D-lactate Dehydrogenase Research Articles

Активация альтернативного дыхания с внутренним акцептором электронов при анаэробной утилизации глюкозы у штаммов Escherichia coli с нарушенной способностью к брожению

The activation of alternative respiration with an internal electron acceptor upon anaerobic utilization of glucose in Escherichia coli strains with the impaired fermentation ability has been studied. It was found out that the respiration processes utilizing pyruvic acid as an endogenous electron acceptor can markedly contribute to the maintenance of the anaerobic redox balance in E. coli strains deficient in mixed acid fermentation pathways. The sequential inactivation of the pathways of anaerobic dissimilation of pyruvate and impairment of the functionality of the reductive branch of the tricarboxylic acid cycle led to the increase in the contribution of the respiratory formation of lactic acid and alanine to the biosynthesis of the reduced products of anaerobic glucose utilization by the strains from 11 to 54%. The analysis of the enantiomeric composition of the lactic acid and alanine secreted by the strains demonstrated that D-lactate dehydrogenase (Dld), L-lactate dehydrogenase (LctD), and D-alanine dehydrogenase (DadA) participated in the biosynthesis of the respective compounds. Escherichia coli, glucose, fermentation, respiration, metabolic engineering, pyruvate, lactate, alanine. The work was supported by a grant from the Russian Foundation for Basic Research (Project no. 18-04-01222)

Enhancement of lactate fraction in poly(lactate-co-3-hydroxybutyrate) synthesized by Escherichia coli harboring the D-lactate dehydrogenase gene from Lactobacillus acetotolerans HT.

For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.

Metabolic engineering of Enterobacter aerogenes to improve the production of 2,3-butanediol

The enhanced production of 2,3-butanediol was investigated using a metabolic engineering approach and optimized fermentation conditions. New engineered strains of Enterobacter aerogenes ATCC 29007 were developed by deleting the d-lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), malate dehydrogenase (mdh), and acetaldehyde dehydrogenase (acdh) genes to block the production of lactate, acetate, succinate, and ethanol, respectively. The resulting engineered strain E. aerogenes SUMI02 (ΔldhAΔpta) produced 36.5 g/L of 2,3-butanediol in flask cultivation, an amount 8.11 times greater than that of its wild type counterpart (4.5 g/L). In addition, the 2,3-butanediol production and productivity reached 38.24 g/L and 0.8 g/L/h, respectively, in the batch fermentation using a bioreactor.

Stabilization of d-lactate dehydrogenase diagnostic enzyme via immobilization on pristine and carboxyl-functionalized carbon nanotubes, a combined experimental and molecular dynamics simulation study

The most important mode of enzyme inactivation is thermal inactivation. Immobilization technology is an efficient approach to elongate the life-time of enzymes. d-lactate dehydrogenase (D-LDH) was stabilized at high temperatures with immobilization on CNT and fCNT. The kinetic and thermodynamic parameters, optimum temperature and pH, and the intrinsic fluorescence of free and immobilized enzymes were examined in the present study. Also, an attempt was made to investigate the effect of CNT and fCNT on the adsorption and conformation of d-lactate dehydrogenase using molecular dynamics (MD) simulations. In comparison with free enzyme, the immobilized enzyme displayed an improved stability at high temperatures and, therefore, the immobilized enzyme is suitable for use in the industry because most reactions in the industry happen at high temperatures. Results of the present study showed that the adsorption of enzyme on CNT is mediated through the van der Waals and π-π stacking interactions, whereas in the adsorption of enzyme on fCNT in addition to hydrophobic interactions, the hydrogen bonding between enzyme and functional groups of fCNT is involved. Moreover, RMSD, RMSF and secondary structure analysis indicate that the fCNT protects the conformation of enzyme more than CNT. Therefore, D-LDH can be efficiently immobilized upon the fCNT compared to the pristine CNT.

Efficient l-lactic acid production from corncob residue using metabolically engineered thermo-tolerant yeast

Lactic acid is an important industrial product and the production from inexpensive and renewable lignocellulose can reduce the cost and environmental pollution. In this study, a Kluyveromyces marxianus strain which produced lactic acid efficiently from corncob was constructed. Firstly, two of six different lactate dehydrogenases, which from Plasmodium falciparum and Bacillus subtilis, respectively, were proved to be effective for l-lactic acid production. Then, five single genetic modifications were conducted. The overexpression of Saccharomyces cerevisiae proton-coupled monocarboxylate transporter, K. marxianus 6-phosphofructokinase, or disruption of K. marxianus putative d-lactate dehydrogenase enhanced the l-lactic acid accumulation. Finally, the strain YKX071, obtained via combination of above effective genetic engineering, produced 103.00 g/L l-lactic acid at 42 °C with optical purity of 99.5% from corncob residue via simultaneous saccharification and co-fermentation. This study first developed an effective platform for high optical purity l-lactic acid production from lignocellulose using yeast with inexpensive nitrogen sources.

Reducing 3,4-dihydroxyphenylpyruvic acid to d-3,4-dihydroxyphenyllactic acid via a coenzyme nonspecific d-lactate dehydrogenase from Lactobacillus reuteri.

The purpose of this work was to find an efficient enzyme to synthesize d-3,4-dihydroxyphenyllactic acid (d-DSS). Nineteen lactic acid bacteria strains were screened for production of d-DSS using 3,4-dihydroxyphenylpyruvic acid (DPA) as a substrate. Lactobacillus reuteri JN516 exhibited the highest d-DSS yield. A nonspecific coenzyme, d-lactate dehydrogenase (d-LDH82319), from L. reuteri JN516 with high DPA reducing activity was identified. This enzyme reduced DPA to form d-DSS with excellent optical purity (enantioselectivity >99%). Its molecular weight was 35kDa based on SDS-PAGE migration. The Michaelis-Menten constant (Km ), turnover number (kcat ), and catalytic efficiency (kcat /Km ) of d-LDH82319 for DPA were 0·09mmoll-1 , 2·17s-1 and 24·07(mmoll-1 )-1 s-1 , respectively, with NADH as the coenzyme. The (Km ), (kcat ) and (kcat /Km ) of d-LDH82319 for DPA were 0·10mmoll-1 , 0·13s-1 and 1·30(mmoll-1 )-1 s-1 , respectively, with NADPH as the coenzyme. The optimum temperature and pH of d-LDH82319 were 25°C and pH 8 respectively. Additionally, d-LDH82319 had a broad substrate range for alpha-keto acids, among which the activity of reducing pyruvate was the strongest; therefore, it belongs to the group of d-lactate dehydrogenases. d-LDH82319 and glucose dehydrogenase (GDH) were coexpressed to produce d-DSS from DPA. d-LDH82319 from L. reuteri JN516 with high DPA reducing activity has the characteristics of a nonspecific coenzyme. d-LDH82319 is the first reported coenzyme nonspecific d-lactate dehydrogenase with DPA-reducing activity. The coexpression system provided an effective method to produce d-DSS.

Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae

D-lactic acid (D-LA) is gaining increased attention as it can improve the thermostability of poly lactic acid. Acid-tolerant Saccharomyces cerevisiae is a good host for D-LA fermentation. High catalytic efficiency of D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) is crucial for the production of D-LA in yeast. Here, a synthetic biology approach was used to construct high-producing D-LA strains by redesigning and optimization of D-LDH expression by a combination of different promoters, terminators and D-LDHs. The pyruvate decarboxylase-deficient mutant strain TAMH was used as host strain for optimizing the 40 D-LDH expression cassettes. The TCSt strain harboring the pTCSt plasmid with the TEF1 promoter, E. coliD-LDH and Synth25 synthetic short terminator produced 5.8 g/L D-LA with an optical purity of 99.9%. The production of D-LA was further improved by integrating this high expression cassette into the Ty1 transposable element of the YIP-01 strain with deleted Pdc1and Pdc6. The resulting strain YIP-pTCSt-301(CGMCC2.5726) was screened by a double enzyme-coupled system. Genomes sequencing of the strain revealed three copies of the D-LDH expression cassette. This strain was further improved by deleting the Jen1, Cyb2, Dld1, and Adh1 genes and the resulting strain YIP-J-C-D-A1 (CGMCC2.5783) produced 80.0 g/L D-LA with a yield of 0.6 g/g glucose and a volumetric productivity of 1.1 g/L/h in fed-batch fermentation under non-neutralization conditions.

Enzymological characteristics of a novel archaeal dye-linked D-lactate dehydrogenase showing loose binding of FAD.

A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD.

Structural Basis of Sequential Allosteric Transitions in Tetrameric d-Lactate Dehydrogenases from Three Gram-Negative Bacteria

d-Lactate dehydrogenases (d-LDHs) from Fusobacterium nucleatum (FnLDH) and Escherichia coli (EcLDH) exhibit positive cooperativity in substrate binding, and the Pseudomonas aeruginosa enzyme (PaLDH) shows negatively cooperative substrate binding. The apo and ternary complex structures of FnLDH and PaLDH have been determined together with the apo-EcLDH structure. The three enzymes consistently form homotetrameric structures with three symmetric axes, the P-, Q-, and R-axes, unlike Lactobacillus d-LDHs, P-axis-related dimeric enzymes, although apo-FnLDH and EcLDH form asymmetric and distorted quaternary structures. The tetrameric structure allows apo-FnLDH and EcLDH to form wide intersubunit contact surfaces between the opened catalytic domains of the two Q-axis-related subunits in coordination with their asymmetric and distorted quaternary structures. These contact surfaces comprise intersubunit hydrogen bonds and hydrophobic interactions and likely prevent the domain closure motion during initial substrate binding. In contrast, apo-PaLDH possesses a highly symmetrical quaternary structure and partially closed catalytic domains that are favorable for initial substrate binding and forms virtually no intersubunit contact surface between the catalytic domains, which present their negatively charged surfaces to each other at the subunit interface. Complex FnLDH and PaLDH possess highly symmetrical quaternary structures with closed forms of the catalytic domains, which are separate from each other at the subunit interface. Structure-based mutations successfully converted the three enzymes to their dimeric forms, which exhibited no significant cooperativity in substrate binding. These observations indicate that the three enzymes undergo typical sequential allosteric transitions to exhibit their distinctive allosteric functions through the tetrameric structures.

Catalytic, Computational, and Evolutionary Analysis of the d-Lactate Dehydrogenases Responsible for d-Lactic Acid Production in Lactic Acid Bacteria.

d-Lactate dehydrogenase (d-LDH) catalyzes the reversible reaction pyruvate + NADH + H+ ↔ lactate + NAD+, which is a principal step in the production of d-lactate in lactic acid bacteria. In this study, we identified and characterized the major d-LDH (d-LDH1) from three d-LDHs in Leuconostoc mesenteroides, which has been extensively used in food processing. A molecular simulation study of d-LDH1 showed that the conformation changes during substrate binding. During catalysis, Tyr101 and Arg235 bind the substrates by hydrogen bonds and His296 acts as a general acid/base for proton transfer. These residues are also highly conserved and have coevolved. Point mutations proved that the substrate binding sites and catalytic site are crucial for enzyme activity. Network and phylogenetic analyses indicated that d-LDH1 and the homologues are widely distributed but are most abundant in bacteria and fungi. This study expands the understanding of the functions, catalytic mechanism, and evolution of d-LDH.

Low-pH production of d-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7.

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of d-lactic acid (LA), an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d-lactate dehydrogenase gene (d-LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole-genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d-LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively.

Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.

Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse.

Enhanced biosynthesis of chiral phenyllactic acid from L-phenylalanine through a new whole-cell biocatalyst.

Phenyllactic acid (PLA) is a high-value compound, which was usually produced by lactic acid bacteria (LAB) as biocatalysts and glucose or phenylpyruvic acid (PPA) as starting materials for PLA synthesis in previous studies. However, the PLA produced using LAB is a racemic mixture. Besides, both glucose and PPA were unsatisfactory substrates, as the former could not produce high concentrations of PLA while the latter is not a renewable and green substrate. To overcome these drawbacks, in this study, a new biotransformation process was developed for chiral PLA production from L-phenylalanine via the intermediate PPA using recombinant Escherichia coli co-expressing L-amino acid deaminase, NAD-dependent L-lactate dehydrogenase or NAD-dependent D-lactate dehydrogenase, and formate dehydrogenase. After optimization, the recombinant E. coli produced L- and D-PLA at concentrations of 59.9 and 60.3mM in 6h, respectively. Hence, this process provides an effective and promising alternative method for chiral PLA production.

Efficient production of (R)-(-)-2-hydroxy-4-phenylbutyric acid by recombinant Pichia pastoris expressing engineered D-lactate dehydrogenase from Lactobacillus plantarum with a single-site mutation.

(R)-2-hydroxy-4-phenylbutyric acid (R-HPBA) is a valuable intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by oxidoreductases is an efficient approach for its synthesis. Here, we report a novel biocatalytic approach for asymmetric synthesis of R-HPBA using recombinant Pichia pastoris expressing the Tyr52Leu variant of D-lactate dehydrogenase (D-LDH) from Lactobacillus plantarum. The recombinant yeast cells showed impressive catalytic activity at a high concentration of NaOPBA (380mM, 76g/L) and achieved full conversion starting with 40g/L NaOPBA or even at higher concentration. Under optimized reaction conditions (pH 7.5, 37°C, and 2% glucose), a full conversion with > 95% reaction yield and ~ 100% product enantiomeric excess (ee) was achieved for the preparation of R-HPBA on a 2-g scale. The findings of this study promote both the biotransformation of R-HPBA and an extension of the application of recombinant yeast as biocatalysts.

Simultaneous Detection of Lactate Enantiomers Based on Diffusion-controlled Bioelectrocatalysis.

Amperometric biosensors were constructed for the simultaneous detection of lactate enantiomers. The enantioselectivity of the sensor is based on NAD-dependent l- and d-lactate dehydrogenases that, respectively, oxidize l- and d-lactates into pyruvate. The NADH formed during the enzymatic reduction was catalytically oxidized at Meldola's blue-adsorbed mesoporous electrodes. Stable amperometric measurements were performed in a two-electrode system using Ag|AgCl|sat. KCl as a counter electrode via a salt bridge. The response of the sensor reached a pseudo-steady state within 60 s. The agreement of the sensitivities for l- and d-lactates and the pseudo-steady-state characteristics of the sensors demonstrate that the current is strongly influenced by the diffusion of lactates at the edge of the electrode, enabling reproducible measurements. The pseudo steady-state characteristics are also realized at the chip-type electrode. The sensor was successfully applied for the detection of d- and l-lactates in horse serum.

Prognostic value of D-lactate dehydrogenase in patients with clear cell renal cell carcinoma.

Lactate dehydrogenase (LDH) is an enzyme involved in anaerobic glycolysis and is associated with the prognosis of patients with renal cell carcinoma (RCC). The human genome has four LDH genes: LDHA, LDHB, LDHC and LDHD. In order to determine which of these four LDH genes may predict clear cell RCC (ccRCC), a total of 509 patients with ccRCC from The Cancer Genome Atlas (TCGA) cohort and 192 patients with ccRCC from the Fudan University Shanghai Cancer Centre (FUSCC) cohort were enrolled in the present study. The expression profiles of LDHD genes in the TCGA cohort were obtained from the TCGA RNAseq database. The Cox proportional hazards regression model and Kaplan-Meier curves were used to assess relative factors. The LDH family genes that were revealed to have an association with overall survival (OS) were further validated in the FUSCC cohort. In the TCGA cohort, following Cox proportional hazards analysis, LDHD expression (P=0.0400; hazard ratio, 0.872; 95% confidence interval, 0.764–0.994) was revealed to be predictive of the prognosis of patients with ccRCC. Further analysis revealed that low LDHD expression (P<0.0001) was significantly associated with a poor prognosis in terms of OS. Additionally, the expression of LDHD (P<0.0001) was significantly different in patients with ccRCC compared with paired controls. In the FUSCC cohort, low LDHD expression was also associated with a poor OS (P=0.0103), and the tumour pathological T stage was a factor that influenced the expression of LDHD (P=0.0120). Furthermore, the expression of LDHD influenced the serum LDH level (P=0.0126). The downregulation of LDHD expression may be a predictor of poor prognosis in patients with ccRCC.

D-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum

A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10minat 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH.

Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

BackgroundPromoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Therefore, fine-tuning of metabolic pathway gene expression requires an applicable promoter system. In this study, a dissolved oxygen-dependent nar promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in Escherichia coli. To demonstrate the feasibility of using the synthetic nar promoters in production of biochemicals in E. coli, the d-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated.ResultsThe spacer sequence of 15 bp between the − 35 and − 10 elements of the upstream region of the wild-type nar promoter was randomized, fused to the GFP gene, transduced into E. coli, and screened by flow cytometry. The sorted synthetic nar promoters were divided into three groups according to fluorescence intensity levels: strong, intermediate, and weak. The selected three representative nar promoters of strong, intermediate, and weak intensities were used to control the expression level of the d-lactate and 2,3-BDO biosynthetic pathway enzymes in E. coli. When the ldhD gene encoding d-lactate dehydrogenase was expressed under the control of the strong synthetic nar promoter in fed-batch cultures of E. coli, the d-lactate titers were 105.6 g/L, 34% higher than those using the wild-type promoter (79.0 g/L). When the three 2,3-BDO pathway genes (ilvBN, aldB, and bdh1) were expressed under the control of combinational synthetic nar promoters (strong–weak–strong) in fed-batch cultures of E. coli, the titers of 2,3-BDO were 88.0 g/L, 72% higher than those using the wild-type promoter (51.1 g/L).ConclusionsThe synthetic nar promoters, which were engineered to have strong, intermediate, and weak intensities, were successfully applied to metabolic engineering of d-lactate and 2,3-BDO pathways in E. coli. By controlling expression levels of d-lactate and 2,3-BDO pathway enzymes using the synthetic nar promoters, the production of d-lactate and 2,3-BDO was increased over that using the wild-type promoter by 34 and 72%, respectively. Thus, this synthetic promoter module system will support the improved production of biochemicals and biofuels through fine-tuning of gene expression levels.

Engineering CRISPR interference system in Klebsiella pneumoniae for attenuating lactic acid synthesis

BackgroundKlebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid.ResultsHere, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae. EGFP was recruited as a reporter of this CRISPRi system. Fluorescence assay of this CRISPRi system showed that enhanced green fluorescent protein (EGFP) expression level was repressed by 85–90%. To further test this CRISPRi system, guide RNAs were designed to individually or simultaneously target four lactate-producing enzyme genes. Results showed that all lactate-producing enzyme genes were significantly repressed. Notably, d-lactate dehydrogenase (ldhA) was shown to be the most influential enzyme for lactic acid formation in micro-aerobic conditions, as inhibiting ldhA alone led to lactic acid level similar to simultaneously repressing four genes. In shake flask cultivation, the strain coexpressing puuC (an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde to 3-HP) and dCas9-sgRNA inhibiting ldhA produced 1.37-fold 3-HP relative to the reference strain. Furthermore, in bioreactor cultivation, this CRISPRi strain inhibiting ldhA produced 36.7 g/L 3-HP, but only generated 1 g/L lactic acid. Clearly, this engineered CRISPRi system largely simplified downstream separation of 3-HP from its isomer lactic acid, an extreme challenge for 3-HP bioprocess.ConclusionsThis study offers a deep understanding of lactic acid metabolism in diverse species, and we believe that this CRISPRi system will facilitate biomanufacturing and functional genome studies of K. pneumoniae or beyond.

GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Methylglyoxal(MG) is a potent cytotoxin that is produced as a byproduct of various metabolic reactions in the cell. The major enzymes for MG detoxification are Glyoxalase I(GLYI), Glyoxalase II(GLYII) and D-lactate dehydrogenase(D-LDH). These three enzymes work together and convert MG into D-pyruvate, which directly goes to TCA cycle. Here, a comparative study of the ability of MG detoxification of these three enzymes has been done in both E. coli and yeast. Ectopic expression of these three genes from Arabidopsis in E. coli in presence of different abiotic stress revealed the contribution of each of these genes in detoxifying MG. Yeast mutants of MG detoxification enzymes were also grown in different stress conditions to record the effect of each gene. These mutants were also used for complementation assays using the respective MG detoxifying genes from Arabidopsis in presence of various stress conditions. The MG content and the corresponding growth of cells was measured in all the bacterial as well as yeast strains. This study reveals differential contribution of MG detoxification enzymes in mitigating MG levels and alleviating stress in both prokaryotes as well as eukaryotes. GLYI and D-LDH were found to be key enzymes in MG detoxification under various abiotic stresses.