l-Lactate dehydrogenase from Cyanidioschyzon merolae shows high catalytic efficiency for pyruvate reduction and is inhibited by ATP

l-Lactate is a commodity chemical used in various fields. Microorganisms have produced l-lactate via lactic fermentation using saccharides derived from crops as carbon sources. Recently, l-lactate production using microalgae, whose carbon source is carbon dioxide, has been spotlighted because the prices of the crops have increased. A red alga Cyanidioschyzon merolae produce l-lactate via lactic fermentation under dark anaerobic conditions. The l-lactate titer of C. merolae is higher than those of other microalgae but lower than those of heterotrophic bacteria. Therefore, an increase in the l-lactate titer is required in C. merolae. l-Lactate dehydrogenase (l-LDH) catalyzes the reduction of pyruvate to l-lactate during lactic fermentation. C. merolae possesses five isozymes of l-LDH. The results of previous transcriptome analysis suggested that l-LDHs are the key enzymes in the lactic fermentation of C. merolae. However, their biochemical characteristics, such as catalytic efficiency and tolerance for metabolites, have not been revealed. We compared the amino acid sequences of C. merolae l-LDHs (CmLDHs) and characterized one of the isozymes, CmLDH1. BLAST analysis revealed that the sequence similarities of CmLDH1 and the other isozymes were above 99%. The catalytic efficiency of CmLDH1 under its optimum conditions was higher than those of l-LDHs of other organisms. ATP decreased the affinity and turnover number of CmLDH1 for NADH. These findings contribute to understanding the characteristics of l-LDHs of microalgae and the regulatory mechanisms of lactic fermentation in C. merolae.


Introduction
Lactate/lactic acid is one of the commodity chemicals used for different fields such as foods, cosmetics, and medicines (Abdel- Rahman et al. 2013).Lactate has enantiomers, l-lactate and d-lactate.Both enantiomers are required for manufacturing bioplastic derived from lactate, namely polylactide (Tsuji 2005;Tsuji et al. 2006).Industrial lactate production uses lactic fermentation by microorganisms such as lactic acid bacteria, whose carbon sources are saccharides derived from crops (Ghaffar et al. 2014).However, the prices of the crops have increased because the prices are affected by population growth, soaring crude oil prices, and biofuel production (Bilgili et al. 2020).In recent years, when global warming accelerated, metabolite production from carbon dioxide using eukaryotic microalgae and cyanobacteria is spotlighted.Eukaryotic microalgae and cyanobacteria can produce lactate using carbon dioxide fixed via photosynthesis as the sole carbon source, minimizing the costs of carbon sources such as saccharides (Abdel-Rahman et al. 2013).
Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs (pH 1-3 and 40-50 °C) and does not possess a cell wall (De Luca et al. 1978).The genome sequences of the nucleus, mitochondria, and chloroplast in C. merolae are completely elucidated (Ohta et al. 1998(Ohta et al. , 2003;;Matsuzaki et al. 2004;Nozaki et al. 2007).Previous transcriptome analysis indicated that C. merolae performed anaerobic energy conversion, such as lactic fermentation, rather than aerobic respiration at night (Miyagishima et al. 2019).C. merolae produces l-lactate under dark anaerobic conditions (Yoshida et al. 2024).Among eukaryotic microalgae and cyanobacteria, a model cyanobacterium Synechocystis sp.PCC 6803 and Euglena gracilis also produce l-lactate (Angermayr and Hellingwerf 2013;Tomita et al. 2016).In Synechocystis sp.PCC 6803, genetic manipulation is necessary to produce l-lactate because wild-type does not produce l-lactate (Angermayr and Hellingwerf 2013).The l-lactate titer (3.2 g/L) and productivity (16.0-19.4mg/L/h) of C. merolae are higher than those of the Synechocystis sp.PCC 6803 mutant (1.8 g/L and 2.7 mg/L/h, respectively) (Yoshida et al. 2024;Angermayr and Hellingwerf 2013).l-Lactate production in Euglena gracilis is not efficient because its l-lactate titer is occasionally below 10 mg/L (Tomita et al. 2016).Thus, C. merolae is a candidate for a host of l-lactate production from carbon dioxide.However, the l-lactate titer and productivity of C. merolae are lower than those of heterotrophic bacteria (Abdel-Rahman et al. 2013), and a further increase in the l-lactate titer of C. merolae is required.
In this study, we compared the amino acid sequences of five CmLDHs (CmLDH1-5) and biochemically analyzed one of the isozymes, CmLDH1.

Preparation of a vector used for the expression of CmLDH1 in Escherichia coli
The sequence of the gene encoding CmLDH1 (CMA145C) was acquired from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https:// www.genome.jp/ kegg/ kegg_ ja.html).The sequence was synthesized by Eurofins Genomics Japan (Tokyo, Japan), and the synthesized sequence was introduced into the BamHI-XhoI site of vector pGEX6P-1 (G.E.Healthcare Japan, Tokyo, Japan).The vector was transformed into competent cells of Escherichia coli BL21 (DE3) (BioDynamics Laboratory Inc., Tokyo, Japan).After the transformation of the E. coli, the E. coli cells were cultured in an LB medium (2.4 L) at 30 °C with shaking (150 rpm).During the cultivation, the expression of the recombinant CmLDH1 was induced by 5 µM isopropyl β-d-1-thiogalactopyranoside (Wako Chemicals, Osaka, Japan) overnight.

Affinity purification of a glutathione-S-transferase (GST) -tagged CmLDH1
The E. coli cells in 600 mL culture were suspended in 10 mL phosphate-buffered saline/tween (PBS-T) (0.137 M NaCl, 0.27 mM KCl, 8.1 mM Na 2 HPO 4 .12H 2 O, 1.47 mM KH 2 PO 4 , and 0.001% Tween 20).The cells were sonicated twelve times for 15 s at 20% intensity using model VC-750 (EYELA, Tokyo, Japan).After centrifugation at 14,200 × g for 15 min at 4 °C, 800 µL of Glutathione Sepharose 4B resin (G.E.Healthcare Japan, Tokyo, Japan) was added to the supernatant.The sample was gently shaken on ice for 60 min.After that, 10 mM MgSO 4 •7H 2 O and 5 mM ATP were added to the sample, and the mixture was shaken for 30 min at 37 °C.The mixture was centrifugated at 5800 × g for 2 min at 4 °C to remove the supernatant.The resin was washed with 3 mL PBS-T five times and 700 µL of PBST five times.The GST-CmLDH1 was eluted by 500 µL glutathione-S-transferase (GST) elution buffer [50 mM Tris-HCl (pH 9.6) and 10 mM reduced glutathione] five times.Then, the GST-CmLDH1 was concentrated in a Vivaspin 500 MWCO 30000 device (Sartorius, Göttingen, Germany).
The concentration of purified GST-CmLDH1 was measured by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted using 8% gels, and the gels were stained by QuickBlue stain reagent (BioDynamics Inc., Tokyo, Japan).

Enzyme assay
The reaction catalyzed by CmLDH1 proceeded in 1 mL assay solution [100 mM sodium acetate (pH 4.0-5.5),Tris-HCl (pH 7.0-8.0),or the phosphate-citrate buffer (pH 4.0-8.0),different concentrations of sodium pyruvate, NADH, and CmLDH1].After incubating the assay solution without sodium pyruvate and NADH at different temperature for 5 min, sodium pyruvate and NADH was added to the assay solution to initiate the enzymatic reaction.During the reaction, the decrease in the NADH concentration, namely the change of the absorbance at 340 nm, was monitored for 1 min using a Hitachi U-3900H spectrophotometer (Hitachi High-Tech., Tokyo, Japan).The enzymatic activity of 1 unit was defined as the amount of enzyme that converts 1 μmol of substrate per minute.The V max (the maximum reaction velocity) and S 0.5 (the substrate concentration at 1/2 V max ) of CmLDH1 were calculated by curve fitting of the hill equation (Dixon and Webb 1979) (below) using the KaleidaGraph ver.4.5 software.
The k cat (turnover number) were calculated from V max .

Cultivation of C. merolae and measurement of CmLDH activity in the cell extracts
Cyanidioschyzon merolae NIES-3377 (from the National Institute for Environmental Studies) was cultivated in 70 mL of Modified Allen's medium containing 20 mM (NH 4 ) 2 SO 4 (pH 2.5) at 40 °C (Minoda et al. 2004).During the cultivation, the cultures were bubbled with 1% (v/v) CO 2 in the air under a white light (25 μmol/m 2 /s photons).After 3 days of the cultivation, the cell density (OD 730 ) was measured by a Shimadzu UV-2400 spectrophotometer (Shimadzu, Kyoto, Japan).C. merolae cells were recultivated for 3 days from OD 730 = 0.4.Measurement of CmLDH activity in cell extracts of C. merolae were performed as described previously (Yoshida et al. 2024).After 3 days of the cultivation, C. merolae cells [OD 730 × culture volume (mL) = 100] were collected by centrifugation at 5800 × g for 2 min.The cells were resuspended in 1 mL of PBS-T [0.137 M NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 ⋅12H 2 O, 1.47 mM KH 2 PO 4 , 0.005% (w/v) Tween-20] and sonicated on ice by a model VC-750 sonicator (EYELA, Tokyo, Japan) at 20% intensity for 10 s.The sonication was repeated five times.The mixture was centrifugated at 17,400 × g for 5 min at 4 °C.The total protein concentration in the supernatant was measured by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA), and 200 μg of total proteins was used for enzyme assay.

Results
We compared the amino acid sequences of five CmLDH isozymes (CmLDH1-5) (Fig. 1).l-LDH is a paralog of MDH, and the five amino acid residues determine l-LDH or MDH (Yin and Kirsch 2007).CmLDH isozymes excluding CmLDH4 possessed the five amino acid residues that are widely conserved in l-LDHs (aa47: Valine, aa115: glutamine, aa119: glutamate, aa258: Alanine, aa262: Isoleucine) (Yin and Kirsch 2007) (Fig. 1).The N-terminal sequence of CmLDH4 was 80 residues shorter than those of the other CmLDHs (Fig. 1).Therefore, CmLDH4 did not possess one of the amino acid residues determining l-LDH or MDH (aa47) (Fig. 1).The BLAST analysis when CmLDH1 was set at a query sequence revealed that the sequence identities and similarities (positives) of CmLDH1 and the other isozymes were 99% and ≥ 99%, respectively (Table 1).CmLDH isozymes excluding CmLDH4 possessed identical amino acid sequences without five amino acids residues (aa19, 26, 144, 150, and 300) (Fig. 1).The five residues were not included in the substrate binding site defined in Homo sapience LDH (Pineda et al. 2007) and the NADH binding site defined in Bacillus stearothermophilus LDH (Wigley et al. 1992) (Fig. 1).CmLDH4 did not possess one of the amino acids residues composing the NADH binding site defined in B. stearothermophilus LDH (aa68) (Fig. 1).An amino acid sequence of CmLDH1 has been used to determine the localization of CmLDHs in the cells as representative CmLDH (Moriyama et al. 2015).Hence, we biochemically characterized CmLDH1 in this study.
Under 57 °C and pH 4.5, the five metabolites decreased CmLDH1 activity (Fig. 5a).Under 57 °C and pH 7.0, ATP and ADP (particularly ATP) decreased CmLDH1 activity (Fig. 5b).ATP also decreased CmLDH1 activity under 30-50 °C and CmLDH activity in cell extracts of C. merolae (Fig. 6).ATP increased the S 0.5 of CmLDH1 for NADH and decreased the k cat and k cat /S 0.5 of CmLDH1 for pyruvate and NADH (Table 2).Under 57 °C and pH 7.0, CmLDH1 activity did not change and decreased in the presence of 1 mM and 5 mM AMP, respectively (Fig. 5b).Under 57 °C and pH 7.0, CmLDH1 activity did not change and increased in the presence of 1 mM and 5 mM FBP, respectively (Fig. 5b).Under 57 °C and pH 7.0, CmLDH1 activity did not change and decreased in the presence of 1 mM and 5 mM phosphoenolpyruvate (PEP), respectively (Fig. 5b).

Discussion
In this study, we compared the amino acid sequences of five CmLDH isozymes and examined the biochemical properties of CmLDH1, such as catalytic efficiency and tolerance to effectors.

Table 2 Kinetic parameters of CmLDH1
The measurement conditions were summarized in the legend of Fig. 3. Data exhibit average ± standard deviation obtained from three independent experiments Kinetic parameters for NADH in the presence of > 0.5 mM ATP cannot be measured because of low activity S 0.5 the substrate concentration at 1/2 V max , k cat turnover number, k cat /S 0.5 catalytic efficiency, n H Hill coefficient Asterisks exhibit significant differences between kinetic parameters in the presence and absence of ATP (Welch's t-test: *P < 0.05, **P < 0.005)  were 2 mM and 0.15 mM, respectively.The amount of CmLDH1 was 3 pmol.The t 1/2 (time where the residual activity was 50%) was calculated by a linear equation obtained from all the values.Data exhibit average ± standard deviation obtained from three independent experiments CmLDHs excluding CmLDH4 possessed almost identical amino acid sequences (Fig. 1 and Table 1).Although the N-terminal sequence of CmLDH4 was shorter than those of the other CmLDHs, the upstream sequence of CmLDH4 was similar to the N-terminal sequence of the other CmLDHs (Fig. 1).In Cyanidiophyceae, including C. merolae, gene The concentration of sodium pyruvate was 0.13 mM (S 0.5 at pH 4.5).The concentration of NADH was 0.05 mM because the absorbance change in the presence of inhibitors was not detected when the concentrations of both substrates were S 0.5 .The amount of CmLDH1 was 1 pmol.b CmLDH1 activities in the presence of different metabolites under 57 °C and pH 7.0.The sodium pyruvate and NADH concentrations were 0.20 mM (S 0.5 at pH 7.0) and 0.05 mM, respectively.
The amount of CmLDH1 was 1 pmol.CmLDH1 activity in Fig. 5 was represented by relative activity when the activity in the absence of metabolites was 100%.All data in Fig. 5 exhibit average ± standard deviation from three independent experiments.Asterisks exhibit significant differences between CmLDH1 activities in the presence and absence of metabolites (Welch's t-test: *P < 0.05, **P < 0.005).All metabolites used in this experiment as effectors are sodium salt.FBP: Fructose-1,6-bisphosphate, PEP Phosphoenolpyruvate Fig. 6 Effect of ATP on CmLDH activities.a CmLDH1 activities in the presence of 1 mM ATP at different temperatures.The pH was fixed at pH 7.0.The sodium pyruvate and NADH concentrations were 0.20 mM and 0.05 mM, respectively.The amount of CmLDH1 was 1 pmol.b CmLDH activities in cell extracts of C. merolae in the presence and absence of 1 mM ATP.The sodium pyruvate and NADH concentrations were 0.20 mM and 0.05 mM, respectively.The amount of total proteins was 200 μg.CmLDH activity in Fig. 6 was represented by relative activity when the activity without ATP was 100%.All data in Fig. 6 exhibit average ± standard deviation obtained from three independent experiments.Asterisks exhibit significant differences between CmLDH activities in the presence and absence of ATP (Welch's t-test: **P < 0.005) duplications are observed in subtelomeric regions, and the composition of the duplicated genes varies depending on the lineages (Cho et al. 2023).In C. merolae genome, all genes encoding CmLDHs are in the subtelomeric regions (Nozaki et al. 2007).These results suggest that genes encoding CmLDHs were generated by gene duplication in the subtelomeres.Among CmLDHs, only CmLDH4 did not possess amino acid residues equivalent to positions 47 and 68 of CmLDH1 (Fig. 1), suggesting that CmLDH4 cannot catalyze pyruvate reduction.
The catalytic efficiency of CmLDH1 for both substrates at pH 4.5 was higher than those of l-LDHs from other organisms (4 species) (Table 3).The catalytic efficiency of CmLDH1 for pyruvate at pH 7.0 was higher than those of l-LDHs from Cryptosporidium parvum, Limosilactobacillus fermentum, and Sporolactobacillus inulinus and similar to that of Enterococcus Mundtii (pH 7.5, 3 mM FBP) (Table 3).The catalytic efficiency of CmLDH1 for NADH at pH 7.0 was higher than those of l-LDHs from Cryptosporidium parvum and Limosilactobacillus fermentum and similar to that of Enterococcus Mundtii (pH 7.5, 3 mM FBP) (Table 3).These comparisons suggest that CmLDH1 is a high-activity l-LDH.Although absolute concentrations (molar concentrations) of pyruvate and NADH in C. merolae have been not reported, those of yeast have been reported as those of unicellular eukaryotes (pyruvate: 9.4 mM, NADH: 0.11 mM) (Park et al. 2016).These concentrations of pyruvate and NADH were markedly higher than the S 0.5 of CmLDH1 (pyruvate: 0.13-0.20 mM, NADH: 0.0064-0.011mM) (Table 2).This result suggests that CmLDH1 shows high activity similar to V max in the cells.Absolute quantification of intracellular metabolites of C. merolae is also necessary to determine the CmLDH1 activity in the cells accurately in the future.Previous microarray analysis revealed that the expression levels of genes encoding CmLDH and glycolysis enzymes rather than the tricarboxylic acid cycle enzymes increase at night (Miyagishima et al. 2019), suggesting that lactic fermentation is one of the main energy conversions at night in C. merolae.The high catalytic activity of CmLDH1 might enable C. merolae to perform efficient lactate fermentation at night.The stability of CmLDH1 was higher at pH 7.0 than at pH 4.5 (Fig. 4).Unlike l-lactate production at neutral pH in C. merolae, that at acidic pH leads to a decrease in intracellular pH and reaches a plateau at an early period (Yoshida et al. 2024).This might be due to the low stability of CmLDHs at acidic pH.
CmLDH1 activity was inhibited by ATP, ADP, and AMP (particularly ATP) in vitro (Fig. 5).These metabolites inhibit l-LDHs from sweet potato roots, Lactuca sativa L, and Staphylococcus epidermidis (Oba et al. 1977;Betsche 1981;Götz and Schleifer 1975).In C. merolae, the concentration of ATP is similar to that of ADP and higher than that of AMP (Miyagishima et al. 2019).Also, the absolute concentration of ATP in yeast (1.9 mM) (Park et al. 2016) is higher than the ATP concentration where ATP inhibited both CmLDHs in cell extracts of C. merolae and purified CmLDH1 (1 mM) (Figs. 5 and 6).These results suggest that among the adenine nucleotides, ATP mainly acts as an inhibitor of CmLDH1 in vivo.In L. sativa LDH, ATP decreases the affinity for NADH and acts as a competitive inhibitor for NADH (Betsche 1981).In CmLDH1, ATP decreased not only the affinity but also the k cat for NADH (Table 2).This suggests that ATP acts as a mixed inhibitor for NADH and does not bind to the NADH binding site in CmLDH1 (Fig. 1).C. merolae keeps the adenylate energy charge (balance of adenine nucleotides) almost constant throughout the day/night cycle (Miyagishima et al. 2019).Therefore, we presume that ATP generated via lactic fermentation strongly inhibits CmLDHs to avoid the overproduction of ATP at night.
CmLDH1 activity was affected by FBP and PEP in vitro (Fig. 5).FBP inhibited and slightly activated CmLDH1 activity at pH 4.5 and 7.0, respectively (Fig. 5).The pH of cytosol in C. merolae is neutral pH (Zenvirth et al. 1985), suggesting that FBP activates CmLDH activity in vivo.The activation of l-LDHs by FBP has been confirmed in bacteria (Lactococcus lactis, Lactobacillus plantarum, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus mundtii, B. stearothermophilus) (Gaspar et al. 2007;Feldman-Salit et al. 2013;Matoba et al. 2014;Flores and Ellington 2005).The activities of l-LDHs from L. lactis, L. plantarum, S. pyogenes, and E. faecalis increase 1000, 1.05, 83, and 7.8-fold in the presence of 3 mM FBP (Gaspar et al. 2007;Feldman-Salit et al. 2013).B. stearothermophilus LDH activity increases 15-fold in the presence of 5 mM FBP (Flores Ellington 2005).Although CmLDH1 activity increased 1.2fold in the presence of 5 mM FBP (Fig. 5b), the absolute concentration of FBP in yeast (4 mM) is below 5 mM (Park et al. 2016).These results suggest that FBP is not essential for the catalytic activity of CmLDH1.CmLDHs did not possess histidine at position 201 composing the FBP binding site defined in B. stearothermophilus LDH (Fig. 1).This might be why CmLDH1 activity hardly depended on FBP.CmLDH1 activity did not change and decreased in the presence of 1 mM and 5 mM PEP at pH 7.0, respectively (Fig. 5b).The inhibition of l-LDHs by PEP has been confirmed in Cupriavidus necator, Ipomoea batatas, and Solanum tuberosum (Steinbüchel and Schlegel 1983;Oba et al. 1977;Davies and Davies 1972).The absolute concentration of PEP in yeast (0.029 mM) is below 1 mM (Park et al. 2016), suggesting that PEP hardly affects CmLDH1 activity in vivo.This study revealed the biochemical properties of CmLDH1.Our findings contribute to understanding the biochemical characteristics of l-LDHs in microalgae and the regulatory mechanism of lactic fermentation in C. merolae.CmLDH1 was inhibited by ATP (Figs. 5 and 6).Therefore, the relief of the inhibition by novel culture methods and genetic manipulation of C. merolae might lead to an increase in l-lactate production of C. merolae.

Fig. 1
Fig. 1 Comparison of amino acid sequences of LDHs and E. coli MDH.Amino acid sequences of LDHs and E. coli MDH were aligned using CLC Sequence Viewer ver.8.0.The eMDH, HLDH, and BLDH are E. coli MDH, Homo sapience LDH, and Bacillus stearothermophilus LDH, respectively.The order of amino acid residues of these enzymes is based on that of CmLDH1.The orange squares represent the amino acid residues that differ between CmLDHs (aa19, 26, 144, 150, and 300).The blue squares represent

Fig. 2 Fig. 3
Fig. 2 Temperature and pH dependence of CmLDH1 activity.a Result of SDS-PAGE after purification of CmLDH1.b Effects of temperature (top) and pH (bottom) on CmLDH1 activity.Regarding the measurement of temperature dependence of CmLDH1 activity, pH was fixed at pH 4.5.Regarding the measurement of pH depend-

Fig. 4
Fig. 4 pH stability of CmLDH1.CmLDH1 activities are represented by residual activities, and the activity without incubation at pH 4.5 or 7.0 was 100%.The blue and orange makers indicate activities after incubation at pH 4.5 and 7.0, respectively.The temperature was set at 57 °C.The sodium pyruvate and NADH concentrations

Fig. 5
Fig. 5 Effects of metabolites on CmLDH1 activity.a CmLDH1 activities in the presence of different metabolites under 57 °C and pH 4.5.The concentration of sodium pyruvate was 0.13 mM (S 0.5 at pH 4.5).The concentration of NADH was 0.05 mM because the absorbance change in the presence of inhibitors was not detected when the concentrations of both substrates were S 0.5 .The amount of CmLDH1 was 1 pmol.b CmLDH1 activities in the presence of different metabolites under 57 °C and pH 7.0.The sodium pyruvate and NADH concentrations were 0.20 mM (S 0.5 at pH 7.0) and 0.05 mM, respectively.

Table 1
Result of the BLAST analysis of CmLDHsAn amino acid sequence of CmLDH1 was used as a query sequence.The BLAST search was performed in the Kyoto Encyclopedia of Genes and Genomes database (https:// www.genome.jp/ kegg/ genome.html) Identities exhibit the ratio of identical amino acid residues.Positives exhibit the ratio of amino acid residues whose chemical characteristics are similar to amino acid residues in a query sequence

Table 3
The catalytic efficiencies of l-LDHs from various organisms