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Low-biomass pyruvate production with engineered Vibrio natriegens is accompanied by parapyruvate formation

Microbial Cell Factories volume 24, Article number: 73 (2025) Cite this article

Abstract

Background

Pyruvate is a precursor for various compounds in the chemical, drug, and food industries and is therefore an attractive target molecule for microbial production processes. The fast-growing bacterium Vibrio natriegens excels with its specific substrate uptake rate as an unconventional chassis for industrial biotechnology. Here, we aim to exploit the traits of V. natriegens for pyruvate production in fermentations with low biomass concentrations.

Results

We inactivated the pyruvate dehydrogenase complex in V. natriegens Δvnp12, which harbors deletions of the prophage regions vnp12. The resulting strain V. natriegens Δvnp12 ΔaceE was unable to grow in minimal medium with glucose unless supplemented with acetate. In shaking flasks, the strain showed a growth rate of 1.16 ± 0.03 h− 1 and produced 4.0 ± 0.3 gPyr L− 1 within 5 h. We optimized the parameters in an aerobic fermentation process and applied a constant maintenance feed of 0.24 gAc h− 1 which resulted in a maximal biomass concentration of only 6.6 ± 0.4 gCDW L− 1 and yielded highly active resting cells with a glucose uptake rate (qS) of 3.5 ± 0.2 gGlc gCDW−1 h− 1. V. natriegens Δvnp12 ΔaceE produced 41.0 ± 1.8 gPyr L− 1 with a volumetric productivity of 4.1 ± 0.2 gPyr L− 1 h− 1. Carbon balancing disclosed a gap of 30%, which we identified partly as parapyruvate. Deletion of ligK encoding the HMG/CHA aldolase in V. natriegens Δvnp12 ΔaceE did not impact biomass formation but plasmid-based overexpression of ligK negatively affected growth and led to a 3-fold higher parapyruvate concentration in the culture broth. Notably, we also identified parapyruvate in supernatants of a pyruvate-producing Corynebacterium glutamicum strain. Cell-free bioreactor experiments mimicking the biological process also resulted in parapyruvate formation, pointing to a chemical reaction contributing to its synthesis.

Conclusions

We engineered metabolically highly active resting cells of V. natriegens producing pyruvate with high productivity at a low biomass concentration. However, we also found that pyruvate production is accompanied by parapyruvate formation in V. natriegens as well as in a pyruvate producing C. glutamicum strain. Parapyruvate formation seems to be a result of chemical pyruvate conversion and might be supported biochemically by an aldolase reaction.

Introduction

Pyruvate, in its protonated form called pyruvic acid, is a key intermediate in the central carbon metabolism and serves as precursor for various catabolic and anabolic routes such as the tricarboxylic acid (TCA) cycle or the biosynthesis of branched-chain amino acids. The three-carbon carboxylic acid has industrial relevance and is a precursor for various compounds, such as chemicals, fuels, pharmaceuticals, and polymers [1,2,3]. Examples of pyruvate-derived molecules which have been produced by microbial fermentation include lactate [4], butanol [5], isobutanol [6], 2,3-butandiol [7], alanine [8], l-valine [9], N-acetylneuraminic acid [10], and l-DOPA [11]. Additionally, pyruvate was identified as one of the most relevant precursors for a variety of non-native commercial products in Escherichia coli [12]. Pyruvate is also used as a dietary supplement due to its neuroprotective effects [13, 14] and might have beneficial effects on exercise performance [14, 15]. Pyruvate is produced predominantly by chemical processes such as dehydration and decarboxylation of tartaric acid [16]. Chemical pyruvate production suffers from the associated costs [17] and is ecologically questionable, as substrates are partially fossil-resource-derived. Therefore, extensive research for biotechnological pyruvate production from renewable resources has been carried out and several microorganisms such as E. coli [18], Corynebacterium glutamicum [19], Lactococcus lactis [20], as well as yeasts such as Saccharomyces cerevisiae [21], Yarrowia lipolytica [22], Candida glabrata [23], and engineered derivatives have been exploited for this purpose.

Under aerobic conditions, a major fraction of the intracellular pyruvate pool is utilized for the synthesis of acetyl-CoA. This oxidative decarboxylation is catalyzed by the pyruvate dehydrogenase complex (PDHC) which consists of three subunits [24]. Consequently, several metabolic engineering approaches have focused on abolishing or reducing the flux towards acetyl-CoA. This flux reduction was achieved by deletion of the aceE gene encoding the E1 subunit of the PDHC [25,26,27,28], silencing aceE expression by antisense RNA [29] or CRISPRi [30], enzyme engineering [31], promoter modification [32], and implementing cofactor auxotrophies [18, 22, 23]. Further strain engineering strategies aim to reduce by-product formation by deleting competing pathways [1,2,3]. Depending on the host organism, targets for gene deletion include for example the lactate dehydrogenase [19, 27, 28, 33], pyruvate oxidase [26,27,28], pyruvate decarboxylase [21], phosphoenolpyruvate synthase [27, 28, 33], and alanine aminotransferase [19]. Another way to improve pyruvate production is an increased glycolytic flux. Deletion of the F1-ATPase increased the glycolytic flux in E. coli [28, 34] and an ATPase inhibitor increased pyruvate yield in C. glabrata [35].

Parapyruvate, or 4-hydroxy-4-methyl-2-oxoglutarate (HMG), is a six-carbon dicarboxylic acid formed from two pyruvate molecules via aldol condensation [36]. It is commonly found as impurity in pyruvate supplements [37, 38]. In vitro, parapyruvate can be derived from pyruvate by alkaline treatments [38, 39]. Enzymatic degradation of parapyruvate to pyruvate was demonstrated by the aldolase LigK [40, 41]. In vivo, LigK is part of the protocatechuate (PCA) 4,5-cleavage pathway, catalyzing the last step from 4-carboxy-4-hydroxy-2-oxoadipate (CHA) to pyruvate and oxaloacetate [42]. However, in vivo parapyruvate formation by LigK has not been described as far as we know.

Biotechnological processes rely on the formation and maintenance of the biocatalyst, which may utilize a significant portion of the supplied carbon source and therefore reduce the achievable product yield. Moreover, high biomass concentrations may negatively impact downstream processing and separating biomass from the product and disposal of it are additional costs to consider [43,44,45]. Therefore, lowering biomass concentrations could be an option to reduce the overall process costs. Still, in order to achieve economic viability, key performance indicators (KPIs) of the fermentation, such as titer, productivity, and product yield, need to be high [46]. To maximize the KPIs with low biomass concentration, metabolically highly active cells are necessary, which is inherently linked to the substrate uptake rate of the biocatalyst. Therefore, a suitable candidate for this approach is V. natriegens, a non-pathogenic marine bacterium known for its high growth rate that can reach values of about 1.9 h− 1 in minimal medium with glucose as substrate [47]. The biomass-specific glucose uptake rate of V. natriegens during exponential aerobic growth is 3.9 gGlc gX−1 h− 1, which is twice as high compared to established bacteria such as E. coli and Bacillus subtilis [48]. V. natriegens is a recently emerging unconventional host for biotechnological applications with rising research interest (reviewed in [49,50,51]). Plentiful genetic engineering tools have already been established and applied to V. natriegens, and the pool of parts for synthetic biology is steadily increasing, enabling rapid metabolic engineering of this bacterium [52, 53]. Previously, V. natriegens was engineered for pyruvate production [33] and pyruvate-derived products, such as 2,3-butanediol [54, 55], succinate [56, 57], alanine [48], and l-DOPA [58].

In this study, we explore the possibility for pyruvate production with V. natriegens in a low-biomass setup through metabolic engineering and process development. Furthermore, we highlight the formation of parapyruvate during pyruvate production and investigate the involvement of LigK in vivo, as well as chemical formation of parapyruvate.

Results

Pyruvate production in a low-biomass process

In a previous work, it was shown that V. natriegens harbors two prophage regions. Deletion of these regions led to a more stable and stress resistant growth behavior. Therefore, this prophage-free strain V. natriegens Δvnp12 was used as base for further strain engineering [59]. To disrupt the metabolic flux from pyruvate to acetyl-CoA, the aceE gene (PN96_01335) was deleted. Then, we cultivated the resulting strain V. natriegens Δvnp12 ΔaceE in VN minimal medium and characterized growth, substrate consumption and product formation (see Fig. 1). Deletion of the aceE gene caused the strain to lose the ability to grow on glucose as sole carbon and energy source (see Fig. 1A, B). Additional supplementation of acetate to the medium restored the growth of V. natriegens Δvnp12 ΔaceE, resulting in a growth rate (µ) of 1.16 ± 0.03 h− 1 compared to 1.43 ± 0.01 h− 1 of V. natriegens Δvnp12 under the same conditions (see Fig. 1C, D). While V. natriegens Δvnp12 accumulated acetate over time, the PDHC-deficient strain consumed the acetate simultaneously with the glucose. This strain’s growth was arrested once all acetate was taken up. V. natriegens Δvnp12 ΔaceE produced 4.0 ± 0.3 gPyr L− 1 after 5 h in shaking flasks, which corresponds to a product yield (YP/S) of 0.54 ± 0.03 gPyr gGlc−1 (see Fig. 1D). Accordingly, analysis of intracellular metabolites showed that V. natriegens Δvnp12 ΔaceE exhibits a 6-fold higher concentration of pyruvate in the exponential growth phase compared to V. natriegens Δvnp12.

Fig. 1
figure 1

Growth characteristics of V. natriegens Δvnp12 and V. natriegens Δvnp12 ΔaceE. Plotted are the cell dry weight (CDW, grey, square), pyruvate (Pyr, blue, triangle), acetate (Ace, green, diamond) and glucose (Glc, orange, circle) concentrations over time. V. natriegens Δvnp12 (A and C) and V. natriegens Δvnp12 ΔaceE (B and D) were cultivated in 50 mL minimal medium, either with 7.5 g glucose L-1 as sole carbon source (A and B) or additionally with 1 g acetate L-1 (C and D). Strains were incubated in baffled shaking flasks at 37 °C. Data shown are means and standard deviations of independent triplicates

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V. natriegens Δvnp12 ΔaceE was then cultivated in a controlled bioreactor setup in 500 mL minimal medium with an initial substrate concentration of 100 g glucose L− 1. Biomass formation was limited by addition of 2 g acetate L− 1 to the medium. Upon depletion of acetate after 5 h, the strain reached a maximum biomass concentration (cCDW) of 3.4 ± 0.1 gCDW L− 1 and showed a µ of 0.88 ± 0.02 h− 1 in the exponential growth phase (see Fig. 2A; Table 1). Glucose consumption and pyruvate production continued in the stationary phase to a titer of 22.2 ± 0.9 gPyr L− 1 after 10 h. The qS decreased from 3.2 ± 0.3 gGlc gCDW−1 h− 1 in the exponential phase to 1.9 ± 0.1 gGlc gCDW−1 h− 1 in the stationary phase.

To enhance glucose utilization in the stationary phase, an acetate feed was applied (Fig. 2B). Considering a maintenance requirement of 0.063 gGlc gCDW−1 h− 1 [60], we applied a constant feed of 0.24 gAc h− 1 from the start of the process. The acetate feed prolonged the growth phase with a µ of 0.80 ± 0.03 h− 1 to reach a peak biomass of 6.6 ± 0.4 gCDW L− 1. Moreover, the applied feed maintained the qS of the exponential phase of 3.2 ± 0.2 gGlc gCDW−1 h− 1 in the stationary phase with 3.5 ± 0.2 gGlc gCDW−1 h− 1 (see Table 1). Unlike the initial process, the acetate was not completely depleted from the medium when cell growth stopped and remained at a steady level. The highest pyruvate titer of 41.0 ± 1.8 gPyr L− 1 of the process was reached after 10 h with an YP/S of 0.51 ± 0.02 gPyr gGlc−1 and a volumetric productivity of 4.1 ± 0.2 gPyr L− 1 h− 1.

Analyzing the carbon balance after 10 h of cultivation revealed a gap of unaccounted carbon (see Fig. 2C). The amount increased with the feed applied to the process from about 13–30% and signals a so far unknown carbon sink. One additional compound that could be found in HPLC analysis of the supernatants was parapyruvate. The presence of parapyruvate in the sample was further verified by liquid chromatography–coupled quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS). For the reactor sample, at 12.4 min an m/z peak of 175.0251 was detected, corresponding to an C6H7O6− ion (∂ 1.37 ppm), which was confirmed to represent parapyruvate by the purchased authentic standard (Rt 12.4 min, m/z of 175.0250, ∂ 0.94 ppm). However, absolute quantification was difficult as purchased standards showed additional peaks beside the main parapyruvate peak in the HPLC analysis (see Figure S1). Based on the theoretical concentration calculated with the area of the compound’s main peak, we estimated a concentration of 7 g parapyruvate L− 1 after 10 h in the process with acetate feed. This amount represents 24% of the previous unaccounted carbon fraction and about 17% of the total pyruvate yield.

Table 1 KPIs of V. natriegens Δvnp12 ΔaceE in fermentations without and with acetate feed (see also Fig. 2)
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V. natriegens is known to be sensitive to oxidative stress [50], and we speculated whether this susceptibility could negatively impact fermentations with resting cells as described above. Recently, the heterologous expression of a catalase improved the tolerance against cold-induced loss of viability as a result of oxidative stress [61]. Therefore, we integrated the katG gene, encoding a catalase from E. coli, into the well-tested dns locus [62, 63] in the genome of V. natriegens Δvnp12 ΔaceE to investigate whether katG expression would benefit fermentations with resting cells as alternative to the acetate feed. The resulting strain V. natriegens Δvnp12 ΔaceE Δdns::katGEc was cultivated like the reference strain V. natriegens Δvnp12 ΔaceE in 500 mL minimal medium containing glucose and 2 g acetate L− 1 without additional feed. After 12 h cultivation time, no prolonged metabolic activity was observed and a large amount of leftover glucose remained again (see Figure S2). Noteworthy, compared to the parental strain, fermentations with V. natriegens Δvnp12 ΔaceE Δdns::katGEc showed a significant 19% increase in the pyruvate titer from 24.5 ± 0.7 gPyr L− 1 to 30.3 ± 2.0 gPyr L− 1 (p < 0.05) and the pyruvate yield also increased by 30% from 0.45 ± 0.05 gPyr gGlc−1 to 0.64 ± 0.05 gPyr gGlc−1 (p < 0.05).

Fig. 2
figure 2

Pyruvate production in batch fermentations with V. natriegens Δvnp12 ΔaceE. Plotted are the cell dry weight (CDW, grey, square), pyruvate (Pyr, blue, triangle), acetate (Ace, green, diamond) and glucose (Glc, orange, circle) concentrations over time. Fermentations were performed in 500 mL minimal medium at 37 °C with (A) 100 g glucose L-1 and 2 g acetate L-1 as initial substrates and (B) using an additional feed of 0.24 gAc h-1. (C) Carbon distribution after 10 h in C-mol of found compound per C-mol of overall substrate for both bioreactor setups. Data shown are means and standard deviations of independent triplicates

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The aldolase LigK can form parapyruvate in vivo

As enzymatic formation of parapyruvate was shown in vitro by the aldolase LigK [64], the genome of V. natriegens was screened for the PCA 4,5-cleavage pathway. This pathway consists of six enzymes [42] and all the required genes are present in the genome of V. natriegens, namely ligA, ligB, ligI, ligU, ligJ and ligK in a cluster (PN96_18525 to PN96_18550) and ligC close by (PN96_18475) (see Figure S3).

To investigate in vivo parapyruvate production in V. natriegens via LigK, the corresponding gene was deleted or overexpressed with the plasmid pEKEx2 under the control of an inducible tac promoter, both in wild-type (WT) and V. natriegens Δvnp12 ΔaceE. In the WT background, no differences in growth behavior were observed in both the deletion and in the uninduced and induced overexpression strains (see Fig. 3A). Similarly, V. natriegens Δvnp12 ΔaceE ΔligK did not exhibit a growth rate reduction with a µ of 1.13 ± 0.04 h− 1 compared to the parental strain, which had a µ of 1.15 ± 0.01 h− 1. However, unlike the WT, V. natriegens Δvnp12 ΔaceE showed a significant reduction of the growth rate upon overexpression of ligK, with a µ of 0.97 ± 0.02 h− 1 and 0.63 ± 0.01 h− 1 under uninduced and induced conditions, respectively (see Fig. 3B). To exclude the possibility of an unannotated aldolase being able to catalyze the last step of PCA degradation, V. natriegens WT and V. natriegens ΔligK were grown on 10 mM PCA as sole carbon source. The WT strain showed biomass formation from PCA while the deletion strain did not, confirming the disruption of the pathway by inactivation of LigK (see Fig. 3C).

Fig. 3
figure 3

Growth of V. natriegens WT (A, black, square) and V. natriegens Δvnp12 ΔaceE (B, black, square) compared to respective ligK deletion strains (blue, circle), as well as both backgrounds with the overexpression plasmid pEKEx2-ligK uninduced (orange, triangle) and induced (red, inverted triangle). Cultivations were performed in baffled shaking flasks containing 50 mL minimal medium with 7.5 g glucose L-1 (A and B) and 1 g acetate L-1 (B) at 37 °C. (C) Growth of V. natriegens WT and V. natriegens ΔligK with 10 mM PCA as only carbon and energy source in baffled shaking flasks containing 50 mL minimal medium at 37 °C. Data shown are means and standard deviations of independent triplicates. Asterisk indicates statistical significance of p < 0.05

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Then, V. natriegens Δvnp12 ΔaceE ΔligK and V. natriegens Δvnp12 ΔaceE (pEKEx2-ligK) were cultured in a bioreactor to investigate the impact of LigK on pyruvate and parapyruvate formation. As observed in the shaking flasks, the ligK deletion mutant showed no differences compared to the parental strain V. natriegens Δvnp12 ΔaceE. In contrast, V. natriegens Δvnp12 ΔaceE (pEKEx2-ligK) with induced expression showed a strong shift in pyruvate and parapyruvate formation. Compared to V. natriegens Δvnp12 ΔaceE, the pyruvate titer of the strain harboring pEKEx2-ligK decreased by 78%, reaching 8.8 ± 1.4 gPyr L− 1 after 10 h process time (see Fig. 4A). Moreover, the parapyruvate amount of the overexpression strain was 3-fold higher (about 40 g L− 1) after 20 h compared to V. natriegens Δvnp12 ΔaceE and V. natriegens Δvnp12 ΔaceE ΔligK (see Fig. 4B). This result indicates that LigK can catalyze the formation of parapyruvate under excess pyruvate conditions in vivo but also shows that another (bio)chemical mechanism is responsible for parapyruvate production in the tested strains (see Fig. 4).

To determine whether parapyruvate is a specific trait of V. natriegens, we also analyzed C. glutamicum, which does not harbor LigK and the PCA 4,5-cleavage pathway. LC/Q-TOF-MS analysis identified parapyruvate in the supernatant of the pyruvate-producing strain C. glutamicum ΔaceE Δpqo ΔldhA ΔC–T ilvN ΔalaT ΔavtA [19] but not in the supernatant of C. glutamicum WT after 72 h of incubation (data not shown). This result indicates that pyruvate production might be generally accompanied by parapyruvate formation.

Fig. 4
figure 4

(A) Pyruvate and (B) parapyruvate formation in bioreactor cultivations of V. natriegens Δvnp12 ΔaceE (black, square), V. natriegens Δvnp12 ΔaceE ΔligK (blue, circle) and induced V. natriegens Δvnp12 ΔaceE (pEKEx2-ligK) (red, inverted triangle). Strains were grown in 500 mL minimal medium at 37 °C with 100 g glucose L-1, 2 g acetate L-1 and a feed of 0.24 gAc h-1. Data points with error bars are means and standard deviations of independent triplicates. Data points without error bars represent means of duplicates

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Cell-free parapyruvate formation

Since the deletion of ligK did not reduce the parapyruvate formation, we explored other possibilities for its formation in the bioreactor. Parapyruvate is known to be formed chemically under alkaline conditions in vitro [38, 39]. In all bioreactor experiments, the pH was titrated to 7.5 with 25% ammonia solution to counteract the decrease in pH as a result of pyruvate production. The droplet-based addition might create micro-environments with high alkaline pH conditions, fostering parapyruvate formation. Therefore, we performed cell-free bioreactor experiments mimicking the microbial production process. A constant pyruvic acid feed was applied that acidified the medium demanding for pH control by titration with 25% ammonia solution. Pyruvate and parapyruvate concentrations were measured over time and showed a similar trend in the cell-free environment (see Fig. 5A) and the production process (see Fig. 5B). After 6 h in the cell-free experiments, the feed was stopped, but stirring continued for two more hours (see Fig. 5A, arrow 1). In this time, the pyruvate concentration started to decrease, while the parapyruvate titer still increased. After 8 h, the pH of the reactor was adjusted to 10 (see Fig. 5A, arrow 2) which resulted in a steep decrease in both pyruvate and parapyruvate concentration.

Fig. 5
figure 5

Pyruvate (Pyr, blue, triangle) and parapyruvate (red, circle) formation in the bioreactor over time. (A) Cell-free 500 mL minimal medium with a pyruvic acid feed of 7.5 g L-1 h-1. At 6 h (arrow 1), the feed was stopped and at 8 h (arrow 2), the pH was adjusted to 10. (B) V. natriegens Δvnp12 ΔaceE in 500 mL minimal medium at 37 °C with 100 g glucose L-1, 2 g acetate L-1 and a feed of 0.24 gAc h-1. Data shown are means and standard deviations of independent triplicates

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Discussion

In this study, we engineered V. natriegens for pyruvate production, which we coupled to a metabolic switch for controlling biomass formation. This aim was achieved by deleting the aceE gene encoding the E1 subunit of the PDHC. As a result, the cells were only able to grow in the presence of glucose as long as acetate was available, which fed the TCA cycle with acetyl-CoA. The parental strain V. natriegens Δvnp12 showed exponential growth in medium with glucose and acetate, but acetate was not metabolized in parallel (see Fig. 1C), indicating typical catabolite repression. Due to an overflow metabolism, V. natriegens Δvnp12 additionally produced acetate under unlimited growth in the shaking flasks (see Fig. 1C). In contrast, the PDHC-deficient strain V. natriegens Δvnp12 ΔaceE consumed glucose and acetate in parallel, showing that the requirement for acetyl-CoA overcomes the global regulation by catabolite repression. Similarly, in PDHC-negative E. coli and Pseudomonas putida catabolite repression is relieved [27, 65], and glucose and acetate are utilized simultaneously [27]. PDHC-deficient strains of C. glutamicum, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa also exhibit an acetate auxotrophic phenotype [25, 66,67,68].

This phenotype allowed us to control biomass formation of V. natriegens Δvnp12 ΔaceE by adjusting the amount of acetate in the medium enabling a zero-growth production phase [69]. However, after the depletion of acetate, glucose consumption decreased rapidly from a qS of 3.2 ± 0.3 gGlc gCDW−1 h− 1 in the exponential growth phase to 1.9 ± 0.1 gGlc gCDW−1 h− 1 in the stationary phase accompanied by a stop of pyruvate production after 10 h (see Fig. 2A; Table 1). To prolong the production phase, we applied a constant and low acetate feed of 0.24 gAc h− 1 which is in the range of the maintenance requirements determined for growth on glucose [60]. Interestingly, the acetate feed stimulated glucose utilization, and V. natriegens Δvnp12 ΔaceE showed a qS of 3.5 ± 0.2 gGlc gCDW−1 h− 1 under non-growing conditions that equals the qS of the exponential growth phase of this process (see Table 1). This high qS is an excellent basis for engineering zero-growth production processes for pyruvate-derived products with high productivity. Accordingly, V. natriegens Δvnp12 ΔaceE achieved a volumetric productivity of 4.1 ± 0.2 gPyr L− 1 h− 1 in our setup. This productivity exceeds that of other pyruvate production processes with engineered E. coli [2, 3], C. glutamicum [19], yeast [2, 3] and a recently constructed V. natriegens strain with attenuated PDHC activity [33] (see Table 2).

Table 2 Selection of glucose-based pyruvate production processes by different microorganisms
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We applied the constant acetate feed from the onset of the process. Although acetate was not fully depleted in the medium, the cells stopped growing after 6 h at a biomass concentration of 6.6 ± 0.4 gCDW L− 1 (see Fig. 2B). This growth arrest is probably not a result of a nutrient limitation since the WT of V. natriegens reached higher biomass concentrations in the same medium (data not shown). Therefore, another unknown regulatory mechanism such as quorum sensing [57], or an inhibitory effect of parapyruvate, as discussed below, may account for this behavior. A similar growth arrest was observed with a PDHC-deficient E.coli [28] and relieved by addition of betaine as osmoprotectant. As marine organism, V. natriegens has a variety of osmotic stress response systems [50]. Therefore, future studies may investigate the impact of osmotic pressure on pyruvate production.

Under optimized conditions, V. natriegens Δvnp12 ΔaceE produced 41.0 ± 1.8 gPyr L− 1 within 10 h with a product yield of 0.51 ± 0.02 gPyr gGlc−1 (see Table 1). Compared to other glucose-based microbial production processes, this product yield is at the lower end of the spectrum with values of up to 0.87 gPyr gGlc−1 [2, 3] and might be improved by applying an additional nitrogen limitation as reported for a PDHC-deficient E. coli strain overproducing pyruvate [28]. In our study, the carbon balance indicated the presence of a significant portion of unaccounted carbon in the fermentations of V. natriegens Δvnp12 ΔaceE, with and without acetate feed (see Fig. 2C). We did not observe typical pyruvate-derived side products such as lactate, alanine or valine, but identified parapyruvate as major carbon sink during pyruvate production in the bioreactor which accounted for 7% of the overall carbon. It was shown that parapyruvate can disrupt mitochondrial activity by inhibiting the α-ketoglutarate dehydrogenase complex [37, 71, 72]. Parapyruvate formation in the bioreactor might also impact the metabolism of V. natriegens, and could reasonably explain the growth arrest in the presence of acetate (see Fig. 2B) or the decreasing biomass concentration in the stationary phase (see Fig. 2A, B). To reduce parapyruvate formation, the aldolase LigK was inactivated, as the enzyme was shown to catalyze parapyruvate degradation and synthesis in vitro [64]. The deletion of ligK in V. natriegens Δvnp12 ΔaceE did not affect biomass, pyruvate or parapyruvate formation. However, the overexpression of ligK had a negative impact on growth and increased parapyruvate concentrations in the supernatant (see Figs. 3 and 4). Therefore, it is likely that the LigK enzyme is not expressed in V. natriegens Δvnp12 ΔaceE under the applied process conditions. LigK is part of the PCA 4,5-cleavage pathway and transcription is activated in other organisms by the regulator LigR in the presence of PCA or gallate [73]. A LigR homolog (PN96_18485) is also present in the genome of V. natriegens. These findings indicate potential challenges to use microorganisms harboring the PCA 4,5-cleavage pathway for pyruvate production from lignin-derived aromatic compounds because of potential LigK expression and increased parapyruvate formation.

Enzymatic parapyruvate formation by an unknown aldolase cannot be ruled out completely, but no other enzymes were identified with similarity to LigK in V. natriegens using BLAST [74]. It is unlikely that there is another aldolase catalyzing parapyruvate formation, since no growth on PCA was observed with V. natriegens Δvnp12 ΔaceE ΔligK. In addition, cell-free experiments showed that parapyruvate is formed chemically in the bioreactor. Pyruvate can be converted to parapyruvate under various conditions, including a basic environment [38, 39] or by various inorganic catalysts such as ammonia [75] and cations [76, 77]. Parapyruvate was also found in fermentations with Alcanivorax borkumensis in minimal medium using pyruvate as sole carbon source [78] and during chemical production of pyruvate from H2 and CO2 using Ni3Fe particles [79]. As we also found parapyruvate in C. glutamicum cultures producing pyruvate, it appears to be a general by-product in microbial fermentations with high pyruvate concentrations. Notably, although several microbial systems have been engineered for pyruvate production, no study has reached the maximal theoretical product yield [3] indicating a general unaccounted byproduct such as parapyruvate. Parapyruvate can polymerize [38] and lactonize into zymonic acid [75] and different tautomers and hydrates [80] further complicating precise quantification of all aldol condensation products of pyruvic acid present in fermentation samples. Accordingly, after increasing the pH in the cell-free experiments not only the pyruvate but also the parapyruvate concentration decreased rapidly (see Fig. 5A). Bioreactors can have spatial heterogeneities [81] and our setup includes titration by dropping 25% ammonia solution from the top into the culture broth. This procedure could potentially lead to short term microenvironments with higher pH, fostering the aforementioned effects. Possibly, parapyruvate could be broken down to pyruvate in downstream processing by applying heat [82] but it could also decarboxylate to methylsuccinate [36]. The feasibility of downstream processing requires further research, but still neglects negative effects of parapyruvate on the cells during the process, when it is formed. Therefore, product recovery strategies [83] or fermentations at lower pH might inherently be better suited for pyruvate production, such as yeast processes at pH 4.5–5.5 [21,22,23].

Conclusion

In this study, we expand the current research on pyruvate production with V. natriegens highlighting the potential of low-biomass fermentations with this chassis due to its outstanding substrate uptake rates. Major byproduct formation of parapyruvate was discovered to take place during pyruvate production with V. natriegens and other bacteria. Overexpression of the HMG/CHA aldolase (LigK) increased parapyruvate production from pyruvate during bioreactor experiments. However, our results indicate that parapyruvate formation mainly occurs chemically in a pH-dependent fashion and parapyruvate and its derivatives might therefore be present in most biotechnological pyruvate processes.

Materials and methods

Microorganisms and cultivation conditions

The bacterial strains and plasmids used in this study are listed in Table 3. For cloning purposes, E. coli was cultivated in 2xYT [84] and V. natriegens in modified LBv2 medium [57]. Unless otherwise specified, strains were incubated at 37 °C, with liquid cultures shaken at 180 rpm (Ø 25 mm, Multitron®2; INFORS GmbH, Bottmingen, Switzerland). Solid media for plates were prepared by adding 15 g agar L− 1. Strains were stored at -80 °C as 30% (v v− 1) glycerol stocks from a grown liquid culture.

Table 3 Strains and plasmids used in this study
Full size table

For cultivations, V. natriegens glycerol stocks were streaked out on LBv2 agar plates. Single colonies were incubated overnight in test tubes containing 5 mL LBv2. Precultures in 500 mL baffled shaking flasks containing 50 mL VN medium at pH 7.5 [48] supplemented with 7.5 g glucose L− 1, were inoculated with 500 µL of the overnight culture. Cells were harvested after 3 to 4 h and used to inoculate main cultures. Shaking flask main cultures were inoculated with an OD600 of 0.1 and contained VN medium with 7.5 g glucose L− 1. VN medium for cultivations with aceE-deficient V. natriegens strains were additionally supplemented with 1 g acetate L− 1. Growth experiments with PCA were prepared accordingly with 10 mM PCA as sole carbon source in the main culture. Reactor cultivations were performed in a DASGIP Parallel Bioreactor System (Eppendorf, Jülich, Germany) with a vessel volume of 2 L using 0.5 L VN medium without MOPS, supplemented with 100 g glucose L− 1 and 2 g acetate L− 1 and inoculated with a starting OD600 of 0.5. The pH was maintained at 7.5 using a two-sided pH regulation using 13.3 M NH4OH and 1.1 M H3PO4. If specified, a constant acetate feed of 0.24 gAc h− 1 was applied. Reactors were aerated with pressurized air at 1 vvm and the agitation rate was adjusted between 400 and 1500 rpm to keep the dissolved oxygen (DO) above 50%. Cell-free experiments were performed in the bioreactor using the same settings except neither biomass nor acetate feed was added, and instead, a pyruvic acid feed of 7.5 g L− 1 h− 1 was applied. Where appropriate, media were supplemented with kanamycin (E. coli 50 µg mL− 1; V. natriegens 200 µg mL− 1) and chloramphenicol (E. coli 15 µg mL− 1; V. natriegens 6 µg mL− 1). Gene expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranosid (IPTG).

C. glutamicum was streaked out on a 2xYT plate from glycerol cultures. Single colonies were used to inoculate 5 mL 2xYT with 5 g acetate L− 1 in test tubes and cultivated over the day at 30 °C and 120 rpm. Precultures in 500 mL baffled shaking flasks containing 50 mL 2xYT supplemented with 5 g acetate L− 1 were inoculated with the whole culture of the test tube and cultivated over night at 30 °C and 120 rpm. Main cultures at 30 °C and 120 rpm were inoculated to an OD600 of 1 in CGXII medium [85] containing 40 g glucose L− 1, 10 g acetate L− 1 and 2 mM alanine.

Strain construction

Genome reference and gene identifiers are used from GenBank assembly accession GCA_001456255.1 [91]. Oligonucleotides (Table S1) were purchased from either Sigma-Aldrich Chemie GmbH (Steinheim, Germany) or Eurofins MWG Operon (Ebersberg, Germany). Enzymes were purchased from New England Biolabs GmbH (Frankfurt am Main, Germany) and handled according to the manufacturer’s recommendations. The kits NucleoSpin® Microbial DNA, NucleoSpin® Plasmid and NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) were used for the isolation and purification of genomic DNA, plasmid DNA and PCR fragments according to the manufacturer’s protocols. Electro-competent E. coli and V. natriegens cells were prepared as described [61, 84]. Sequencing was performed by Microsynth Seqlab GmbH (Göttingen, Germany).

Construction of pDM4-ΔaceE (primers #1/#2, #3/#4 and #5/#6), pST_116-ΔligK (primers #9/#10 and #13/#14), and pST_116-Δdns (primers #11/#12 and #13/#14) and subsequent deletion of aceE (primers #7/#8) and ligK (primers #15/#16, #17/#18 and #19/#20) was performed as previously described [57]. For the katG integration, first, the vector pJH001 containing 3 kb sequences up- and downstream of dns gene flanking a dropout sfgfp was constructed. As parts for pJH001, ColE1 origin of replication and sfgfp were amplified from pST_116 using primer pairs #33/#34 and #29/#30, respectively. Carbenecillin resistance gene was amplified from pET-22b(+) using primers pairs #21/#22 and #23/#24, removing a BsaI recognition site by silent mutation. dns flanking sequences were amplified from V. natriegens’ genomic DNA with primer pair #31/#32 for the upstream region and primer pairs #25/#26 and #27/#28 for the downstream region, removing an Esp3I recognition site. All primers added Esp3I recognition sites with matching overhangs and the plasmid was cloned by Golden Gate assembly using Esp3I as described [92]. pJH001- katGEc-1xFLAG was constructed by amplifying the backbone from pJH001 including the sfgfp promotor and terminator with primer pair #35/#36 and katG was amplified from E. coli DH5α using primer pair #37/#38 introducing a 1xFLAG tag. The plasmid was cloned by Golden Gate assembly as described before. The transfer DNA (tDNA) was amplified using primer pair #39/#40 und subsequent integration via NT-CRISPR was performed as described before [63]. Integration was verified using primer pair #41/#42. Functional catalase activity was confirmed by adding 30% H2O2 to cell material and checking for visible oxygen formation.

For the overexpression of ligK, plasmid pEKEx2 was linearized with BamHI and EcoRI. The coding sequence of ligK was amplified from V. natriegens genomic DNA using primer pair #43/#44. Then, the fragment was assembled with the linearized plasmid pEKEx2 by Gibson Assembly [93]. Correct assemblies were screened in transformed E. coli by colony PCR with primer pair #45/#46 and subsequentially confirmed via Sanger sequencing before being introduced into V. natriegens by electroporation.

Analytical methods

The biomass concentration was monitored by measuring the optical density (OD) at 600 nm with a spectrophotometer (Ultrospec® 10, Biochrom, Holliston, MA, USA). OD values were converted into biomass concentrations applying a correlation factor of 0.28 gCDW L− 1 per OD. Growth rates were calculated by fitting a linear regression line to the exponential growth phase in a semi-logarithmic plot and maximizing the coefficient of determination (R2). Determination of sugars and organic acids was performed by high-performance liquid chromatography (HPLC) with an Agilent 1260 infinity II series device (Agilent Technologies, Waldbronn, Germany) using a Hi-Plex H column (7.7 × 300 mm, 8 μm) and Hi-Plex Hguard cartridge (3.0 × 5.0 mm, 8 μm) as previously described [94]. Parapyruvate was purchased from Sigma-Aldrich with a stated quality of 95-105% by titration with HClO4.

For extraction of intracellular metabolites, strains were grown to an OD600 of 5 in shaking flasks. Cells were harvested from 2 mL culture by centrifugation (30 s, 20000 × g, 4 °C). The supernatant was removed, and cells were washed with 1 mL ice-cold 0.9% NaCl. After another centrifugation (30 s, 20000 × g, 4 °C) the supernatant was discarded and the cells were quenched in liquid nitrogen. Subsequently, cells were thawed on ice and resuspended in 0.5 mL ice-cold methanol. The suspension was vortexed and frozen in liquid nitrogen, before being incubated at -20 °C to thaw, vortexed and frozen in liquid nitrogen again. This was repeated until the cells incubated four times at -20 °C. The suspension was then centrifuged (10 min, 20000 × g, 4 °C) and supernatant stored at -80 °C until further analysis.

Sample preparation for the LC/Q-TOF-MS was performed as described [95]. 20 µL sample, either extracts of intracellular metabolites or culture supernatant, was mixed with 1 µL 1 M ammonium acetate (pH 9.2), 4 µL α-aminobutyrate (internal standard) and 15 µL Milli-Q H2O by vortexing. Subsequently, 60 µL acetonitrile was added, samples were vortexed and chilled on ice for 10 min before being centrifuged (10 min, 20000 × g, 4 °C). 90 µL supernatant were transferred to glass vials for measurement.

LC/Q-TOF-MS analysis was performed as described before [96] using a 1290 series UHPLC and 6546 LC/Q-TOF (Agilent Technologies, Waldbronn, Germany) equipped with a InfinityLab Poroshell 120 HILIC-Z (150 × 2.1 mm, 2.7 μm particle size). As eluent A, 1 M ammonium acetate was diluted in Milli-Q H2O (pH 9.2) and mixed with acetonitrile 1:9 (v/v) to a final concentration of 10 mM. Eluent B was 1 M ammonium acetate diluted in Milli-Q H2O (pH 9.2) and mixed with acetonitrile 9:1 (v/v) to a final concentration of 10 mM. MassHunter LC/MS Data Acquisition (v10.1), Qualitative Analysis software (v10.0), Masshunter Profnder (v10.0) and Mass Profiler Professional from Agilent Technologies were used for data acquisition and evaluation, respectively. Identity of pyruvate (Rt = 5.6 min, m/z: 87.009) was confirmed with an authentic standard.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Maleki N, Eiteman M. Recent progress in the microbial production of pyruvic acid. Fermentation. 2017;3:8. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/fermentation3010008.

    Article  CAS  Google Scholar 

  2. Luo Q, Ding N, Liu Y, Zhang H, Fang Y, Yin L. Metabolic engineering of microorganisms to produce pyruvate and derived compounds. Molecules. 2023;28:1418. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/molecules28031418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yuan W, Du Y, Yu K, Xu S, Liu M, Wang S, et al. The production of pyruvate in biological technology: a critical review. Microorganisms. 2022;10:2454. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/microorganisms10122454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cubas-Cano E, González-Fernández C, Ballesteros M, Tomás-Pejó E. Biotechnological advances in lactic acid production by lactic acid bacteria: lignocellulose as novel substrate. Biofuels Bioprod Bioref. 2018;12:290–303. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/bbb.1852.

    Article  CAS  Google Scholar 

  5. Reiße S, Haack M, Garbe D, Sommer B, Steffler F, Carsten J, et al. in vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization. Front Bioeng Biotechnol. 2016;4:74. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fbioe.2016.00074

    Article  PubMed  PubMed Central  Google Scholar 

  6. Blombach B, Eikmanns BJ. Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. Bioeng Bugs. 2011;2:346–50. https://doiorg.publicaciones.saludcastillayleon.es/10.4161/bbug.2.6.17845

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lee JW, Lee Y-G, Jin Y-S, Rao CV. Metabolic engineering of non-pathogenic microorganisms for 2,3-butanediol production. Appl Microbiol Biotechnol. 2021;105:5751–67. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00253-021-11436-2.

    Article  CAS  PubMed  Google Scholar 

  8. Sharma A, Noda M, Sugiyama M, Kaur B, Ahmad A. Metabolic engineering of Pediococcus acidilactici BD16 for heterologous expression of synthetic AlaD gene cassette and ʟ-alanine production in the recombinant strain using fed-batch fermentation. Foods. 2021;10:1964. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/foods10081964

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Oldiges M, Eikmanns BJ, Blombach B. Application of metabolic engineering for the biotechnological production of ʟ-valine. Appl Microbiol Biotechnol. 2014;98:5859–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00253-014-5782-8.

    Article  CAS  PubMed  Google Scholar 

  10. Zhao M, Zhu Y, Wang H, Zhang W, Mu W. Recent advances on N-acetylneuraminic acid: physiological roles, applications, and biosynthesis. Synth Syst Biotechnol. 2023;8:509–19. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.synbio.2023.06.009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kumagai H, Katayama T, Koyanagi T, Suzuki H. Research overview of ʟ-DOPA production using a bacterial enzyme, tyrosine phenol-lyase. Proc Jpn Acad Ser B Phys Biol Sci. 2023;99:75–101. https://doiorg.publicaciones.saludcastillayleon.es/10.2183/pjab.99.006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang X, Tervo CJ, Reed JL. Metabolic assessment of E. coli as a biofactory for commercial products. Metab Eng. 2016;35:64–74. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ymben.2016.01.007.

    Article  CAS  PubMed  Google Scholar 

  13. Zilberter Y, Gubkina O, Ivanov AI. A unique array of neuroprotective effects of pyruvate in neuropathology. Front Neurosci. 2015;9:17. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fnins.2015.00017.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Olek RA, Kujach S, Radak Z. Current knowledge about pyruvate supplementation: a brief review. Sports Med Health Sci. 2024;6:295–301. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.smhs.2024.02.007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jäger R, Metzger J, Lautmann K, Shushakov V, Purpura M, Geiss K-R, Maassen N. The effects of creatine pyruvate and creatine citrate on performance during high intensity exercise. J Int Soc Sports Nutr. 2008;5:4. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1550-2783-5-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Howard JW, Fraser WA. Preparation of pyruvic acid. Org Synth Coll. 1932;1:475–80.

    Google Scholar 

  17. Li Y, Chen J, Lun SY. Biotechnological production of pyruvic acid. Appl Microbiol Biotechnol. 2001;57:451–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s002530100804.

    Article  CAS  PubMed  Google Scholar 

  18. Yokota A, Shimizu H, Terasawa Y, Takaoka N, Tomita F. Pyruvic acid production by a lipoic acid auxotroph of Escherichia coli W1485. Appl Microbiol Biotechnol. 1994;41:638–43. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s002530050192.

    Article  CAS  Google Scholar 

  19. Wieschalka S, Blombach B, Eikmanns BJ. Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol. 2012;94:449–59. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00253-011-3843-9.

    Article  CAS  PubMed  Google Scholar 

  20. Suo F, Liu J, Chen J, Li X, Solem C, Jensen PR. Efficient production of pyruvate using metabolically engineered Lactococcus lactis. Front Bioeng Biotechnol. 2020;8:611701. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fbioe.2020.611701.

    Article  PubMed  Google Scholar 

  21. van Maris AJA, Geertman J-MA, Vermeulen A, Groothuizen MK, Winkler AA, Piper MDW, et al. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol. 2004;70:159–66. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.70.1.159-166.2004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Morgunov IG, Kamzolova SV, Perevoznikova OA, Shishkanova NV, Finogenova TV. Pyruvic acid production by a thiamine auxotroph of Yarrowia lipolytica. Process Biochem. 2004;39:1469–74. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S0032-9592(03)00259-0.

    Article  CAS  Google Scholar 

  23. Yonehara T, Miyata R. Fermentative production of pyruvate from glucose by Torulopsis glabrata. J Ferment Bioeng. 1994;78:155–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0922-338X(94)90255-0.

    Article  CAS  Google Scholar 

  24. Eikmanns BJ, Blombach B. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering. J Biotechnol 2014;192 Pt B:339–45. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jbiotec.2013.12.019

  25. Blombach B, Schreiner ME, Holátko J, Bartek T, Oldiges M, Eikmanns BJ. ʟ-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol. 2007;73:2079–84. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.02826-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tomar A, Eiteman MA, Altman E. The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli. Appl Microbiol Biotechnol. 2003;62:76–82. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00253-003-1234-6.

    Article  CAS  PubMed  Google Scholar 

  27. Zelić B, Gerharz T, Bott M, Vasić-Rački Đ, Wandrey C, Takors R. Fed-batch process for pyruvate production by recombinant Escherichia coli YYC202 strain. Eng Life Sci. 2003;3:299–305. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/elsc.200301756

    Article  CAS  Google Scholar 

  28. Zhu Y, Eiteman MA, Altman R, Altman E. High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain. Appl Environ Microbiol. 2008;74:6649–55. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.01610-08.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nakashima N, Ohno S, Yoshikawa K, Shimizu H, Tamura T. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli. Appl Environ Microbiol. 2014;80:564–73. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.02376-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ziegler M, Hägele L, Gäbele T, Takors R. CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy. Eng Life Sci. 2022;22:70–84. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/elsc.202100021.

    Article  CAS  PubMed  Google Scholar 

  31. Moxley WC, Eiteman MA. Pyruvate production by Escherichia coli by use of pyruvate dehydrogenase variants. Appl Environ Microbiol. 2021;87:e0048721. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.00487-21.

    Article  PubMed  Google Scholar 

  32. Akita H, Nakashima N, Hoshino T. Pyruvate production using engineered Escherichia coli. AMB Express. 2016;6:1–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13568-016-0259-z.

    Article  CAS  Google Scholar 

  33. Wu F, Wang S, Peng Y, Guo Y, Wang Q. Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production. Microb Cell Fact. 2023;22:172. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-023-02185-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jensen PR, Michelsen O. Carbon and energy metabolism of atp mutants of Escherichia coli. J Bacteriol. 1992;174:7635–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/jb.174.23.7635-7641.1992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhou J, Huang L, Liu L, Chen J. Enhancement of pyruvate productivity by inducible expression of a F0F1-ATPase inhibitor INH1 in Torulopsis glabrata CCTCC M202019. J Biotechnol. 2009;144:120–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jbiotec.2009.09.005.

    Article  CAS  PubMed  Google Scholar 

  36. Rios AC, Bera PP, Moreno JA, Cooper G. Pyruvate aldol condensation product: a metabolite that escaped synthetic preparation for over a century. ACS Omega. 2020;5:15063–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acsomega.0c00877

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chang S-C, Lee I, Ting H, Chang Y-J, Yang N-C. Parapyruvate, an impurity in pyruvate supplements, induces senescence in human fibroblastic Hs68 cells via inhibition of the α-ketoglutarate dehydrogenase complex. J Agric Food Chem. 2018;66:7504–13. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acs.jafc.8b01138

    Article  CAS  PubMed  Google Scholar 

  38. Margolis SA, Coxon B. Identification and quantitation of the impurities in sodium pyruvate. Anal Chem. 1986;58:2504–10. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/ac00125a033.

    Article  CAS  Google Scholar 

  39. Lee I, Yang N-C. Using Taguchi method to determine the optimum conditions for synthesizing parapyruvate. Molecules. 2022;27:1840. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/molecules27061870.

    Article  CAS  Google Scholar 

  40. Tack BF, Chapman PJ, Dagley S. Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase. J Biol Chem. 1972;247:6444–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/s0021-9258(19)44712-1.

    Article  CAS  PubMed  Google Scholar 

  41. Shannon LM, Marcus A. γ-Methyl-γ-hydroxy-α-ketoglutaric aldolase. I. Purification and properties. J Biol Chem. 1962;237:3342–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S0021-9258(19)70819-9.

    Article  CAS  PubMed  Google Scholar 

  42. Kamimura N, Takahashi K, Mori K, Araki T, Fujita M, Higuchi Y, Masai E. Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism. Environ Microbiol Rep. 2017;9:679–705. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1758-2229.12597.

    Article  CAS  PubMed  Google Scholar 

  43. Rinke Dias de Souza N, Groenestege M, Spekreijse J, Ribeiro C, Matos CT, Pizzol M, Cherubini F. Challenges and opportunities toward a sustainable bio-based chemical sector in Europe. WIREs Energy Environ. 2024;13:e534. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/wene.534.

    Article  CAS  Google Scholar 

  44. Straathof AJJ. The proportion of downstream costs in fermentative production processes. In: Moo-Young M, editor. Comprehensive Biotechnology: Elsevier; 2011. pp. 811–814. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/B978-0-08-088504-9.00492-X

  45. Manandhar A, Shah A. Techno-economic analysis of bio-based lactic acid production utilizing corn grain as feedstock. Processes. 2020;8:199. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pr8020199.

    Article  Google Scholar 

  46. Konzock O, Nielsen J. TRYing to evaluate production costs in microbial biotechnology. Trends Biotechnol. 2024;42:1339–47. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tibtech.2024.04.007.

    Article  CAS  PubMed  Google Scholar 

  47. Forsten E, Gerdes S, Petri R, Büchs J, Magnus J. Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes. BMC Biotechnol. 2024;24:63. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12896-024-00897-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hoffart E, Grenz S, Lange J, Nitschel R, Müller F, Schwentner A, et al. High substrate uptake rates empower Vibrio natriegens as production host for industrial biotechnology. Appl Environ Microbiol. 2017;83:e01614–17. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.01614-17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Thoma F, Blombach B. Metabolic engineering of Vibrio natriegens. Essays Biochem. 2021;65:381–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1042/EBC20200135.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Hädrich M, Schulze C, Hoff J, Blombach B. Vibrio natriegens: application of a fast-growing halophilic bacterium. Advances in biochemical engineering/biotechnology. Berlin, Heidelberg: Springer; 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/10_2024_271.

    Chapter  Google Scholar 

  51. Blombach B, Grünberger A, Centler F, Wierckx N, Schmid J. Exploiting unconventional prokaryotic hosts for industrial biotechnology. Trends Biotechnol. 2022;40:385–97. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tibtech.2021.08.003.

    Article  CAS  PubMed  Google Scholar 

  52. Hoff J, Daniel B, Stukenberg D, Thuronyi BW, Waldminghaus T, Fritz G. Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis. Environ Microbiol. 2020;22:4394–408. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1462-2920.15128.

    Article  PubMed  Google Scholar 

  53. Hong C, Kim Y, Lee H, Yun S, Lim HG, Yang J, Jang S. Vibrio species as next-generation chassis for accelerated synthetic biology. Biotechnol Bioproc E. 2024;29:241–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12257-024-00023-7.

    Article  CAS  Google Scholar 

  54. Erian AM, Freitag P, Gibisch M, Pflügl S. High rate 2,3-butanediol production with Vibrio natriegens. Bioresource Technol Rep. 2020;10:100408. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biteb.2020.100408.

    Article  Google Scholar 

  55. Meng W, Zhang Y, Ma L, Lü C, Xu P, Ma C, Gao C. Non-sterilized fermentation of 2,3-butanediol with seawater by metabolic engineered fast-growing Vibrio natriegens. Front Bioeng Biotechnol. 2022;10:955097. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fbioe.2022.955097.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Thoma F, Schulze C, Gutierrez-Coto C, Hädrich M, Huber J, Gunkel C, et al. Metabolic engineering of Vibrio natriegens for anaerobic succinate production. Microb Biotechnol. 2022;15:1671–84. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1751-7915.13983.

    Article  CAS  PubMed  Google Scholar 

  57. Schulze C, Hädrich M, Borger J, Rühmann B, Döring M, Sieber V, et al. Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens. Microb Biotechnol. 2024;17:e14277. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1751-7915.14277.

    Article  CAS  PubMed  Google Scholar 

  58. Liu X, Han X, Peng Y, Tan C, Wang J, Xue H, et al. Rapid production of ʟ-DOPA by Vibrio natriegens, an emerging next-generation whole-cell catalysis chassis. Microb Biotechnol. 2022;15:1610–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1751-7915.14001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Pfeifer E, Michniewski S, Gätgens C, Münch E, Müller F, Polen T, et al. Generation of a prophage-free variant of the fast-growing bacterium Vibrio natriegens. Appl Environ Microbiol. 2019;85:e00853–19. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.00853-19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Linton JD, Harrison DE, Bull AT. Molar growth yields, respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a Chemostat. Arch Microbiol. 1977;115:135–42. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/BF00406366.

    Article  CAS  PubMed  Google Scholar 

  61. Weinstock MT, Hesek ED, Wilson CM, Gibson DG. Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods. 2016;13:849–51. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nmeth.3970.

    Article  CAS  PubMed  Google Scholar 

  62. Dalia TN, Hayes CA, Stolyar S, Marx CJ, McKinlay JB, Dalia AB. Multiplex genome editing by natural transformation (MuGENT) for synthetic biology in Vibrio natriegens. ACS Synth Biol. 2017;6:1650–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acssynbio.7b00116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Stukenberg D, Hoff J, Faber A, Becker A. NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens. Commun Biol. 2022;5:265. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s42003-022-03150-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Maruyama K. Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate. J Biochem. 1990;108:327–33. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/oxfordjournals.jbchem.a123201.

    Article  CAS  PubMed  Google Scholar 

  65. Moreno R, Yuste L, Morales G, Rojo F. Inactivation of Pseudomonas Putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds. Microb Biotechnol. 2024;17:e14514. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1751-7915.14514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Schreiner ME, Fiur D, Holátko J, Pátek M, Eikmanns BJ. E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: molecular analysis of the gene and phylogenetic aspects. J Bacteriol. 2005;187:6005–18. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/jb.187.17.6005-6018.2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Jeyaseelan K, Guest JR. Isolation and properties of pyruvate dehydrogenase complex mutants of Pseudomonas aeruginosa PAO. J Gen Microbiol. 1980;120:385–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1099/00221287-120-2-385.

    Article  CAS  PubMed  Google Scholar 

  68. Langley D, Guest JR. Biochemical and genetic characterics of deletion and other mutant strains of Salmonella typhimurium LT2 lacking alpha-keto acid dehydrogenase complex activities. J Gen Microbiol. 1974;82:319–35. https://doiorg.publicaciones.saludcastillayleon.es/10.1099/00221287-82-2-319.

    Article  CAS  PubMed  Google Scholar 

  69. Lange J, Takors R, Blombach B. Zero-growth bioprocesses: a challenge for microbial production strains and bioprocess engineering. Eng Life Sci. 2017;17:27–35. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/elsc.201600108.

    Article  CAS  PubMed  Google Scholar 

  70. Liu L, Xu Q, Li Y, Shi Z, Zhu Y, Du G, Chen J. Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata. Biotechnol Bioeng. 2007;97:825–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/bit.21290.

    Article  CAS  PubMed  Google Scholar 

  71. Montgomery CM, Webb JL. Metabolic studies on heart mitochondria. II. The inhibitory action of parapyruvate on the tricarboxylic acid cycle. J Biol Chem. 1956;221:359–68. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S0021-9258(18)65255-X.

    Article  CAS  PubMed  Google Scholar 

  72. Lee I, Song T-Y, Chen C-L, Yang J-J, Yang N-C. Parapyruvate induces neurodegeneration in C57BL/6JNarl mice via inhibition of the α-ketoglutarate dehydrogenase complex. ACS Omega. 2024;9:5919–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acsomega.3c09469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kamimura N, Takamura K, Hara H, Kasai D, Natsume R, Senda T, et al. Regulatory system of the protocatechuate 4,5-cleavage pathway genes essential for lignin downstream catabolism. J Bacteriol. 2010;192:3394–405. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/jb.00215-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/25.17.3389.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Wolff L. II. Ueber die Parabrenztraubensäure. Justus Liebigs Ann Chem. 1899;305:154–65. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jlac.18993050203

    Article  CAS  Google Scholar 

  76. Gallo AA, Sable HZ. Rate enhancement of pyruvate aldolization by divalent cations: a model for class II aldolases. Biochim Biophys Acta. 1973;302:443–56. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0005-2744(73)90173-3.

    Article  CAS  PubMed  Google Scholar 

  77. Tallman DE, Leussing DL. Pyruvate dimerization catalyzed by nickel(II) and zinc(II). II. Kinetics. J Am Chem Soc. 1969;91:6256–62. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/ja01051a012.

    Article  CAS  Google Scholar 

  78. Karmainski T, Dielentheis-Frenken MRE, Lipa MK, Phan ANT, Blank LM, Tiso T. High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation. Front Bioeng Biotechnol. 2023;11:1325019. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fbioe.2023.1325019.

    Article  PubMed  PubMed Central  Google Scholar 

  79. Beyazay T, Belthle KS, Farès C, Preiner M, Moran J, Martin WF, Tüysüz H. Ambient temperature CO2 fixation to pyruvate and subsequently to citramalate over iron and nickel nanoparticles. Nat Commun. 2023;14:570. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-023-36088-w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Perkins RJ, Shoemaker RK, Carpenter BK, Vaida V. Chemical equilibria and kinetics in aqueous solutions of zymonic acid. J Phys Chem A. 2016;120:10096–107. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acs.jpca.6b10526.

    Article  CAS  PubMed  Google Scholar 

  81. Nadal-Rey G, McClure DD, Kavanagh JM, Cornelissen S, Fletcher DF, Gernaey KV. Understanding gradients in industrial bioreactors. Biotechnol Adv. 2021;46:107660. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biotechadv.2020.107660.

    Article  CAS  PubMed  Google Scholar 

  82. Meyerhof O, Kaplan A. The mechanism of the cyanide inhibition of fermentation. Arch Biochem Biophys. 1952;37:375–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0003-9861(52)90199-9.

    Article  CAS  PubMed  Google Scholar 

  83. Zelić B, Gostović S, Vuorilehto K, Vasić-Racki D, Takors R. Process strategies to enhance pyruvate production with Recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis. Biotechnol Bioeng. 2004;85:638–46. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/bit.10820.

    Article  CAS  PubMed  Google Scholar 

  84. Green MR, Sambrook J. Molecular cloning: A laboratory manual. 4th ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2012.

    Google Scholar 

  85. Thoma F, Appel C, Russ D, Huber J, Werner F, Blombach B. Improving growth properties of Corynebacterium glutamicum by implementing an iron-responsive protocatechuate biosynthesis. Microb Biotechnol. 2023;16:1041–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/1751-7915.14244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Grant SG, Jessee J, Bloom FR, Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990;87:4645–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.87.12.4645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Simon R, Priefer U, Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotechnol. 1983;1:784–91. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nbt1183-784

    Article  CAS  Google Scholar 

  88. Abe S, Takayama K-I, Kinoshita S. Taxonomical studies on glutamic acid-producing bacteria. J Gen Appl Microbiol. 1967;13:279–301. https://doiorg.publicaciones.saludcastillayleon.es/10.2323/jgam.13.279.

    Article  Google Scholar 

  89. Milton DL, O’Toole R, Horstedt P, Wolf-Watz H. Flagellin A is essential for the virulence of Vibrio anguillarum. J Bacteriol. 1996;178:1310–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/jb.178.5.1310-1319.1996.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke KU, Sahm H. Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiol (Reading Engl). 1994;140:1817–28. https://doiorg.publicaciones.saludcastillayleon.es/10.1099/13500872-140-8-1817

    Article  CAS  Google Scholar 

  91. Lee HH, Ostrov N, Wong BG, Gold MA, Khalil AS, Church GM. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. Nat Microbiol. 2019;4:1105–13. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41564-019-0423-8.

    Article  CAS  PubMed  Google Scholar 

  92. Stukenberg D, Hensel T, Hoff J, Daniel B, Inckemann R, Tedeschi JN, et al. The Marburg collection: a golden gate DNA assembly framework for synthetic biology applications in Vibrio natriegens. ACS Synth Biol. 2021;10:1904–19. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acssynbio.1c00126.

    Article  CAS  PubMed  Google Scholar 

  93. Gibson DG. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 2011;498:349–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/B978-0-12-385120-8.00015-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Siebert D, Altenbuchner J, Blombach B. A timed off-switch for dynamic control of gene expression in Corynebacterium glutamicum. Front Bioeng Biotechnol. 2021;9:704681. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fbioe.2021.704681.

    Article  PubMed  PubMed Central  Google Scholar 

  95. Teleki A, Takors R. Quantitative profiling of endogenous metabolites using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Methods Mol Biol. 2019;1859:185–207. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/978-1-4939-8757-3_10.

    Article  CAS  PubMed  Google Scholar 

  96. Siebert D, Glawischnig E, Wirth M-T, Vannahme M, Salazar-Quirós Á, Weiske A, et al. A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production. Microb Cell Fact. 2024;23:62. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02337-w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Open Access funding enabled and organized by Projekt DEAL.

This work was supported by the BL1408/2 − 1 grant (Deutsche Forschungsgemeinschaft) and by the TUM WACKER Institute for Industrial Biotechnology.

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  1. Microbial Biotechnology, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Straubing, Germany

    Maurice Hädrich, Clarissa Scheuchenegger, Sören-Tobias Vital, Christoph Gunkel, Susanne Müller, Josef Hoff, Jennifer Borger, Erich Glawischnig, Felix Thoma & Bastian Blombach

  2. SynBiofoundry@TUM, Technical University of Munich, Straubing, Germany

    Maurice Hädrich, Christoph Gunkel, Josef Hoff & Bastian Blombach

  3. Munich Institute of Integrated Materials, Energy and Process Engineering, Technical University of Munich, Garching, Germany

    Maurice Hädrich, Josef Hoff & Bastian Blombach

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  1. Maurice Hädrich
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BB acquired funding of the project. MH, CS, FT and BB conceived and designed the experiments. MH, CS, CG, SV, SM, JH, JB and EG performed the experiments and analyzed the data. MH and BB drafted the manuscript. All authors reviewed and approved the final manuscript.

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Hädrich, M., Scheuchenegger, C., Vital, ST. et al. Low-biomass pyruvate production with engineered Vibrio natriegens is accompanied by parapyruvate formation. Microb Cell Fact 24, 73 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02693-1

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Microbial Cell Factories

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