Synergistic effects of the KDM4C inhibitor SD70 and the menin inhibitor MI-503 against MLL::AF9-driven acute myeloid leukaemia.

MLL-rearranged (MLL-r) leukaemia is observed in approximately 10% of acute myeloid leukaemia (AML) and is associated with a relatively poor prognosis, highlighting the need for new treatment regimens. MLL fusion proteins produced by MLL rearrangements recruit KDM4C to mediate epigenetic reprogramming, which is required for the maintenance of MLL-r leukaemia. In this study, we used a combinatorial drug screen to selectively identify synergistic treatment partners for the KDM4C inhibitor SD70. The results showed that the drug combination of SD70 and MI-503, a potent menin-MLL inhibitor, induced synergistically enhanced apoptosis in MLL::AF9 leukaemia cells without affecting normal CD34+ cells. In vivo treatment with SD70 and MI-503 significantly prolonged survival in AML xenograft models. Differential gene expression analysis by RNA-seq following combined pharmacological inhibition of SD70 and MI-503 revealed changes in numerous genes, with MYC target genes being the most significantly downregulated. Taken together, these data provide preclinical evidence that the combination of SD70 and MI-503 is a potential dual-targeted therapy for MLL::AF9 AML.

Coexistence of Ferromagnetism and Ferroelectricity in Cu-Intercalated Bilayer CrI3.

Design of two-dimensional (2D) multiferroic materials with two or more ferroic orders in one structure is highly desired in view of the development of next-generation electronic devices. Unfortunately, experimental or theoretical discovery of 2D intrinsic multiferroic materials is rare. Using first-principles calculation methods, we report the realization of multiferroics that couple ferromagnetism and ferroelectricity by intercalating Cu atoms in bilayer CrI3, Cux@bi-CrI3 (x = 0.03, 0.06, and 0.25). Our results show that the intercalation of Cu atoms leads to the inversion symmetry breaking of bilayer CrI3 and produces intercalation density-dependent out-of-plane electric polarization, around 18.84-90.31 pC·cm-2. Moreover, the switch barriers of Cux@bi-CrI3 in both polarization states are small, ranging from 0.31 to 0.69 eV. Furthermore, the magnetoelectric coupling properties of Cux@bi-CrI3 can be modulated via varying the metal ion intercalation density, and half-metal to semiconductor transition can be occurred by decreasing the intercalation density of metal ions. Our work paves a practical path for 2D magnetoelectron coupling devices.

Simulation on two-phase refrigerant compression in the cylinder of rotary compressors using CFD method.

The two-phase compression process in the rotary compressor often occurs, such as defrosting and startup processes, which has a significant impact on the performance and reliability of air conditioning systems. In this paper, the CFD simulations predicting the two-phase refrigerant compression process in the compressor cylinder are conducted using the commercial software ANSYS Fluent. The dynamic mesh for the fluid domain and phase change model for the refrigerant are considered in the simulation. Effects of initial liquid volume fraction, refrigerant type and compressor type on the two-phase compression characteristics using R290 as refrigerant are carried out. Variations of the pressure, temperature, gas fraction distribution and evaporation rate in the cylinder are discussed. The results show that most liquid accumulates near the leakage gap and the bottom of the compression chamber during the two-phase compression process. The peak pressure during the two-phase compression decreases with the increase of the liquid volume fraction. The evaporation rate of R32 in the cylinder is much higher than that of R290. The maximum pressure of the reciprocating compressor is 2.26 times higher than that of the rotary compressor.

Twist family BHLH transcription factor 1 is required for the maintenance of leukemia stem cell in MLL-AF9+ acute myeloid leukemia.

Leukemia stem cells (LSC) are a rare population capable of limitless self-renewal and are responsible for the initiation, maintenance, and relapse of leukemia. Elucidation of the mechanisms underlying the regulation of LSC function could provide novel treatment strategies. Here, we show that TWIST1 is extremely highly expressed in the LSC of MLL-AF9+ acute myeloid leukemia (AML), and its upregulation is positively regulated by KDM4C in a H3K9me3 demethylation-dependent manner. We further demonstrate that TWIST1 is essential for the viability, dormancy, and self-renewal capacities of LSC, and that it promotes the initiation and maintenance of MLL-AF9-mediated AML. In addition, TWIST1 directly interacts and collaborates with HOXA9 in inducing AML in mice. Mechanistically, TWIST1 exerts its oncogenic function by activating the WNT5a/RAC1 axis. Collectively, our study uncovers a critical role of TWIST1 in LSC function and provides new mechanistic insights into the pathogenesis of MLL-AF9+ AML.

Two-Dimensional Transition Metal Boride TMB12 (TM = V, Cr, Mn, and Fe) Monolayers: Robust Antiferromagnetic Semiconductors with Large Magnetic Anisotropy.

Currently, two-dimensional (2D) materials with intrinsic antiferromagnetism have stimulated research interest due to their insensitivity to external magnetic fields and absence of stray fields. Here, we predict a family of stable transition metal (TM) borides, TMB12 (TM = V, Cr, Mn, Fe) monolayers, by combining TM atoms and B12 icosahedra based on first-principles calculations. Our results show that the four TMB12 monolayers have stable antiferromagnetic (AFM) ground states with large magnetic anisotropic energy. Among them, three TMB12 (TM=V, Cr, Mn) monolayers display an in-plane easy magnetization axis, while the FeB12 monolayer has an out-of-plane easy magnetization axis. Among them, the CrB12 and the FeB12 monolayers are AFM semiconductors with band gaps of 0.13 eV and 0.35 eV, respectively. In particular, the AFM FeB12 monolayer is a spin-polarized AFM material with a Néel temperature of 125 K. Moreover, the electronic and magnetic properties of the CrB12 and the FeB12 monolayers can be modulated by imposing external biaxial strains. Our findings show that the TMB12 monolayers are candidates for designing 2D AFM materials, with potential applications in electronic devices.

The effect of switchable electronic polarization states on the electronic properties of two-dimensional multiferroic TMBr2/Ga2SSe2 (TM = V-Ni) heterostructures.

Multiferroic van der Waals (vdW) heterostructures (HSs) prepared by combining different ferroic materials offer an exciting platform for next-generation nanoelectronic devices. In this work, we investigate the magnetoelectric coupling properties of multiferroic vdW HSs consisting of a magnetic TMBr2 (TM = V-Ni) monolayer and a ferroelectric Ga2SSe2 monolayer using first-principles theory calculations. It is found that the magnetic orderings in the magnetic TMBr2 layers are robust and the band alignment of these TMBr2/Ga2SSe2 HSs can be altered by reversing the polarization direction of the ferroelectric layer. Among them, VBr2/Ga2SSe2 and FeBr2/Ga2SSe2 HSs can be switched from a type-I to a type-II semiconductor, which allows the generation of spin-polarized and unpolarized photocurrent. Besides, CrBr2/Ga2SSe2, CoBr2/Ga2SSe2 and NiBr2/Ga2SSe2 exhibit a type-II band alignment in reverse ferroelectric polarization states. Moreover, the magnetic configuration and band alignment of these TMBr2/Ga2SSe2 HSs can be further modulated by applying an external strain. Our findings suggest the potential of TMBr2/Ga2SSe2 HSs in 2D multiferroic and spintronic applications.

Genome-Wide Characterization and Expression Analysis of Pathogenesis-Related 1 (PR-1) Gene Family in Tea Plant (Camellia sinensis (L.) O. Kuntze) in Response to Blister-Blight Disease Stress.

Pathogenesis-related 1 (PR-1) proteins, which are defense proteins in plant-pathogen interactions, play an important role in the resistance and defense of plants against diseases. Blister blight disease is caused by Exobasidium vexans Massee and a major leaf disease of tea plants (Camellia sinensis (L.) O. Kuntze). However, the systematic characterization and analysis of the PR-1 gene family in tea plants is still lacking, and the defense mechanism of this family remains unknown. In this study, 17 CsPR-1 genes were identified from the tea plant genome and classified into five groups based on their signal peptide, isoelectric point, and C-terminus extension. Most of the CsPR-1 proteins contained an N-terminal signal peptide and a conserved PR-1 like domain. CsPR-1 genes comprised multiple cis-acting elements and were closely related to the signal-transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. These characteristics imply an important role of the genes in the defense of the tea plant. In addition, the RNA-seq data and real-time PCR analysis demonstrated that the CsPR-1-2, -4, -6, -7, -8, -9, -10, -14, -15, and -17 genes were significantly upregulated under tea blister-blight stress. This study could help to increase understanding of CsPR-1 genes and their defense mechanism in response to tea blister blight.

Association between pharmacokinetics of lenvatinib in healthy subjects and genetic polymorphisms of ABCB1 3435C>T and ABCB1 2677G>T/A.

The aim of this study was to evaluate the impact of genetic polymorphisms in the pharmacokinetics of metabolism and transportation of lenvatinib in the Chinese population.Sixty-three healthy Chinese individuals were recruited and administered with a single dose of 4 mg lenvatinib. Allelic discriminations for 10 SNPs of CYP3A4 (20230 G>A(*1G)), CYP3A5 (6986 A>G(*3)), ABCB1 (1236 C>T, 2677 G>T/A, 3435 C>T), ABCG2 (421 C>A, 34 G>A), ABCC2 (-24 C>T, 1249 G>A, 3972 C>T) were performed. The concentrations of lenvatinib in the plasma were determined by UPLC-MS/MS.Under the fasting condition, individuals carrying of ABCB1 3435 C>T genotype presented lower Cmax (p < 0.01) and λz (p < 0.05), but higher t1/2 (p < 0.05) than those carrying C/C and T/T genotypes. For ABCB1 2677 G>T/A variant, individuals with the G/T and A/G genotype showed higher AUC (p < 0.05) and t1/2 (p < 0.01), but lower λz (p < 0.05) than those carrying G/G genotypes. Individuals with the A/T, A/A and T/T genotype had higher AUC, but no significant differences (p > 0.05) were observed. They also had higher t1/2 (p < 0.01) and lower λz (p < 0.01) than those carrying G/G genotypes.Under the fed condition, no difference in any pharmacokinetic parameters were observed with any polymorphisms in the 10 fragments.Data in this paper had demonstrated that polymorphisms ABCB1 3435 C>T and ABCB1 2677 G>T/A were associated with the pharmacokinetic variability of lenvatinib.

Analgesic and physiological effect of electroacupuncture combined with epidural lidocaine in goats.

OBJECTIVE: To investigate physiological and antinociceptive effects of electroacupuncture (EA) with lidocaine epidural nerve block in goats. STUDY DESIGN: Prospective experimental trial. ANIMALS: Forty-eight hybrid male goats weighing 27 ± 2 kg. METHODS: The goats were randomly assigned to six groups: L2.2, epidural lidocaine (2.2 mg kg-1); L4.4, epidural lidocaine (4.4 mg kg-1); EA; EA-L1.1, EA with epidural lidocaine (1.1 mg kg-1); EA-L2.2, EA with epidural lidocaine (2.2 mg kg-1); and EA-L4.4, EA with epidural lidocaine (4.4 mg kg-1). EA was administered for 120 minutes. Epidural lidocaine was administered 25 minutes after EA started. Nociceptive thresholds of flank and thigh regions, abdominal muscle tone, mean arterial pressure (MAP), heart rate (HR), respiratory frequency (fR) and rectal temperature were recorded at 30, 60, 90, 120, 150 and 180 minutes. RESULTS: Lidocaine dose-dependently increased nociceptive thresholds. There were no differences in nociceptive thresholds between L4.4 and EA from 30 to 120 minutes. The threshold in EA-L2.2 was lower than in EA-L4.4 from 30 to 120 minutes, but higher than in EA-L1.1 from 30 to 150 minutes or in L4.4 from 30 to 180 minutes. The abdominal muscle tone in EA-L2.2 was higher at 30 minutes, but lower at 90 and 120 minutes than at 0 minutes. There were no differences in muscle tone between L4.4 and L2.2 or EA-L4.4, and between any two of the three EA-lidocaine groups from 0 to 180 minutes. The fR and HR decreased in L4.4 at 60 and 90 minutes compared with 0 minutes. No differences in fR, HR, MAP and temperature among the groups occurred from 30 to 180 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: EA combined with 2.2 mg kg-1 epidural lidocaine provides better antinociceptive effect than 4.4 mg kg-1 epidural lidocaine alone in goats. EA provided antinociception and allowed a decrease in epidural lidocaine dose.

Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display.

Vaccine reverse engineering is emerging as an important approach to vaccine antigen identification, recently focusing mainly on structural characterization of interactions between neutralizing monoclonal antibodies (mAbs) and antigens. Using mAbs that bind unknown antigen structures, we sought to probe the intrinsic features of antibody antigen-binding sites with a high complexity peptide library, aiming to identify conformationally optimized mimotope antigens that capture mAb-specific epitopes. Using a high throughput sequencing-enhanced messenger ribonucleic acid (mRNA) display approach, we identified high affinity binding peptides for a hepatitis C virus neutralizing mAb. Immunization with the selected peptides induced neutralizing activity similar to that of the original mAb. Antibodies elicited by the most commonly selected peptides were predominantly against specific epitopes. Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available. RESEARCH IN CONTEXT: We used a large number of randomly produced small proteins ("peptides") to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

Herpes simplex virus 2 expresses a novel form of ICP34.5, a major viral neurovirulence factor, through regulated alternative splicing.

Herpes simplex virus 1 (HSV-1) and HSV-2, two closely related neurotropic human herpesviruses, achieve neurotropism through ICP34.5, a major viral neurovirulence factor. In this report, in addition to the full-length 38-kDa protein (ICP34.5α), we identified a 28-kDa novel form of ICP34.5 (ICP34.5ß) in HSV-2-infected cells. ICP34.5ß is translated from unspliced ICP34.5 mRNA, with the retained intron introducing a premature stop codon. Thus, ICP34.5ß lacks the C-terminal conserved GADD34 domain but includes 19 additional amino acids encoded by the intron. Although a fraction of both HSV-2 ICP34.5 proteins are detected in the nucleolus, ICP34.5α is predominantly located in cytoplasm, and ICP34.5ß is mainly detected more diffusely in the nucleus. ICP34.5ß is unable to counteract PKR-mediated eIF2 phosphorylation but does not interfere with ICP34.5α's function in this process. Efficient expression of ICP34.5ß in cell culture assays is dependent on viral infection or expression of ICP27, a multifunctional immediate-early gene. The effect of ICP27 on the ICP34.5ß protein level is attributed to its selective inhibition of ICP34.5 splicing, which results in increased expression of ICP34.5ß but a reduced level of ICP34.5α. The C- terminal KH3 domain but not the RNA binding domain of ICP27 is required for its specific inhibition of ICP34.5 splicing and promotion of ICP34.5ß expression. Our results suggest that the expression of ICP34.5α and ICP34.5ß is tightly regulated in HSV-2 and likely contributes to viral pathogenesis.

The gastrointestinal manifestations of telomere-mediated disease.

Defects in telomere maintenance genes cause pathological telomere shortening, and manifest in syndromes which have prominent phenotypes in tissues of high turnover: the skin and bone marrow. Because the gastrointestinal (GI) epithelium is highly proliferative, we sought to determine whether telomere syndromes cause GI disease, and to define its prevalence, spectrum, and natural history. We queried subjects in the Johns Hopkins Telomere Syndrome Registry for evidence of luminal GI disease. In sixteen percent of Registry subjects (6 of 38), there was a history of significant GI pathology, and 43 additional cases were identified in the literature. Esophageal stenosis, enteropathy, and enterocolitis were the recurrent findings. In the intestinal mucosa, there was striking villous atrophy, extensive apoptosis, and anaphase bridging pointing to regenerative defects in the epithelial compartment. GI disease was often the first and most severe manifestation of telomere disease in young children. These findings indicate that telomere dysfunction disrupts the epithelial integrity in the human GI tract manifesting in recognizable disease processes. A high index of suspicion should facilitate diagnosis and management.

Frizzled 2 and frizzled 7 function redundantly in convergent extension and closure of the ventricular septum and palate: evidence for a network of interacting genes.

Frizzled (Fz) 2 and Fz7, together with Fz1, form a distinct subfamily within the Frizzled family of Wnt receptors. Using targeted gene deletion, we show that: Fz7(-/-) mice exhibit tail truncation and kinking with 100% penetrance and ventricular septal defects (VSDs) with ~15% penetrance; Fz2(+/-);Fz7(-/-) mice exhibit VSDs with ~50% penetrance and cleft palate with less than 10% penetrance; and Fz2(-/-);Fz7(-/-) mice exhibit convergent extension defects and mid-gestational lethality with 100% penetrance. When Fz2 and/or Fz7 mutations are combined with mutations in Vangl2, Dvl3, Wnt3a, Wnt5a or Wnt11, an increased frequency of VSDs is observed with Dvl3, Wnt3a and Wnt11; an increased frequency of palate closure defects is observed with Vangl2; and early lethality and enhanced tail shortening are observed with Wnt5a. To assess the signaling pathways that underlie these and other Frizzled-mediated genetic interactions, we used transfected mammalian cells to analyze (1) canonical Wnt signaling induced by all pairwise combinations of the ten mouse Frizzleds and the 19 mouse Wnts and (2) localization of each Frizzled at cell-cell junctional complexes formed by mouse Celsr1, a likely indicator of competence for planar cell polarity signaling. These in vitro experiments indicate that Fz2 and Fz7 are competent to signal via the canonical pathway. Taken together, the data suggest that genetic interactions between Fz2, Fz7 and Vangl2, Dvl3 and Wnt genes reflect interactions among different signaling pathways in developmental processes that are highly sensitive to perturbations in Frizzled signaling.

Telomere length is a determinant of emphysema susceptibility.

RATIONALE: Germline mutations in the enzyme telomerase cause telomere shortening, and have their most common clinical manifestation in age-related lung disease that manifests as idiopathic pulmonary fibrosis. Short telomeres are also a unique heritable trait that is acquired with age. OBJECTIVES: We sought to understand the mechanisms by which telomerase deficiency contributes to lung disease. METHODS: We studied telomerase null mice with short telomeres. MEASUREMENTS AND MAIN RESULTS: Although they have no baseline histologic defects, when mice with short telomeres are exposed to chronic cigarette smoke, in contrast with controls, they develop emphysematous air space enlargement. The emphysema susceptibility did not depend on circulating cell genotype, because mice with short telomeres developed emphysema even when transplanted with wild-type bone marrow. In lung epithelium, cigarette smoke exposure caused additive DNA damage to telomere dysfunction, which limited their proliferative recovery, and coincided with a failure to down-regulate p21, a mediator of cellular senescence, and we show here, a determinant of alveolar epithelial cell cycle progression. We also report early onset of emphysema, in addition to pulmonary fibrosis, in a family with a germline deletion in the Box H domain of the RNA component of telomerase. CONCLUSIONS: Our data indicate that short telomeres lower the threshold of cigarette smoke-induced damage, and implicate telomere length as a genetic susceptibility factor in emphysema, potentially contributing to its age-related onset in humans.

Phenotypes in mTERT⁺/⁻ and mTERT⁻/⁻ mice are due to short telomeres, not telomere-independent functions of telomerase reverse transcriptase.

Telomerase is essential for telomere length maintenance. Mutations in either of the two core components of telomerase, telomerase RNA (TR) or the catalytic protein component telomerase reverse transcriptase (TERT), cause the genetic disorders dyskeratosis congenita, pulmonary fibrosis, and other degenerative diseases. Overexpression of the TERT protein has been reported to have telomere length-independent roles, including regulation of the Wnt signaling pathway. To examine the phenotypes of TERT haploinsufficiency and determine whether loss of function of TERT has effects other than those associated with telomere shortening, we characterized both mTERT⁺/⁻ and mTERT⁻/⁻ mice on the CAST/EiJ genetic background. Phenotypic analysis showed a loss of tissue renewal capacity with progressive breeding of heterozygous mice that was indistinguishable from that of mTR-deficient mice. mTERT⁻/⁻ mice, from heterozygous mTERT⁺/⁻ mouse crosses, were born at the expected Mendelian ratio (26.5%; n = 1,080 pups), indicating no embryonic lethality of this genotype. We looked for, and failed to find, hallmarks of Wnt deficiency in various adult and embryonic tissues, including those of the lungs, kidneys, brain, and skeleton. Finally, mTERT⁻/⁻ cells showed wild-type levels of Wnt signaling in vitro. Thus, while TERT overexpression in some settings may activate the Wnt pathway, loss of function in a physiological setting has no apparent effects on Wnt signaling. Our results indicate that both TERT and TR are haploinsufficient and that their deficiency leads to telomere shortening, which limits tissue renewal. Our studies imply that hypomorphic loss-of-function alleles of hTERT and hTR should cause a similar disease spectrum in humans.

Short telomeres compromise ß-cell signaling and survival.

The genetic factors that underlie the increasing incidence of diabetes with age are poorly understood. We examined whether telomere length, which is inherited and known to shorten with age, plays a role in the age-dependent increased incidence of diabetes. We show that in mice with short telomeres, insulin secretion is impaired and leads to glucose intolerance despite the presence of an intact ß-cell mass. In ex vivo studies, short telomeres induced cell-autonomous defects in ß-cells including reduced mitochondrial membrane hyperpolarization and Ca(2+) influx which limited insulin release. To examine the mechanism, we looked for evidence of apoptosis but found no baseline increase in ß-cells with short telomeres. However, there was evidence of all the hallmarks of senescence including slower proliferation of ß-cells and accumulation of p16(INK4a). Specifically, we identified gene expression changes in pathways which are essential for Ca(2+)-mediated exocytosis. We also show that telomere length is additive to the damaging effect of endoplasmic reticulum stress which occurs in the late stages of type 2 diabetes. This additive effect manifests as more severe hyperglycemia in Akita mice with short telomeres which had a profound loss of ß-cell mass and increased ß-cell apoptosis. Our data indicate that short telomeres can affect ß-cell metabolism even in the presence of intact ß-cell number, thus identifying a novel mechanism of telomere-mediated disease. They implicate telomere length as a determinant of ß-cell function and diabetes pathogenesis.

Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder.

Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

Consequences of cps mutation of Klebsiella pneumoniae on 1,3-propanediol fermentation.

The filtration in 1,3-propanediol (1,3-PD) downstream process is influenced by the large amounts of capsular polysaccharides (CPS) produced by Klebsiella pneumoniae CGMCC 1.6366. The morphological and fermentation properties were investigated with the CPS-deficient mutant K. pneumoniae CGMCC 1.6366 CPS. Similar biomass was obtained with CGMCC 1.6366, and the mutant strain in batch cultures indicating the cell growth was slightly inhibited by CPS defection. The viscosity of fermentation broth by mutant strain decreased by 27.45%. The flux with ceramic membrane filter was enhanced from 168.12 to 303.6 l h(-1) m(-2), exhibiting the great importance for downstream processing of 1,3-PD fermentation. The products spectrum of mutant isolate changed remarkably regarding to the concentration of fermentation products. The synthesis of important 1,3-PD and 2,3-butanediol was enhanced from 9.73 and 4.06 g l(-1) to 10.37 and 4.77 g l(-1) in batch cultures. The noncapsuled K. pneumoniae provided higher 1,3-PD yield of 0.54 mol mol(-1) than that of encapsuled wild parent in batch cultures. The fed-batch fermentation of mutant strain resulted in 1,3-PD concentration, yield, and productivity of 78.13 g l(-1), 0.53 mol mol(-1), and 1.95 g l(-1) h(-1), respectively.

Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae HR526, a new isolated 1,3-propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D-lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D-lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments using pure glycerol as feedstock, the 1,3-PD concentrations, conversion, and productivity increased from 95.39 g L(-1), 0.48 and 1.98 g L(-1) h(-1) to 102. 06 g L(-1), 0.52 mol mol(-1) and 2.13 g L(-1) h(-1), respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55 mol mol(-1) to a maximum of 0.65 mol mol(-1). Lactate would not accumulate until 1,3-PD exceeded 84 g L(-1), and the final lactate concentration decreased dramatically from more than 40 g L(-1) to <3 g L(-1). Enzymic measurements showed LDH activity decreased by 89-98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD(+) enhanced more than 50% in the late-exponential phase as the D-lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production.

Physiologic mechanisms of sequential products synthesis in 1,3-propanediol fed-batch fermentation by Klebsiella pneumoniae.

The glycerol fed-batch fermentation by Klebsiella pneumoniae CGMCC 1.6366 exhibited the sequential synthesis of products, including acetate, 1,3-propanediol (1,3-PD), 2,3-butanediol, ethanol, succinate, and lactate. The dominant flux distribution was shifted from acetate formation to 1,3-PD formation in early- exponential growth phase and then to lactate synthesis in late-exponential growth phase. The underlying physiological mechanism of the above observations has been investigated via the related enzymes, nucleotide, and intermediary metabolites analysis. The carbon flow shift is dictated by the intrinsic physiological state and enzymatic activity regulation. Especially, the internal redox state could serve as a rate-controlling factor for 1,3-PD production. The q(1,3-PD) formation was the combined outcomes of regulations of glycerol dehydratase activity and internal redox balancing. The q(ethanol)/q(acetate) ratios demonstrated the flexible adaptation mechanism of K. pneumoniae preferring ATP generation in early-exponential growth phase. A low PEP to pyruvate ratio corresponded LDH activity increase, leading to lactate accumulation in stationary phase.