D-lactate Dehydrogenase Research Articles

Structural Insights into Malic Enzyme Variants Favoring an Unnatural Redox Cofactor.

The use of nicotinamide cytosine dinucleotide (NCD), a biocompatible nicotinamide adenosine dinucleotide (NAD) analogue, is of great scientific and biotechnological interest. Several redox enzymes have been devised to favor NCD, and have been successfully applied in creating NCD-dependent redox systems. However, molecular interactions between cofactor and protein have still to be disclosed in order to guide further engineering efforts. Here we report the structural analysis of an NCD-favoring malic enzyme (ME) variant derived from Escherichia coli. The X-ray crystal structure data revealed that the residues located at position 346 and 401 in ME acted as the "gatekeepers" of the adenine moiety binding cavity. When Arg346 was substituted with either acidic or aromatic residues, the corresponding mutants showed substantially reduced NCD preference. Inspired by these observations, we generated Lactobacillus helveticus derived d-lactate dehydrogenase variants at Ile177, the counterpart to Arg346 in ME, and found a similar trend in terms of cofactor preference changes. As many NAD-dependent oxidoreductases share key structural features, our results provide guidance for protein engineering to obtain more NCD-favoring variants.

Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit

ABSTRACT D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, Km value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that ldLDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.

Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor.

Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD-pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D-lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D-lactate at the expense of formate.

Stabilization of recombinant d-Lactate dehydrogenase enzyme with trehalose: Response surface methodology and molecular dynamics simulation study

In this work, the effect of trehalose was investigated on the structural stability of the d-Lactate dehydrogenase (DLDH) enzyme. Initially, response surface methodology (RSM) was applied to optimize different process variables for the stabilization of DLDH using trehalose. The stabilization of DLDH with trehalose was examined under various experimental conditions, including trehalose concentration, pH, temperature, and incubation time. The effect of the processing parameters was tested using the RSM method and a central composite design (CCD) model. According to the ANOVA results, there was a close correlation between the predicted and experimental values of the response parameter. The optimum values of trehalose concentration, pH, temperature, and incubation time were found to be 1.25 M, 8, 30 °C, and 25 min, respectively, for achieving the maximum activity. Then, the kinetic and thermodynamic parameters of DLDH in the absence and presence of trehalose were examined. The DLDH half-life at 40 °C was 28.88 min, while, in the presence of trehalose, the enzyme had a longer half-life (33.01 min) at this temperature. Also, in the end, molecular dynamics (MD) simulations of the DLDH enzyme was performed at the optimum conditions to obtain fundamental insights into the conformational transitions of the DLDH enzyme in the absence and presence of trehalose.

Kinetic characterization and structure analysis of an altered polyol dehydrogenase with d‐lactate dehydrogenase activity

During adaptive metabolic evolution a native glycerol dehydrogenase (GDH) acquired a d-lactate dehydrogenase (LDH) activity. Two active-site amino acid changes were detected in the altered protein. Biochemical studies along with comparative structure analysis using an X-ray crystallographic structure model of the protein with the two different amino acids allowed prediction of pyruvate binding into the active site. We propose that the F245S alteration increased the capacity of the glycerol binding site and facilitated hydrogen bonding between the S245 γ-O and the C1 carboxylate of pyruvate. To our knowledge, this is the first GDH to gain LDH activity due to an active site amino acid change, a desired result of in vivo enzyme evolution.

Bacterial Genes of Metabolite Biomarkers of Autism Spectrum Disorders in Gut Microbiota of Young Children: Detection by Real-Time PCR and In Silico Analysis

Two different approaches, real-time PCR and in silico analysis, were used to study the abundance of bacterial genes encoding the enzymes producing biomarker metabolites in metagenomes of gut microbiota of children with autism spectrum disorders (ASD) and healthy children. As a result of the analysis of the published data, the following biomarker metabolites of ASD were chosen for the research: p-cresol, indole, propionic acid, and D-lactic acid. For the real-time PCR and the transcriptomic analysis, primer sequences were used specifically developed for conservative regions of the genes involved in production of these bacterial metabolites. Nucleotide sequences of the conservative regions were also united into a catalog and were searched in metagenomic assemblies as part of in silico analysis. Comparison of the results revealed a decrease in abundance of the genes in the metagenomes of children with ASD during both PCR and bioinformatics analyses. In addition, real-time PCR made it possible to detect a significant increase in abundance of the genes encoding p-hydroxyphenylacetate decarboxylase and D-lactate dehydrogenase in Bacteroides fragilis and Alistipes finegoldii species and methylmalonyl-CoA decarboxylase in B. fragilis, Alistipes shahii, and Eubacterium rectali species. A low level of the transcripts in total RNA was found in microbiota of a child with severe form of ASD.

S2782 D-Lactic Acidosis in Short Bowel Syndrome

INTRODUCTION: Lactic acidosis (LA) in a patient presenting with abdominal pain raises concern for bowel ischemia. The physician is prompted to administer intravenous fluids, locate the ischemic area, and consider invasive procedures. However, different isomers of lactic acid exist. Elevation of D-lactic acid reveals an alternate diagnosis. Understanding D-lactic acidosis (D-LA) can guide physicians towards effective treatment and prevent harm from unnecessary imaging and invasive intervention. CASE DESCRIPTION/METHODS: 43 year old male with Crohn's Disease (CD) and bowel resection presents with 2 weeks of diffuse abdominal pain. On examination he was hypertensive and somnolent with slurred speech. His abdomen was soft but tender, with scarring from abdominal surgeries. Blood work revealed leukocytosis and anion-gap metabolic acidosis (AGMA) with LA. Testing for ketoacidosis and toxic ingestion was negative. Gastrointestinal pathogen panel was negative. Computed tomography (CT) was negative for bowel thickening or enhancement, and CT angiography was negative for mesenteric thrombosis. Endoscopy did not reveal active CD. Lactic acid remained elevated despite intravenous fluids. The patient was noted consuming juice and several packets of sugar with tea, raising concern for D-LA. After restricting carbohydrates, lactic acid and AGMA rapidly normalized. A blood sample sent out from time of admission showed elevated D-lactic acid. DISCUSSION: This patient's neurologic symptoms, AGMA and LA, simple carbohydrate intake and prior bowel resection raised concern for D-LA, a rare condition observed in patients with short bowel syndrome (SBS). Decreased intestinal absorption in SBS results in delivery of carbohydrates to the colon, where fermentation by colonic flora form L- and D-Lactic acid and other organic acids. Systemic absorption can cause AGMA, LA and neurologic symptoms, such as encephalopathy, dysarthria and ataxia. Diagnosis involves clinical suspicion, AGMA that resolves with carbohydrate restriction, and neurologic symptoms. D-lactate dehydrogenase assay confirms diagnosis. Treatment involves correction of acidosis and oral carbohydrate restriction. Enteric antibiotic use has been proposed to reduce acid-forming bacteria. Physicians should suspect D-LA in patients with CD post-bowel resection and otherwise unexplained AGMA, as this could prevent harm through inappropriate treatment including intravenous antibiotics and corticosteroids, radiation exposure, and invasive endoscopic evaluation.

Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.

Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.

A D-lactate dehydrogenase from rice is involved in conferring tolerance to multiple abiotic stresses by maintaining cellular homeostasis

D-lactate dehydrogenase (D-LDH) converts D-lactate (the end product of glyoxalase system) to pyruvate and thereby completes the detoxification process of methylglyoxal. D-LDH detoxifies and diverts the stress induced toxic metabolites, MG and D-lactate, towards energy production and thus, protects the cell from their deteriorating effects. In this study, a D-LDH enzyme from rice (OsD-LDH2, encoded by Os07g08950.1) was characterized for its role in abiotic stress tolerance. For this, a combination of in silico, molecular, genetic and biochemical approaches was used. The kinetic analysis revealed OsD-LDH2 to be the most efficient D-LDH enzyme in comparison to D-LDHs from other plant species. Heterologous overexpression of OsD-LDH2 provides tolerance against multiple abiotic stresses in E. coli, yeast and plant system. The analysis of D-LDH mutant and OsD-LDH2 overexpressing transgenic plants uncovered the crucial role of D-LDH in mitigation of abiotic stresses. OsD-LDH2 overexpressing plants maintained lower level of ROS and other toxic metabolites along with better functioning of antioxidant system. This is the first report on correlation of D-LDH with multiple abiotic stress tolerance. Overall, OsD-LDH2 emerged as a promising candidate which can open a new direction for engineering stress tolerant crop varieties by maintaining their growth and yield in unfavorable conditions.

Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1.

This study evaluates whole-genome sequence of Lactobacillus reuteri PNW1 and identifies its safety genes that may qualify it as a putative probiotic. It further extracted the bacteriocin produced by the strain and tested its effectiveness against pathogenic STEC E. coli O177. The genomic DNA was sequenced on illuminal Miseq instrument and the sequenced data was assessed for quality reads before assembled with SPAdes. The draft assembly was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further downstream analyses were carried out using appropriate bioinformatic tools. Production of biogenic amines was biochemically confirmed through HPLC analysis. The assembled genome was 2,430,215 bp long in 420 contigs with 39% G+C content. Among all known genes, putatively responsible for the production of toxic biochemicals, only arginine deiminase (EC3.5.3.6) was spotted. Coding sequences (CDS) putative for D-lactate dehydrogenase (EC1.1.1.28), L-lactate dehydrogenase (EC1.1.1.27) and bacteriocin helveticin J were found within the genome together with plethora of other probiotic important genes. The strain harbours only resistant genes putative for Lincosamide (lnuC) and Tetracycline resistant genes (tetW). There was no hit found for virulence factors and probability of the strain being a human pathogen was zero. Two intact prophage regions were detected within the genome of L. reuteri PNW1 and nine CDS were identified for insertion sequence by OASIS which are belong to seven different families. Five putative CDS were identified for the CRISPR, each associated with Cas genes. Maximum zone of inhibition exhibited by the bacteriocin produced L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after being partially purified. With the strain predicted as non-human pathogen, coupled with many other identified desired features, L. reuteri PNW1 stands a chance of making good and safe candidates for probiotic, though further in-vivo investigations are still necessary.

Major role of lactate dehydrogenase D-LDH1 for the synthesis of lactic acid in Fructobacillus tropaeoli CRL 2034.

The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points •F. tropaeoli CRL 2034 harbors three ldh genes in its genome. •ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. •Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. •Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

Female genital tract (FGT) inflammation increases HIV infection susceptibility. Non-optimal cervicovaginal microbiota, characterized by depletion of Lactobacillus species and increased bacterial diversity, is associated with increased FGT cytokine production. Lactobacillus species may protect against HIV partly by reducing FGT inflammation. We isolated 80 lactobacilli from South African women with non-optimal (Nugent 4–10; n = 18) and optimal microbiota (Nugent 0–3; n = 14). Cytokine production by vaginal epithelial cells in response to lactobacilli in the presence and absence of Gardnerella vaginalis was measured using Luminex. Adhesion to vaginal epithelial cells, pH, D/L-lactate production and lactate dehydrogenase relative abundance were assessed. Lactobacilli from women with non-optimal produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1α, IL-1β and MIP-1α/β production significantly elevated. Overall, lactobacilli suppressed IL-6 (adjusted p < 0.001) and IL-8 (adjusted p = 0.0170) responses to G. vaginalis. Cytokine responses to the lactobacilli were inversely associated with lactobacilli adhesion to epithelial cells and D-lactate dehydrogenase relative abundance. Thus, while cervicovaginal lactobacilli reduced the production of the majority of inflammatory cytokines in response to G. vaginalis, isolates from women with non-optimal microbiota were more inflammatory and produced less lactic acid than isolates from women with optimal microbiota.

Engineering d-Lactate Dehydrogenase to Favor an Non-natural Cofactor Nicotinamide Cytosine Dinucleotide.

Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d-lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d-lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.

A combined theoretical and experimental study to improve the thermal stability of recombinant D‐lactate dehydrogenase immobilized on a novel superparamagnetic Fe3O4NPs@metal–organic framework

AbstractLactate dehydrogenase (LDH) is an enzyme that catalyzes the reduction of nicotinamide adenine dinucleotide (NADH) and pyruvate to nicotinamide adenine dinucleotide (NAD+) and D‐lactate in the final step of anaerobic glycolysis. This enzyme belongs to the family of oxidoreductases. Humans possess two isoforms of LDH enzyme: NAD‐dependent L‐lactate dehydrogenase (L‐LDH) and NAD‐dependent D‐lactate dehydrogenase (D‐LDH). D‐LDH is released during tissue damage, and is a sign of diseases such as kidney stones, heart failure, and some types of cancers and appendicitis. Accordingly, the design and construction of biosensors for the determination of lactate levels are important. The thermal sensitivity of D‐LDH and low protein production in the host bacteria limit the use of this protein in certain applications. To solve these problems, two solutions were used in this study. First, the codon‐optimized 1008 bp D‐LDH gene fused with a histidine tag was cloned at the NcoI/XhoI sites and expressed in E. coli BL21. Second, a new metal–organic framework (Fe3O4NPs@Ni‐MOF) was synthesized and used for immobilization and stabilization of D‐LDH. Fe3O4NPs@Ni‐MOF core‐shell nanocomposites were characterized by Fourier transform infrared spectroscopy, vibrating sample magnetometer, scanning electron microscopy, X‐ray diffraction, and the Brunauer–Emmett–Teller method. In comparison with the free enzyme, the immobilized enzyme presented better stability at high temperatures. The immobilization of the enzyme could be useful because most reactions happen at high temperatures in industry. To examine the effect of Fe3O4NPs@Ni‐MOF on the adsorption and conformation of D‐LDH at the atomistic level, a molecular dynamics simulation was carried out. Our study showed that the interaction between Fe3O4NPs@Ni‐MOF and D‐LDH involved van der Waals interactions, hydrophobic interaction energies, cation–π interaction between the Ni ions of the MOF with the enzyme residues and also, the hydrogen bond interactions between enzyme and heteroatoms in the MOF. Root mean square fluctuation and secondary structure analysis showed that Fe3O4NPs@Ni‐MOF protected the conformation of the enzyme.

3-phenyllactic acid productionby free-whole-cells of Lactobacillus crustorum in batch and continuous fermentation systems.

3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve3-PLA yields in batch and continuous fermentation systems using of free-whole-cellsofthe threeL.crustorum strains. The fermentation conditions of free-whole-cells of thethree L. crustorum strains for3-PLA productionwereoptimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P<0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, thestrain produced 15·2mmoll-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5mmoll-1 h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.

From methylglyoxal to pyruvate: a genome-wide study for the identification of glyoxalases and D-lactate dehydrogenases in Sorghum bicolor

BackgroundThe glyoxalase pathway is evolutionarily conserved and involved in the glutathione-dependent detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis. It acts via two metallo-enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), to convert MG into D-lactate, which is further metabolized to pyruvate by D-lactate dehydrogenases (D-LDH). Since D-lactate formation occurs solely by the action of glyoxalase enzymes, its metabolism may be considered as the ultimate step of MG detoxification. By maintaining steady state levels of MG and other reactive dicarbonyl compounds, the glyoxalase pathway serves as an important line of defence against glycation and oxidative stress in living organisms. Therefore, considering the general role of glyoxalases in stress adaptation and the ability of Sorghum bicolor to withstand prolonged drought, the sorghum glyoxalase pathway warrants an in-depth investigation with regard to the presence, regulation and distribution of glyoxalase and D-LDH genes.ResultThrough this study, we have identified 15 GLYI and 6 GLYII genes in sorghum. In addition, 4 D-LDH genes were also identified, forming the first ever report on genome-wide identification of any plant D-LDH family. Our in silico analysis indicates homology of putatively active SbGLYI, SbGLYII and SbDLDH proteins to several functionally characterised glyoxalases and D-LDHs from Arabidopsis and rice. Further, these three gene families exhibit development and tissue-specific variations in their expression patterns. Importantly, we could predict the distribution of putatively active SbGLYI, SbGLYII and SbDLDH proteins in at least four different sub-cellular compartments namely, cytoplasm, chloroplast, nucleus and mitochondria. Most of the members of the sorghum glyoxalase and D-LDH gene families are indeed found to be highly stress responsive.ConclusionThis study emphasizes the role of glyoxalases as well as that of D-LDH in the complete detoxification of MG in sorghum. In particular, we propose that D-LDH which metabolizes the specific end product of glyoxalases pathway is essential for complete MG detoxification. By proposing a cellular model for detoxification of MG via glyoxalase pathway in sorghum, we suggest that different sub-cellular organelles are actively involved in MG metabolism in plants.

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39kDa. It is active at 40°C and pH 6.5 and stable in a neutral to alkaline environment below 35°C. The kinetic constants, including maximal reaction rate (V max), apparent Michaelis-Menten constant (K m), turnover number (K cat) and catalytic efficiency (K cat/K m) for phenylpyruvic acid were 1.95 U mg-1, 2.83mM, 12.29s-1, and 4.34mM-1s-1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5gl-1 d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2gl-1 d-1. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals.

Malic Enzyme Facilitates d-Lactate Production through Increased Pyruvate Supply during Anoxic Dark Fermentation in Synechocystis sp. PCC 6803.

d-Lactate is one of the most valuable compounds for manufacturing biobased polymers. Here, we have investigated the significance of endogenous malate dehydrogenase (decarboxylating) (malic enzyme, ME), which catalyzes the oxidative decarboxylation of malate to pyruvate, in d-lactate biosynthesis in the cyanobacterium Synechocystis sp. PCC6803. d-Lactate levels were increased by 2-fold in ME-overexpressing strains, while levels in ME-deficient strains were almost equivalent to those in the host strain. Dynamic metabolomics revealed that overexpression of ME led to increased turnover rates in malate and pyruvate metabolism; in contrast, deletion of ME resulted in increased pool sizes of glycolytic intermediates, probably due to sequential feedback inhibition, initially triggered by malate accumulation. Finally, both the loss of the acetate kinase gene and overexpression of endogenous d-lactate dehydrogenase, concurrent with ME overexpression, resulted in the highest production of d-lactate (26.6 g/L) with an initial cell concentration of 75 g-DCW/L after 72 h fermentation.

Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae.

d-Phenyllactic acid is a versatile natural organic acid, which is used as an antiseptic agent, monomer of the biodegradable material poly-phenyllactic acid and in the synthesis chiral intermediate of pharmaceuticals. In this report, the novel NADH-dependent d-lactate dehydrogenase LrLDH was identified by screening a shotgun genome of Lactobacillus rossiae. To improve cofactor regeneration, the Exiguobacterium sibiricum glucose dehydrogenase EsGDH was overexpressed together with LrLDH in E. coli BL21(DE3)-pCDFDuet-1-gdh-ldh. The total enzyme activity in the fermentation broth of E. coli BL 21(DE3)-pCDFDuet-1-gdh-ldh peaked at 2359.0Ul-1 when induced by 10gl-1 lactose at 28°C and 150rpm for 14h. The biocatalytic reduction of sodium phenylpyruvate to d-phenyllactic acid was successfully carried out using whole cells of the engineered E. coli. Under the optimized biocatalysis conditions, 50gl-1 sodium phenylpyruvate was completely converted to d-phenyllactic acid with a space-time yield and enantiomeric excess of 262.8gl-1day-1 and > 99.5%, respectively. To our best knowledge, it is the highest productivity reported to date, with great potential for the mass production of d-phenyllactic acid.