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Enhancing D-lactic acid production from methane through metabolic engineering of Methylomonas sp. DH-1

Microbial Cell Factories volume 24, Article number: 70 (2025) Cite this article

Abstract

Background

Methane is an abundant and low-cost carbon source with great potential for conversion into value-added chemicals. Methanotrophs, microorganisms that utilize methane as their sole carbon and energy source, present a promising platform for biotechnological applications. This study aimed to engineer Methylomonas sp. DH-1 to enhance D-LA production through metabolic pathway optimization during large-scale cultivation.

Results

In this study, we regulated the expression of D-lactate dehydrogenase (D-LDH) using a Ptac promoter with IPTG induction to mitigate the toxic effects of lactate accumulation. To further optimize carbon flow away from glycogen, the glgA gene was deleted. However, this modification led to growth inhibition, especially during scale-up, likely due to the accumulation of ADP-glucose caused by the rewired carbon flux under carbon-excess conditions. Deleting the glgC gene, which encodes glucose 1-phosphate adenylyltransferase, alleviated this issue. The final optimized strain, JHM805, achieved a D-LA production of 6.17 g/L in a 5-L bioreactor, with a productivity of 0.057 g/L/h, marking a significant improvement in D-LA production from methane.

Conclusions

The metabolic engineering strategies employed in this study, including the use of an inducible promoter and alleviation of ADP-glucose accumulation toxicity, successfully enhanced the ability of the strain to produce D-LA from methane. Furthermore, optimizing the bioreactor fermentation process through methane and nitrate supplementation resulted in a significant increase in both the titer and productivity, exceeding previously reported values.

Background

Methane, a cost-effective and abundant carbon feedstock, is readily available for various natural and industrial processes. Advanced technologies, such as hydraulic fracturing and horizontal drilling, have improved methane extraction from shale gas, enhancing its use as a renewable feedstock for producing various value-added chemicals. [1,2,3,4]. Additionally, as the main component of biogas, methane can be sustainably derived from biomass, agricultural waste, and other organic materials, offering an eco-friendly option for biochemical and industrial applications [5, 6]. Given methane’s higher greenhouse gas potential compared to CO2, its utilization can also contribute to mitigating global warming.

The biological conversion of methane offers several advantages, including operation under mild conditions and absence of toxic by-products. Methanotrophic bacteria are promising biocatalysts because they utilize methane as their sole energy and carbon source. Significant efforts have been made to develop efficient genetic manipulation tools for these organisms. These tools include conjugation and electroporation methods for gene deletion and foreign DNA introduction [7,8,9], and negative selection systems such as sacB and mutant pheS counter-selection methods for multiple gene deletions [10, 11]. Additionally, marker-free chromosomal editing using the CRISPR/Cas9 system has been successfully implemented in both type I methanotroph Methylococcus capsulatus Bath and type II methanotroph Methylocystis parvus OBBP [12].

With the development of efficient genetic manipulation tools, methanotrophs have been increasingly explored in recent years to produce valuable chemicals from methane [13]. The chemicals produced from methane range from simple low-carbon compounds, such as methanol and acetate [14,15,16] to value-added medium-carbon chemicals, such as ectoine and cadaverine [17, 18]. More complex substances, such as terpenoids, fatty acids [19, 20], polyhydroxyalkanoates (PHA), and biodegradable thermoplastic polyesters [21,22,23,24], can also be produced. However, the titers of these products remain low compared to the results of conventional sugar fermentation through metabolic engineering of model microorganisms, such as Escherichia coli and Saccharomyces cerevisiae. The challenges of producing valuable compounds using engineered methanotrophic bacteria extend beyond the lack of information; they also include the genetic instability of strains when exposed to toxic chemicals. These limitations make the scaling up of bioprocesses for industrial applications particularly challenging. Many studies on fermenter-scale chemical production continue to report low productivity. In particular, few studies have successfully achieved gram-scale production of Methylomonas sp. DH-1 under various culture conditions [25,26,27,28,29], thereby highlighting the limitations of this methanotroph.

Organic acids, such as muconic acid, lactic acid, and 3-hydroxypropionic acid, can be used as building blocks for bioplastics [25, 30, 31]. With growing concerns over the increasing use of petroleum-based plastics, studies on bioplastic production have recently gained significant attention. In particular, poly-lactic acid (PLA), derived from lactic acid (LA), has attracted significant attention as a major bioplastic. Several approaches have been explored to produce LA from methane using type I methanotrophs, but the titer and yield have been limited by the toxicity of lactate within cells. In Methylomicrobium buryatense 5GB1S, the lactate dehydrogenase (LDH) gene was expressed on an episomal plasmid, resulting in 0.8 g/L of LA production in a continuous stirred bioreactor [32]. This titer matched the maximum LA tolerance of the strain, suggesting that intracellular accumulation of LA imposes a strict limitation on the production levels. Subsequent studies in the same strain reported enhancements in LDH expression through the engineering of promoters and ribosome binding sites [33]. Furthermore, in Methylomicrobium alcaliphilum 20zR, genes encoding the pyruvate dehydrogenase complex were deleted to increase pyruvate flux towards LA instead of acetyl-CoA. However, this manipulation failed to improve the titer, indicating that tolerance to LA is the primary bottleneck in LA production [34]. The challenge of LA production stems from the inherent toxicity of weak organic acids in microbial cells. When accumulated intracellularly, LA dissociates into lactate and protons, disrupting cellular homeostasis and normal function. This toxicity makes LA production using methanotrophs highly challenging, emphasizing the need for well-defined strategies to overcome these limitations.

In our previous study, we addressed this problem by performing adaptive laboratory evolution of Methylomonas sp. DH-1, resulting in JHM80, a strain exhibiting tolerance to 8.0 g/L of LA. We expressed the D-specific LDH gene from Leuconostoc mesenteroides (Lm.LDH) using a glgBA promoter while deleting the glgA gene, which encodes glycogen synthase to prevent carbon flux towards glycogen biosynthesis. As a result, this engineered strain produced 1.19 g/L of D-LA [25]. In this study, we used an inducible promoter to regulate the expression of D-LDH in JHM80. Furthermore, we found that deletion of the glgA gene inadvertently led to cell toxicity due to ADP-glucose accumulation. We addressed this growth inhibition by eliminating the glgC gene and optimizing the large-scale fermentation process, ultimately achieving record-high production of 6.17 g/L of D-LA in a 5-L fermenter.

Methods

Strains and culture conditions

All strains used in this study are listed in Table 1. Strains derived from Methylomonas sp. DH-1 were cultured in nitrate mineral salts (NMS) medium (0.49 g/L MgSO4, 1.0 g/L KNO3, 0.23 g/L CaCl2·2H2O, 3.8 mg/L Fe-EDTA, 0.5 mg/L Na2MoO4, 10 μM CuSO4·5H2O, with the addition of 1000X trace element solution, 100X vitamin stock and 100X phosphate stock solution). Cells were grown in 12.5 mL NMS medium in a 125 mL baffled flask or 50 mL NMS medium in a 500 mL baffled flask with rubber-type screw cap supplemented with 20% (v/v) methane and 80% air at 30 °C with shaking at 170 rpm. Proper concentrations of anhydrotetracycline (aTc) or IPTG were added to the medium for induction of D-LDH with 10 μg/mL of kanamycin. All growth and metabolic measurements were performed in biological duplicates unless otherwise specified.

Table 1 Strains used in this study
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Plasmid construction and genetic manipulation of Methylomonas sp. DH-1

Plasmids used in this study to generate strains with chromosomal modifications are listed in Table 2. To construct plasmids for expression of lactate dehydrogenase from L. mesenteroides (Lm.LDH) with inducible promoters, we used pDel2-glgA-Lm.LDH as a parental plasmid which contains Lm.LDH flanked by 1-kb upstream (UglgA) and 1-kb downstream (DglgA) of glgA [25]. To provide restriction enzyme sites for promoter cloning, pDel3-glgA-Lm.LDH that contains MauBI/BamHI sites in front of the Lm.LDH gene was generated by using AccuRapid™ Cloning Kit (Bioneer, Korea). The Ptet and Ptac were cloned between MauBI and BamHI sites, resulting in pDel3-glgA-Ptet-Lm.LDH and pDel3-glgA-Ptac-Lm.LDH respectively. To construct plasmids for gene deletion, previously generated pDel2-fliE plasmid was used [25]. The 1-kb upstream and downstream sequences of target genes were amplified by PCR and cloned into NotI/SpeI and ApaI/SacI sites in pDel2-fliE, respectively.

Table 2 Plasmids used in this study
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Recombinant plasmid DNA was introduced to DH-1 by electroporation and as described before [25]. Competent DH-1 cells were prepared as follows. Cells cultured from NMS plate were harvested with sterilized, ice-cold water and centrifuged at 14,000 rpm for 2 min. Cells were washed twice and resuspended with 200 ~ 300 μL of ice-cold water. 50 μL of cell resuspension and 3 μL of plasmid DNA were mixed gently and transferred to an ice-cold 2-mm-gap cuvette. Electroporation was performed using a Gene Pulser II system (Bio-Rad, USA) at preprogrammed Ec2 setting. Immediately after electrical discharge, 1 mL of room temperature NMS medium was added to cells and transferred to 30 mL serum bottle supplied with additional 2 mL of NMS medium and 20% CH4. After overnight incubation in a shaking incubator, cell pellets were harvested by centrifugation and spread onto NMS plate containing 10 μg/mL of kanamycin or 25 μg/mL of ampicillin.

Fermenter culture

Strain JHM805 was pre-cultured in a 1-L baffled flask containing 200 mL of NMS medium (standard medium with 1 g/L KNO3 or modified medium with 6 g/L KNO3) and 10 µg of kanamycin, supplied with 20% methane and 80% air. The headspace of the flask was purged at 0 h and 24 h during incubation. After incubation in a shaking incubator for 48 h, 200 mL of seed culture was transferred to the bioreactor. Bioreactor fermentation was performed in a 5-L Bioreactor (BioCNS, Daejeon, Republic of Korea) containing 3 L of NMS medium with 10 µg/ml kanamycin and 50 µM IPTG at 30 °C, with an agitation speed of 800 rpm. The gas mixture of 20% methane and 80% air, controlled by a mass flow controller (Brooks Instrument, Hatfield, PA) was supplied using microgas sparger at the rate of 320 mL/min. To maintain pH at the range of 6.9 ~ 7.1, 2 N HCl and 5 N NaOH were used.

Analytical method

Cell growth was analyzed by measuring optical densities at 600 nm. To quantify D-LA, 150 µL of culture supernatant was collected, filtered through 0.22 µm filter, and analyzed via high performance liquid chromatography (HPLC). The separation was performed using a BioRad Aminex HPX-87H column, with 5 mM H2SO4 as the mobile phase at a 0.6 mL/min flow rate. The column temperature was maintained at 60 °C, while the refractive index (RI) detector was set to 35 °C for detection. Consumed methane concentration was analyzed using gas chromatography system (Agilent 7890B, Agilent Corporation, USA) equipped with molecular sieved 5A column and PorapakQ column at 50 °C with argon gas as a carrier gas at a constant pressure of 27 psi. The analytes were detected by thermal conductivity detector (TCD) at 250 °C.

The concentration of nitrate was analyzed by Ion chromatography system (Dionex Aquion, Thermo fischer, USA) equipped with DS6 heated conductivity cell and Dionex Ionpac AS23 RFIC column at 30 °C. 4.5 mM carbonate with 0.8 mM bicarbonate (Dionex AS23 Eluent concentrate, Thermo Fisher, USA) was used as an anion mobile phase.

Results and discussion

Introduction of inducible promoters for fine-tuned expression of D-LDH

In our previous study, we developed the JHM86 strain by integrating the Lm.LDH gene, encoding D-specific LDH (D-LDH), into the chromosome of the LA-tolerant JHM80 strain and simultaneously deleting the glgA gene. This engineered strain, JHM86, which expresses the Lm.LDH gene under the control of the glgBA operon promoter, successfully produced 1.19 g/L of D-LA [25]. To further enhance D-LA production, we substituted the glgBA promoter with the more potent constitutive mxaF promoter. However, this approach failed to produce a viable strain, likely because of severe growth inhibition caused by D-LA toxicity. Based on a previous study [27], several promoters predicted to be weaker than mxaF but stronger than the glgBA promoter were tested to alleviate the growth defect. However, these promoters were unable to improve D-LA production (Supplementary Figure S1).

To address this issue, inducible promoters were introduced to enable controlled expression of target genes at desired points and levels. We tested two inducible promoters to express the D-LDH gene: the tet promoter (Ptet) and the tac promoter (Ptac), which were induced by anhydrotetracycline (aTc) and Isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively (Fig. 1A). The Ptet has previously been used to produce L-LA from methane in M. buryatense, achieving 0.8 g/L of LA in a continuous stirred bioreactor [32]. We inserted DNA fragments containing Ptet-LDH and Ptac-LDH with their repressors into the glgA site of JHM80 to generate JHM803 (JHM80 ΔglgA::Ptet-LDH) and JHM804 (JHM80 ΔglgA::Ptac-LDH), respectively.

Fig. 1
figure 1

D-LA production from methane with a fine-tuned D-LDH expression system. A Metabolic pathway of D-LA production from methane and schematic overview of the fine-tuned D-LDH expression system, controlled by the tet or tac promoter. B Growth and D-LA production of JHM803, expressing D-LDH under the Tet promoter, in media supplemented with the indicated concentrations of aTc. C Growth and D-LA production of JHM804, expressing D-LDH under the tac promoter, in media supplemented with the indicated concentrations of IPTG. The initial inoculation OD600 was 0.2 and error bars indicate standard deviations of two independent experiments

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In JHM803, D-LA production was minimal without aTc induction, demonstrating tight control of the tet promoter (Fig. 1B). D-LA production increased with rising concentrations of aTc, peaking at 29.5 mg/L when induced with 0.5 mg/L of aTc for 48 h (Fig. 1B). The aTc concentration used was half of the maximum tested concentration that did not cause significant growth inhibition (Supplementary Figure S2). In contrast, the tac promoter showed less stringent regulation than the tet promoter. The JHM804 strain produced up to 103.7 mg/L of D-LA at 48 h, even without IPTG induction of D-LDH. When 100 μM IPTG was added, D-LA production increased to 187 mg/L at 72 h, representing a 6.3-fold increase over JHM803 (Fig. 1C). Although the D-LA titers with IPTG induction were similar to those without induction during the first 48 h of cultivation (Fig. 1C), IPTG-induced cells exhibited approximately 22-fold higher D-LA production per cell OD (Supplementary Figure S3). This increase was primarily attributed to severe growth inhibition resulting from the enhanced transcription of D-LDH, leading to lactate toxicity under non-neutralizing conditions. Although the parental strain JHM80 can tolerate up to 8 g/L of externally added LA under neutralizing conditions [25], intracellular production of LA appears to impose greater toxicity, significantly limiting cell growth. The tet promoter is advantageous owing to its tight regulation. However, considering the requirement of strong D-LDH expression to enhance LA production, the tac promoter was selected for further experiments.

Evaluation of the tac promoter for D-LA production with methane feeding

We next assessed JHM804, which utilizes Ptac-controlled D-LDH expression, against our previous JHM86 strain, which expresses D-LDH from the glgBA promoter [25]. To determine the optimal IPTG concentration for D-LA production in fed-batch culture, we cultivated JHM804 in NMS medium with various IPTG concentrations, supplying 20% methane every 24 h. Since the initial tests with 50 μM IPTG caused severe growth inhibition (Fig. 1C), we explored lower IPTG concentrations ranging from 5 to 25 μM, starting with cell inoculation at an OD600 of 0.5. As a control, JHM86 cells were grown in the absence of IPTG. As IPTG concentration increased, the growth of the JHM804 strain was inhibited in a concentration-dependent manner (Fig. 2A). Concurrently, the D-LA production titer per cell OD increased (Fig. 2B), reflecting the effects of lactate toxicity linked to higher D-LDH expression levels. As a result, 5 μM IPTG treatment resulted in the highest D-LA production levels, which were comparable to those observed in JHM86 (Fig. 2C). These results suggest that controlling D-LDH transcription with the tac promoter enables higher expression levels than regulation by the glgBA promoter at IPTG concentrations exceeding 5 μM. While LA toxicity limits D-LA production, the inducible system enables more precise control of D-LDH expression, potentially surpassing the production levels of JHM86 when LA toxicity is mitigated through continuous pH neutralization during bioreactor fermentation. Moreover, continuous exposure to LA stress may increase the risk of genetic mutations. Considering the observed genetic instability and limited tolerance to organic acids in Methylomonas sp. DH-1 [35], we concluded that constitutive D-LA production could be detrimental to cells. Consequently, maintaining a low level of LA production prior to induction may promote stable production of D-LA.

Fig. 2
figure 2

Assessment of D-LA production through regulation of D-LDH expression from the Tac or glgBA promoters. Growth (A) and D-LA (C) production of JHM86, expressing D-LDH under the glgBA promoter, in NMS medium, and JHM804, expressing D-LDH under the Tac promoter, in IPTG-supplemented medium. Methane was supplied every 24 h. Cell growth and D-LA production were measured during the growth. The relative D-LA content (B), calculated as the ratio of LA concentrations to OD600, was compared under different conditions. The initial inoculation OD600 was 0.5, and data from two independent experiments were averaged and displayed with standard deviations

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Disruption of glucose 1-phosphate adenylyltransferase (glgC) to alleviate toxicity of ADP-glucose accumulation due to glgA deletion

Next, we attempted to scale up the culture using a bioreactor to further increase the D-LA titer. Unexpectedly, the JHM804 strain failed to grow when scaled up from a 50 mL flask to a 500 mL flask, even in the absence of the inducer IPTG. Since basal D-LA production was low without IPTG induction, LA toxicity was unlikely to be the main reason for growth inhibition. In contrast, the LA-tolerant parental strain, JHM80, showed normal growth in a 500 mL flask culture (Fig. 3A). This led us to hypothesize that the growth defects observed in larger flasks may be related to the deletion of glgA. Supporting this hypothesis, the previously developed JHM86 strain, which also carried a glgA deletion, failed to grow during scale-up in a bioreactor.

Fig. 3
figure 3

Effect of glgC deletion on cell growth and LA tolerance. A Growth comparison of indicated strains in 125 mL flasks (12.5 mL NMS medium) and 500 mL flasks (50 mL NMS medium) to examine scale-up effects. The initial inoculation OD600 was 0.1. B Metabolic pathway of glycogen synthesis from methane in Methylomonas sp. DH-1, highlighting the key intermediates: F6P, fructose-6-P; G6P, Glucose-6-p; G1P, Glucose-1-p. The carbon flux under carbon excess conditions is illustrated with arrows for the JHM801(ΔglgA) and JHM802 (ΔglgABC) strains, highlighting ADP-glucose accumulation resulting from glgA deletion. C Effect of glgC deletion on cell growth of JHM804 under different culture scales. The initial inoculation OD600 was 0.2 and the strains were grown in 125 mL flask containing 12.5 mL NMS medium or 500 mL flask containing 50 mL NMS medium. Each value represents the average ± standard deviation of two independent experiments. D Effect of IPTG addition (50 µM) on cell growth and D-LA production in JHM805 with methane supplied every 24h, with the initial inoculation OD600 was 0.1. Error bars indicate standard deviations of two independent experiments

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Excessive foam formation was observed in larger flasks, as shown in Supplementary Figure S4. The increased surface area, combined with a larger radius of rotation and higher angular velocity, caused more frequent collisions with the baffles in the flasks. This generated greater turbulence in the culture medium, enhancing gas transfer to the liquid phase. The improved exchange of methane and oxygen, both essential for cell growth, may have resulted in “carbon overflow,” where carbon uptake exceeded the metabolic capacity of the strain. Under such conditions, converting excess carbon into storage pathways such as glycogen serves as a natural “ameliorator” to mitigate metabolic stress [36, 37].

In Methylomonas sp. DH-1, along with the primary metabolism of converting methane to pyruvate, the glycogen synthesis pathway functions as a storage compound to manage excess carbon (Fig. 3B). Methane is metabolized to fructose 6-phosphate (F6P) via the ribulose monophosphate (RuMP) cycle, and F6P is then converted to glucose 1-phosphate (G1P). Glycogen synthesis involves three key enzymes: G1P adenylyltransferase (GlgC), glycogen synthase (GlgA), and glycogen branching enzyme (GlgB) (Fig. 3B). GlgC catalyzes the conversion of G1P to ADP-glucose, which is then utilized by GlgA to form linear α-(1 → 4)-linked glucose chains. Finally, GlgB introduces α-(1 → 6)-linked branches into glucose chains, resulting in a branched glycogen structure.

Therefore, with the deletion of glgA, the glycogen pathway halts at ADP-glucose, particularly during large-scale culture where carbon excess leads the carbon flux to glycogen accumulation (Fig. 3B). In general, the accumulation of sugar phosphates (e.g. galactose-1-phosphate, fructose-1-phosphate, and trehalose-6-phosphate) is recognized as toxic to cells—from E. coli to humans—due to the wasteful consumption of ATP [38]. ADP-glucose, an essential intermediate, plays a crucial role in balancing the ATP and ADP levels within cells. The adenylate energy charge (AEC), calculated as ([ATP] + 0.5 × [ADP])/([ATP] + [ADP] + [AMP]), serves as a key indicator of the cell’s metabolic state and its ability to perform energy-consuming processes. Under optimal growth conditions, the AEC is typically maintained around 0.9 [39, 40]. However, in cells accumulating ADP-glucose, the AEC has been reported to drop to 0.1 [41]. The accumulation of this intermediate has been found to be lethal in cyanobacteria, as deletion of glycogen synthase genes results in toxic ADP-glucose accumulation and cell death [41]. Interestingly, salt-induced stress has been reported to suppress this toxicity by redirecting carbon flux towards the production of osmolyte glucosylglycerol, highlighting the importance of metabolic flexibility in mitigating ADP-glucose accumulation. Despite its significance, comprehensive studies on this intermediate remain limited.

To test the effect of ADP-glucose accumulation, we deleted either the glgA gene alone or the entire glycogen synthesis operon (glgA, glgB, and glgC) in JHM80 and evaluated their growth in a 500 mL flask (Fig. 3A). In a 500 mL flask, the JHM801 strain (JHM80 ΔglgA) exhibited significant growth inhibition, whereas the JHM802 strain (JHM80 ΔglgABC) showed restored growth. The growth defect in the JHM801 strain was also evident in the smaller 125 mL flask culture, although it was less pronounced than in the 500 mL setup. The observation that deleting glgA has a greater negative impact on cell growth as culture volume increases suggests that during scale-up, metabolic flux towards ADP-glucose may intensify due to the redirected carbon flux towards glycogen synthesis in a carbon-rich environment. To address this issue, we alleviated ADP-glucose accumulation in the JHM804 strain by deleting the glgC gene. As expected, the resulting strain, JHM805 (JHM804 ΔglgC), exhibited growth recovery in both 125 mL and 500 mL flask cultures (Fig. 3C). We also evaluated the capacity of JHM805 to produce D-LA. While D-LDH induction with 50 μM IPTG severely inhibited the cell growth of JHM804 (Fig. 1C), JHM805 exhibited almost normal growth and produced up to 0.93 g/L of D-LA (Fig. 3D). These results indicate that deleting glgC not only facilitates culture scale-up but also potentially increases LA tolerance by improving overall cell fitness. Based on our observations, glgC deletion did not significantly impair cell growth or biomass yield, suggesting that metabolic burden was minimal under our experimental conditions. Alternative strategies to mitigate ADP-glucose toxicity, such as inducing salt stress [41] or modifying glycogen metabolism via ADP-glucose-involved genes [42], could also be considered. However, due to the limited knowledge of the Methylomonas sp. DH-1 strain and its expected instability under external stress, we concluded that glgC deletion is the most suitable approach in this context.

In our initial strain design for D-LA production, we deleted glgA gene to prevent competitive use of a carbon source for glycogen synthesis [25]. Notably, certain methanotrophic bacteria can accumulate substantial glycogen levels up to 30% of the dry cell weight (DCW) [43, 44]. Thus, inhibition of glycogen synthesis is an important strategy for increasing cellular lipid or protein levels [20, 42]. However, deleting the glgC gene might be a more effective strategy. This approach not only halts glycogen production but also reduces the accumulation of potentially toxic ADP-glucose. However, considering that M. buryatense exhibits only a minor growth defect during flask or bioreactor cultivation following glgA deletion [8, 20], further studies are needed to elucidate the underlying mechanisms of carbon storage. Interestingly, M. buryatense contains annotated genes for both glycogen and sucrose biosynthesis pathways, whereas Methylomonas sp. DH-1 strain has genes exclusively annotated for glycogen, indicating a potential difference in carbon storage preference. Further investigation into the regulation of carbon storage biosynthesis is necessary to optimize the carbon excess conditions and enhance the production of various chemicals in methanotrophs.

Bioreactor fermentation for D-LA production

The final strain, JHM805, was cultured in a 5-L bioreactor filled with 3 L of NMS medium supplemented with 50 µM IPTG. A mixture of 20% (v/v) methane and 80% air (v/v) was supplied continuously. Throughout cultivation, cell growth, D-LA production, and KNO3 concentrations were monitored. The JHM805 strain exhibited exponential growth until nitrate depletion occurred at 48 h (Fig. 4A). D-LA production increased with cell growth but significantly decreased after nitrate depletion, nearly halting 24 h post-depletion. After 108 h, the D-LA production reached 2.06 g/L (Fig. 4A).

Fig. 4
figure 4

Batch fermentation and nitrate feeding fermentation of JHM805 strain in continuous stirred bioreactor. A Growth and D-LA production of JHM805 strain in a 5L bioreactor containing 3 L NMS medium, supplemented with 10 μg/mL kanamycin and 50 μM of IPTG. A gas mixture of 20% (v/v) of methane and 80% (v/v) of air was continuously supplied and the initial inoculation OD600 was 0.1. Nitrate depletion coincides with the onset of D-LA production, indicating a shift in metabolic flux upon nitrogen limitation. B Growth of JHM805 in NMS media containing various concentrations of KNO3 to evaluate the effect of nitrate availability. The initial inoculation OD600 was 0.2 and error bars indicate the standard deviation of two independent experiments. C JHM805 fermentation in a 5-L bioreactor with modified NMS medium (6 × KNO3) with 10 μg/mL kanamycin and 50 μM of IPTG. Nitrate was replenished before depletion to prevent nitrogen limitation and 20% (v/v) of methane and 80% (v/v) of air were continuously supplied and the initial inoculation OD600 was 0.1. Cell growth, D-LA production, and nitrate consumption were monitored during the growth

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Nitrogen supplementation has been shown to enhance the production of various metabolites, including LA and lipids, in different organisms [45, 46]. In Rhizopus arrhizus, maintaining a low C/N ratio through NH4NO3 supplementation significantly increased LA production [47]. Additionally, nitrogen limitation leads to a high C/N ratio, which often triggers the redirection of carbon flux toward storage compound synthesis rather than product formation [48]. The beneficial effects of nitrogen supplementation are primarily attributed to increased biomass formation and enhanced metabolic activity, as nitrogen is a fundamental component of amino acids, nucleotides, and cofactors essential for cell growth and enzyme production [49]. Particularly in gas-converting microbial processes, increasing biomass is more critical than in conventional fermentations using soluble substrates, considering the challenges of achieving high cell density in gas-fermentation [50]. In methanotrophs, studies have used higher nitrate concentrations than those in standard NMS medium to enhance target material production. In M. capsulatus Bath, 3 × nitrate (3.0 g/L KNO3) was used for bioreactor operation to increase mevalonate production [50]. Additionally, 4 × nitrate supplementation has been used to enhance lipid production [20], while 8 × nitrate supplementation has been applied to achieve higher lactate production with increased cell growth [32] in M. buryatense. As in conventional fermentations using soluble substrates, increasing biomass production by supplying a higher nitrogen source may also be an effective strategy for enhancing target compound production in methanotroph cultures.

Based on these findings, we decided to improve cell growth and D-LA production by supplying more nitrate as an N source. To assess the tolerance of JHM805 to elevated nitrogen levels, we evaluated its growth at 4x, 6x, 8x, and 10x  KNO3 concentrations relative to the standard NMS medium (where 1 g/L KNO3 is equivalent to 9.89 mM). Growth remained unaffected at concentrations up to 6 g/L KNO3 (59.3 mM), indicating that nitrate supplementation does not impose significant inhibitory effects (Fig. 4B).

Consequently, bioreactor cultivation was conducted using 6 g/L KNO3 in the NMS medium. During cell culture, nitrate was added once at a concentration below the inhibitory level, before complete depletion occurred. This approach improved cell growth compared with batch cultivation using 1 g/L KNO3. In the fed-batch culture, the maximum OD600 reached 11.2, which is a 3.35-fold increase compared to the OD600 of 3.34 observed in the batch culture using 1 g/L KNO3 (see Fig. 4A). The D-LA titer reached 6.17 g/L after 108 h of fermentation (Fig. 4C), which is the highest reported among studies on D- or L-LA production in microorganisms using methane or methanol as carbon sources (Table 3). The D-LA titer (g/L) per OD600 was 0.65, which was lower than the 0.83 observed in the bioreactor culture without nitrate supplementation (Fig. 4A and Supplementary Table S1). However, in the modified NMS medium, D-LA productivity reached 0.057 g/L/h. Based on these data, higher cell growth due to increased nitrogen supply likely led to the observed increase in lactate titer and productivity. A higher or additional nitrate supply is unlikely to have shifted the intracellular metabolic flux toward lactate production, given the decreased D-LA titer (g/L) per OD600. The D-LA productivity of 0.057 g/L/h is not only the highest rate ever reported in methanotrophs but also represents a 7.12-fold increase from the 0.008 g/L/h achieved in our previous study [25] (Fig. 4C).

Table 3 Production of LA and other organic acids from methane
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While our study successfully improved D-LA production in Methylomonas sp. DH-1, further optimization strategies can enhance its industrial applicability. A key challenge in D-LA production by this strain is its low tolerance to organic acids, making it essential to explore rational or random metabolic engineering approaches to improve toxicity resistance. Notably, we identified small peptides that are predicted to modulate the substrate specificity of efflux pumps, potentially enhancing acetate export [35]. Engineering these efflux pumps to efficiently remove intracellularly accumulated D-LA could be a promising strategy to further improve production.

Additionally, while we optimized nitrate supplementation for bioreactor cultivation, previous studies suggest that modifying the metal ion composition in the culture medium [51] or supplementing other components (e.g., phosphate and organic acids) [52, 53] could enhance methane consumption and cell growth of methanotrophs. Given that genetic and metabolic engineering of methanotrophs is still in its early stages, many unexplored opportunities remain to further enhance their performance. These findings highlight the potential of methanotroph-based bioproduction of high-value chemicals and provide a strong foundation for future research.

Conclusions

The bioconversion of methane, an abundant carbon source, into D-LA, a biodegradable plastic monomer, represents a promising strategy. Methane-based fermentation offers economic advantages by using a low-cost, widely available feedstock, thereby reducing production costs. In this study, we engineered an LA-tolerant strain of Methylomonas sp. DH-1 to improve LA production by focusing on three critical aspects. First, the use of an inducible promoter for D-LDH expression effectively reduced lactate toxicity, a major challenge in improving production. Second, we addressed the metabolic imbalance during scale-up, particularly ADP-glucose accumulation due to glgA deletion (encoding glycogen synthase). This issue was resolved by deleting the glgC gene, which encodes glucose-1-phosphate (G1P) adenylyltransferase. Finally, this study contributes to the limited research on large-scale production systems utilizing methanotrophs, demonstrating Methylomonas sp. DH-1 as a promising platform for sustainable D-LA production from methane. Our optimized strain achieved a final titer of 6.17 g/L in a 5-L fed-batch fermenter by preventing nitrogen source depletion, marking the highest D-LA titer reported in methanotrophs to date.

Availability of data and materials

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

References

  1. Jew AD, Druhan JL, Ihme M, Kovscek AR, Battiato I, Kaszuba JP, et al. Chemical and reactive transport processes associated with hydraulic fracturing of unconventional oil/gas shales. Chem Rev. 2022;122(9):9198–263.

    Article  CAS  PubMed  Google Scholar 

  2. Kroepsch AC. Horizontal drilling, changing patterns of extraction, and piecemeal participation: urban hydrocarbon governance in Colorado. Energy Policy. 2018;120:469–80.

    Article  Google Scholar 

  3. Chen Y, Xu J. The shale gas boom in the US: Productivity shocks and price responsiveness. J Clean Prod. 2019;229:399–411.

    Article  Google Scholar 

  4. Mohammad N, Mohamad Ishak WW, Mustapa SI, Ayodele BV. Natural gas as a key alternative energy source in sustainable renewable energy transition: a mini review. Front Energy Res. 2021. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fenrg.2021.625023.

    Article  Google Scholar 

  5. Lodato C, Hamelin L, Tonini D, Astrup TF. Towards sustainable methane supply from local bioresources: anaerobic digestion, gasification, and gas upgrading. Appl Energy. 2022;323:119568.

    Article  CAS  Google Scholar 

  6. Kalyuzhnaya M, Kumaresan D, Heimann K, Caetano N, Visvanathan C, Karthik O. Editorial: methane: a bioresource for fuel and biomolecules. Front Environ Sci. 2020. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fenvs.2020.00009.

    Article  Google Scholar 

  7. Ojala DS, Beck DA, Kalyuzhnaya MG. Genetic systems for moderately halo(alkali)philic bacteria of the genus Methylomicrobium. Methods Enzymol. 2011;495:99–118.

    Article  PubMed  Google Scholar 

  8. Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, et al. Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense. Appl Environ Microbiol. 2015;81(5):1775–81.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Yan X, Chu F, Puri AW, Fu Y, Lidstrom ME. Electroporation-based genetic manipulation in type I methanotrophs. Appl Environ Microbiol. 2016;82(7):2062–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ishikawa M, Yokoe S, Kato S, Hori K. Efficient counterselection for Methylococcus capsulatus (Bath) by using a mutated pheS gene. Appl Environ Microbiol. 2018;84(23):e01875-e1918.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nguyen AD, Hwang IY, Lee OK, Kim D, Kalyuzhnaya MG, Mariyana R, et al. Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane. Metab Eng. 2018;47:323–33.

    Article  CAS  PubMed  Google Scholar 

  12. Rumah BL, Claxton Stevens BH, Yeboah JE, Stead CE, Harding EL, Minton NP, et al. In vivo genome editing in type I and II methanotrophs using a CRISPR/Cas9 system. ACS Synth Biol. 2023;12(2):544–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Le Thi Quynh H, Lee EY. Methanotrophs: metabolic versatility from utilization of methane to multi-carbon sources and perspectives on current and future applications. Bioresour Technol. 2023;384:129296.

    Article  Google Scholar 

  14. Patel SKS, Gupta RK, Kalia VC, Lee JK. Synthetic design of methanotroph co-cultures and their immobilization within polymers containing magnetic nanoparticles to enhance methanol production from wheat straw-based biogas. Bioresour Technol. 2022;364:128032.

    Article  CAS  PubMed  Google Scholar 

  15. Sheets JP, Ge X, Li YF, Yu Z, Li Y. Biological conversion of biogas to methanol using methanotrophs isolated from solid-state anaerobic digestate. Bioresour Technol. 2016;201:50–7.

    Article  CAS  PubMed  Google Scholar 

  16. Cai C, Shi Y, Guo J, Tyson GW, Hu S, Yuan Z. Acetate production from anaerobic oxidation of methane via intracellular storage compounds. Environ Sci Technol. 2019;53(13):7371–9.

    Article  CAS  PubMed  Google Scholar 

  17. Cho S, Lee YS, Chai H, Lim SE, Na JG, Lee J. Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z. Biotechnol Biofuels Bioprod. 2022;15(1):5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nguyen TT, Lee OK, Naizabekov S, Lee EY. Bioconversion of methane to cadaverine and lysine using an engineered type II methanotroph, Methylosinus trichosporium OB3b. Green Chem. 2020;22(22):7803–11.

    Article  CAS  Google Scholar 

  19. Nguyen AD, Kim D, Lee EY. Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria, using alpha-humulene as a model compound. Metab Eng. 2020;61:69–78.

    Article  CAS  PubMed  Google Scholar 

  20. Fei Q, Puri AW, Smith H, Dowe N, Pienkos PT. Enhanced biological fixation of methane for microbial lipid production by recombinant Methylomicrobium buryatense. Biotechnol Biofuels Bioprod. 2018;11:129.

    Article  Google Scholar 

  21. Chen X, Rodríguez Y, López JC, Muñoz R, Ni B-J, Sin G. Modeling of polyhydroxyalkanoate synthesis from biogas by methylocystis hirsuta. ACS Sustain Chem Eng. 2020;8(9):3906–12.

    Article  CAS  Google Scholar 

  22. Gesicka A, Oleskowicz-Popiel P, Lezyk M. Recent trends in methane to bioproduct conversion by methanotrophs. Biotechnol Adv. 2021;53:107861.

    Article  CAS  PubMed  Google Scholar 

  23. Pieja AJ, Sundstrom ER, Criddle CS. Cyclic, alternating methane and nitrogen limitation increases PHB production in a methanotrophic community. Bioresour Technol. 2012;107:385–92.

    Article  CAS  PubMed  Google Scholar 

  24. Hyun SW, Krishna S, Chau THT, Lee EY. Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production. Bioresour Technol. 2024;402:130759.

    Article  CAS  PubMed  Google Scholar 

  25. Lee JK, Kim S, Kim W, Kim S, Cha S, Moon H, et al. Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution. Biotechnol Biofuels Bioprod. 2019;12:234.

    Article  Google Scholar 

  26. Nguyen DTN, Lee OK, Hadiyati S, Affifah AN, Kim MS, Lee EY. Metabolic engineering of the type I methanotroph Methylomonas sp. DH-1 for production of succinate from methane. Metab Eng. 2019;54:170–9.

    Article  CAS  PubMed  Google Scholar 

  27. Lee HM, Ren J, Yu MS, Kim H, Kim WY, Shen J, et al. Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production. Biotechnol Biofuels. 2021;14(1):228.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chau THT, Nguyen AD, Lee EY. Boosting the acetol production in methanotrophic biocatalyst Methylomonas sp. DH-1 by the coupling activity of heteroexpressed novel protein PmoD with endogenous particulate methane monooxygenase. Biotechnol Biofuels Bioprod. 2022;15(1):7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jo JH, Park JH, Kim BK, Kim SJ, Park CM, Kang CK, et al. Improvement of succinate production from methane by combining rational engineering and laboratory evolution in Methylomonas sp. DH-1. Microb Cell Fact. 2024;23(1):297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Henard CA, Akberdin IR, Kalyuzhnaya MG, Guarnieri MT. Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts. Green Chem. 2019;21(24):6731–7.

    Article  CAS  Google Scholar 

  31. Nguyen DTN, Lee OK, Lim C, Lee J, Na JG, Lee EY. Metabolic engineering of type II methanotroph, Methylosinus trichosporium OB3b, for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway. Metab Eng. 2020;59:142–50.

    Article  CAS  PubMed  Google Scholar 

  32. Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT. Bioconversion of methane to lactate by an obligate methanotrophic bacterium. Sci Rep. 2016;6:21585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Garg S, Clomburg JM, Gonzalez R. A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1. J Ind Microbiol Biotechnol. 2018;45(6):379–91.

    Article  CAS  PubMed  Google Scholar 

  34. Henard CA, Franklin TG, Youhenna B, But S, Alexander D, Kalyuzhnaya MG, et al. Biogas biocatalysis: methanotrophic bacterial cultivation, metabolite profiling, and bioconversion to lactic acid. Front Microbiol. 2018. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fmicb.2018.02610.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Cha S, Cho YJ, Lee JK, Hahn JS. Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas sp. DH-1. Biotechnol Biofuels Bioprod. 2023;16(1):114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ortega-Martinez P, Nikkanen L, Wey LT, Florencio FJ, Allahverdiyeva Y, Diaz-Troya S. Glycogen synthesis prevents metabolic imbalance and disruption of photosynthetic electron transport from photosystem II during transition to photomixotrophy in Synechocystis sp. PCC 6803. New Phytol. 2024;243(1):162–79.

    Article  CAS  PubMed  Google Scholar 

  37. Grundel M, Scheunemann R, Lockau W, Zilliges Y. Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology (Reading). 2012;158(12):3032–43.

    Article  PubMed  Google Scholar 

  38. Boulanger EF, Sabag-Daigle A, Thirugnanasambantham P, Gopalan V, Ahmer BMM. Sugar-phosphate toxicities. Microbiol Mol Biol Rev. 2021;85(4):e0012321.

    Article  PubMed  Google Scholar 

  39. Atkinson DE. Energy charge of the adenylate pool as a regulatory parameter. Interact Feedback Mod Biochem. 1968;7(11):4030–4.

    CAS  Google Scholar 

  40. De la Fuente IM, Cortés JM, Valero E, Desroches M, Rodrigues S, Malaina I, et al. On the dynamics of the adenylate energy system: homeorhesis vs homeostasis. PLoS ONE. 2014;9(10):e108676.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Diaz-Troya S, Roldan M, Mallen-Ponce MJ, Ortega-Martinez P, Florencio FJ. Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria. J Exp Bot. 2020;71(6):2005–17.

    Article  CAS  PubMed  Google Scholar 

  42. But SY, Suleimanov RZ, Oshkin IY, Rozova ON, Mustakhimov II, Pimenov NV, et al. New solutions in single-cell protein production from methane: construction of glycogen-deficient mutants of methylococcus capsulatus mir. Fermentation. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/fermentation10050265.

    Article  Google Scholar 

  43. Khadem AF, van Teeseling MC, van Niftrik L, Jetten MS, den Camp Op HJ, Pol A. Genomic and physiological analysis of carbon storage in the verrucomicrobial methanotroph “Ca Methylacidiphilum fumariolicum” SolV. Front Microbiol. 2012. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fmicb.2012.00345.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Eshinimaev BT, Khmelenina VN, Sakharovskii VG, Suzina NE, Trotsenko YA. Physiological, biochemical, and cytological characteristics of a haloalkalitolerant methanotroph grown on methanol. Microbiology. 2002;71(5):512–8.

    Article  CAS  Google Scholar 

  45. Lobeda K, Jin Q, Wu J, Zhang W, Huang H. Lactic acid production from food waste hydrolysate by Lactobacillus pentosus: focus on nitrogen supplementation, initial sugar concentration, pH, and fed-batch fermentation. J Food Sci. 2022;87(7):3071–83.

    Article  CAS  PubMed  Google Scholar 

  46. Feng D, Chen Z, Xue S, Zhang W. Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiangensis (Chrysophyta) by nitrogen supplement. Biores Technol. 2011;102(12):6710–6.

    Article  CAS  Google Scholar 

  47. Zhang ZY, Jin B, Kelly JM. Production of lactic acid and byproducts from waste potato starch by Rhizopus arrhizus: role of nitrogen sources. World J Microbiol Biotechnol. 2007;23(2):229–36.

    Article  CAS  Google Scholar 

  48. Sundstrom ER, Criddle CS. Optimization of methanotrophic growth and production of Poly(3-Hydroxybutyrate) in a high-throughput microbioreactor system. Appl Environ Microbiol. 2015;81(14):4767–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Stein LY. Proteobacterial methanotrophs, methylotrophs, and nitrogen. In: Kalyuzhnaya MG, Xing X-H, editors. Methane biocatalysis: paving the way to sustainability. Cham: Springer International Publishing; 2018. p. 57–66.

    Chapter  Google Scholar 

  50. Jang N, Jeong J, Ko M, Song DU, Emelianov G, Kim SK, et al. High Cell-Density Cultivation of Methylococcus capsulatus Bath for Efficient Methane-Derived Mevalonate Production. J Agric Food Chem. 2023;71(12):4924–31.

    Article  CAS  PubMed  Google Scholar 

  51. Ito H, Yoshimori K, Ishikawa M, Hori K, Kamachi T. Switching between methanol accumulation and cell growth by expression control of methanol dehydrogenase in methylosinus trichosporium OB3b mutant. Front Microbiol. 2021. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fmicb.2021.639266.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Veraart AJ, Steenbergh AK, Ho A, Kim SY, Bodelier PLE. Beyond nitrogen: The importance of phosphorus for CH4 oxidation in soils and sediments. Geoderma. 2015;259–260:337–46.

    Article  Google Scholar 

  53. Xing X-H, Wu H, Luo M-F, Wang B-P. Effects of organic chemicals on growth of Methylosinus trichosporium OB3b. Biochem Eng J. 2006;31(2):113–7.

    Article  CAS  Google Scholar 

  54. Hur DH, Na JG, Lee EY. Highly efficient bioconversion of methane to methanol using a novel type I sp. DH-1 newly isolated from brewery waste sludge. J Chem Technol Biot. 2017;92(2):311–8.

    Article  CAS  Google Scholar 

  55. Nguyen TT, Lee EY. Methane-based biosynthesis of 4-hydroxybutyrate and P(3-hydroxybutyrate-co-4-hydroxybutyrate) using engineered Methylosinus trichosporium OB3b. Bioresour Technol. 2021;335:125263.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the National Research Foundation of Korea- (NRF) grant funded by the Korea government (MSIT) (2016M3D3A01913245, RS-2024–00351458 and RS-2024–00466473).

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Author notes

  1. Seungwoo Cha, Jae-Hwan Jo, Jong Kwan Lee, equally contributed to this work.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea

    Seungwoo Cha, Jong Kwan Lee, Wooyoung Park & Ji-Sook Hahn

  2. Bioenergy and Resources Upcycling Research Laboratory, Korea Institute of Energy Research, 152 Gajeong-Ro, Yuseong-Gu, Daejeon, 34129, Republic of Korea

    Jae-Hwan Jo & Min-Sik Kim

  3. Interdisciplinary Program for Agriculture and Life Sciences, Chonnam National University, 77 Yongbong-Ro, Buk-Gu, Gwangju, 61186, Republic of Korea

    Jae-Hwan Jo

  4. Gwangju Clean Energy Research Center, Korea Institute of Energy Research, 270 Samso-Ro, Buk-Gu, Gwangju, 61003, Republic of Korea

    Myounghoon Moon & Gwon Woo Park

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Contributions

S.C.: Supervision, Writing-original draft. J. -H. J.: Investigation, Writing-original draft. J. K. L.: Investigation, Conceptualization, Writing-original draft. W.P.: Writing-original draft. M.M.: Data analysis, Writing-original draft. G. W. P.: Data analysis, Writing-original draft. M. -S. K: Supervision, Funding acquisition. J. -S. H: Conceptualization, Funding acquisition, Supervision, Writing-original draft.

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Cha, S., Jo, JH., Lee, J.K. et al. Enhancing D-lactic acid production from methane through metabolic engineering of Methylomonas sp. DH-1. Microb Cell Fact 24, 70 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02695-z

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Microbial Cell Factories

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