, 12, 101. https://doi.org/10.3390/fermentation12020101

AMA Style

Godar AG, Cristobal F, Taquillo L, Wang X, Nielsen DR. Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation. Fermentation. 2026; 12(2):101. https://doi.org/10.3390/fermentation12020101

Chicago/Turabian Style

Godar, Amanda G., Francesca Cristobal, Luis Taquillo, Xuan Wang, and David R. Nielsen. 2026. "Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation" Fermentation 12, no. 2: 101. https://doi.org/10.3390/fermentation12020101

APA Style

Godar, A. G., Cristobal, F., Taquillo, L., Wang, X., & Nielsen, D. R. (2026). Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation. Fermentation, 12(2), 101. https://doi.org/10.3390/fermentation12020101

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