The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Molecular Weight ; Recombinant Proteins ; biosynthesis ; Temperature ; Thermotoga maritima ; enzymology

The contribution of particles to cardiovascular mortality and morbidity has been enlightened by epidemiologic and experimental studies. However, adverse biological effects of the particles with different sizes on cardiovascular cells have not been well recognized. In this study, sub-cultured human umbilical vein endothelial cells (HUVECs) were exposed to increasing concentrations of pure quartz particles (DQ) of three sizes (DQPM1, <1 μm; DQPM3-5, 3-5 μm; DQPM5, 5 μm) and carbon black particles of two sizes (CB0.1, <0.1 μm; CB1, <1 μm) for 24 h. Cytotoxicity was estimated by measuring the activity of lactate dehydrogenase (LDH) and cell viability. Nitric oxide (NO) generation and cytokines (TNF-α and IL-1β) releases were analyzed by using NO assay and enzyme-linked immunoabsorbent assay (ELISA), respectively. It was found that both particles induced adverse biological effects on HUVECs in a dose-dependent manner. The size of particle directly influenced the biological activity. For quartz, the smaller particles induced stronger cytotoxicity and higher levels of cytokine responses than those particles of big size. For carbon black particles, CB0.1 was more capable of inducing adverse responses on HUVECs than CB1 only at lower particle concentrations, in contrast to those at higher concentrations. Meanwhile, our data also revealed that quartz particles performed stronger cell damage and produced higher levels of TNF-α than carbon black particles, even if particles size was similar. In conclusion, particle size as well as particle composition should be both considered in assessing vascular endothelial cells injury and inflammation responses induced by particles.
Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Interleukin-1beta ; secretion ; L-Lactate Dehydrogenase ; metabolism ; Nitric Oxide ; biosynthesis ; Particle Size ; Particulate Matter ; chemistry ; pharmacology ; Quartz ; chemistry ; Soot ; chemistry ; Time Factors ; Tumor Necrosis Factor-alpha ; secretion

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.
Aerobiosis ; Anaerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Glycerol ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; biosynthesis ; Promoter Regions, Genetic ; genetics ; Temperature

Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.
Corynebacterium glutamicum ; genetics ; metabolism ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Industrial Microbiology ; Isocitrate Lyase ; biosynthesis ; genetics ; L-Lactate Dehydrogenase ; genetics ; Succinic Acid ; metabolism ; Transduction, Genetic

High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (P(ldhA)) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, P(ldhA)-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD::P(ldhA)-BcoaLDH, deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA and deltaldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90 g/Lh and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%.
Bacillus ; enzymology ; Cloning, Molecular ; Escherichia coli ; enzymology ; genetics ; metabolism ; Glycerol ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; genetics ; metabolism

Escherichia coli strain NZN111 is a promising candidate for the fermentative production of succinate. However, because lactate dehydrogenase and pyruvate formate lyase were inactivated in NZN111, this strain had an unbalanced NADH/NAD+ ratio and could not use glucose under anaerobic conditions. In this study, a recombinant strain E. coli NZN111/pTrc99a-pncB was constructed to overexpress the nicotinic acid phosphoribosyl transferase gene (pncB). Under anaerobic conditions with the addition of 0.5 mmol/L nicotinic acid and 0.3 mmol/L isopropyl beta-D-thiogalactopyranoside (IPTG), the specific nicotinic acid phosphoribosyl transferase (NAPRTase, EC 2.4.2.11) activity in the recombinant strain was 11-fold higher than that in E. coli NZN111, the concentration of NAD(H) was increased by 3.85-fold, especially the concentration of NAD+ was increased by 5.17-fold and NADH/NAD+ was decreased from 0.640 to 0.125. The recombinant strain regained the capability of growth and glucose utilization under anaerobic conditions.
Acetyltransferases ; genetics ; metabolism ; Anaerobiosis ; Escherichia coli ; classification ; genetics ; metabolism ; Fermentation ; Genetic Enhancement ; methods ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; NAD ; metabolism ; Nicotinamide Phosphoribosyltransferase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L x h).
Acetyltransferases ; genetics ; Anaerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Knockout Techniques ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; genetics ; Malate Dehydrogenase ; genetics ; metabolism ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Succinic Acid ; metabolism

Alanine Transaminase ; blood ; Animals ; Antibodies ; pharmacology ; Apoptosis ; Collagen Type I ; biosynthesis ; DNA-Binding Proteins ; metabolism ; Disease Models, Animal ; Insulin-Like Growth Factor Binding Protein 1 ; immunology ; L-Lactate Dehydrogenase ; blood ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; drug therapy ; immunology ; metabolism ; Male ; Mice ; Protective Agents ; pharmacology ; Thioacetamide ; Transforming Growth Factor beta ; metabolism