Improved production of propionic acid in Propionibacterium jensenii via combinational overexpression of glycerol dehydrogenase and malate dehydrogenase from Klebsiella pneumoniae.

Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA.

Engineering propionibacteria as versatile cell factories for the production of industrially important chemicals: advances, challenges, and prospects.

Propionibacteria are actinobacteria consisting of two principal groups: cutaneous and dairy. Cutaneous propionibacteria are considered primary pathogens to humans, whereas dairy propionibacteria are widely used in the food and pharmaceutical industries. Increasing attention has been focused on improving the performance of dairy propionibacteria for the production of industrially important chemicals, and significant advances have been made through strain engineering and process optimization in the production of flavor compounds, nutraceuticals, and antimicrobial compounds. In addition, genome sequencing of several propionibacteria species has been completed, deepening understanding of the metabolic and physiological features of these organisms. However, the metabolic engineering of propionibacteria still faces several challenges owing to the lack of efficient genome manipulation tools and the existence of various types of strong restriction-modification systems. The emergence of systems and synthetic biology provides new opportunities to overcome these bottlenecks. In this review, we first introduce the major species of propionibacteria and their properties and provide an overview of their functions and applications. We then discuss advances in the genome sequencing and metabolic engineering of these bacteria. Finally, we discuss systems and synthetic biology approaches for engineering propionibacteria as efficient and robust cell factories for the production of industrially important chemicals.

[Effect of PD-1 deficiency on atherogenic immune responses].

OBJECTIVE: To explore the functional changes of T lymphocyte from PD-1⁻/⁻ mice and the effects on atherogenic immune responses. METHODS: PD-1⁻/⁻ mice were mated with ApoE⁻/⁻ mice to obtain PD-1⁻/⁻ApoE⁻/⁻mice. PD-1⁻/⁻ApoE⁻/⁻ mice were used as control. Both groups had a high-lipid diet for 12 weeks. Then the samples of aortic sinuses were harvested for oil-red and immunohistochemical stains. Samples of spleen and sera were obtained. Numbers of lymphocytes in spleen were evaluated by flow cytometry (FACS). And the cells of proliferation were also measured. The intracellular and serum cytokines were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: After a 12-week high-lipid diet, aortic root revealed more lesions in PD-1⁻/⁻ApoE⁻/⁻ mice than in PD-1⁺/⁺ ApoE⁻/⁻ controls ((0.51 ± 0.08) vs (0.29 ± 0.06) mm², t = 2.36, P < 0.01)). There was a significant increase in CD3 T cells in the lesions of PD-1⁻/⁻ApoE⁻/⁻ mice ((154.88 ± 36.64)/mm² vs (59.38 ± 25.28)/mm², t = 2.14, P < 0.01). In PD-1⁻/⁻ApoE⁻/⁻ mice, the numbers of total cells ((8.59 ± 0.55)× 107) and cells in CD4⁺ and CD8⁺ T lymphocyte subsets ((1.53 ± 0.18)× 107 and (1.41 ± 0.15)×107), increased significantly than those in control mice ((6.29 ± 0.39)×107, (0.94 ± 0.10)×107 and (0.70 ± 0.09)× 107 respectively, t = 3.43, 2.88, 4.03, all P < 0.05). Same changes were detected in both CD3⁺ CD62L(-) and CD3⁺ CD25⁺ cells. PD-1 deficiency in both CD4⁺ and CD8⁺ T cells resulted in enhanced proliferation by either macrophages or dendritic cells as antigen presenting cells (APCs). The supernatant levels of interleukin-2 (IL-2) ((53.38 ± 5.94) pg/ml), interferon-γ (IFN-γ) ((114.50 ± 9.69) pg/ml) and tumor necrosis factor-α (TNF-α) ((326.00 ± 22.25) pg/ml ) from PD-1⁻/⁻ T cells co-cultured with CD3 were significantly higher than those of control T cells ((15.63 ± 2.23), (33.25 ± 4.16) and (64.50 ± 8.17) pg/ml respectively, t = 5.95, 7.70, 11.03, all P < 0.01). However, only the serum levels of TNF-α increased in PD-1⁻/⁻ApoE⁻/⁻mice compared with control. CONCLUSIONS: PD-1 deficiency in T cells increase markedly immune responses. And it is one of the mechanisms in atherogenesis.

Development of a Propionibacterium-Escherichia coli shuttle vector for metabolic engineering of Propionibacterium jensenii, an efficient producer of propionic acid.

Propionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have included Propionibacterium thoenii, P. freudenreichii, and P. acidipropionici. In this report, we show that P. jensenii ATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, a P. jensenii-Escherichia coli shuttle vector was developed for the metabolic engineering of P. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated from P. acidipropionici ATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, a P. jensenii-Escherichia coli shuttle vector was constructed using the pZGX01 plasmid, the E. coli pUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed into P. jensenii ATCC 4868 and 4870, but it also could be transformed into freudenreichii ATCC 6207 subspecies of P. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) from Klebsiella pneumoniae was expressed in P. jensenii ATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineered P. jensenii ATCC 4868 strain reached 28.23 ± 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ± 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering of P. jensenii.

[Correlation of elevated serum CXCL16 and severity of acute coronary syndromes in male elders].

OBJECTIVE: To examine the level of circulating soluble CXC-chemokine ligand 16 (CXCL16) in different types of coronary artery disease and its association with severity of disease. METHODS: A total of 204 male elders, aged 60 - 92 years old, were recruited and divided into control group (n = 40), stable angina pectoris group (SAP, n = 80) and acute coronary syndrome group (ACS, n = 84). enzyme-linked immunosorbent assay (ELISA) was used to examine the concentration of serum soluble CXCL16. RESULTS: As compared with the control (1.87 mg/L) and SAP groups (2.01 mg/L), CXCL16 level was significantly increased in ACS group (2.35 mg/L) P = (0.001, 0.026). Correlation analysis showed that CXCL16 was associated with age (r = 0.152, P = 0.038) and C-reactive protein (CRP) (r = 0.270, P < 0.001). When categorizing CXCL16 into quartiles, logistic analysis adjusted for age and CRP showed that the higher CXCL16 concentrations significantly increased the risk of ACS but not SAP. The odds ratio of group IV OR (95%CI) were 7.91(2.41 - 21.54) versus group I as reference in ACS. CONCLUSION: The elevated level of circulating soluble CXCL16 is associated with severity of disease in male elder ACS patients.

Physiological and Transcriptional Responses of Streptomyces albulus to Acid Stress in the Biosynthesis of ε-Poly-L-lysine.

Streptomyces albulus has commercially been used for the production of ε-poly-L-lysine (ε-PL), a natural food preservative, where acid stress is inevitably encountered in the biosynthesis process. To elucidate the acid tolerance response (ATR), a comparative physiology and transcriptomic analysis of S. albulus M-Z18 at different environmental pH (5.0, 4.0, and 3.0) was carried out. In response to acid stress, cell envelope regulated the membrane fatty acid composition and chain length to reduce damage. Moreover, intracellular pH homeostasis was maintained by increasing H+-ATPase activity and intracellular ATP and amino acid (mainly arginine, glutamate, aspartate and lysine) concentrations. Transcriptional analysis based on RNA-sequencing indicated that acid stress aroused global changes and the differentially expressed genes involved in transcriptional regulation, stress-response protein, transporter, cell envelope, secondary metabolite biosynthesis, DNA and RNA metabolism and ribosome subunit. Consequently, the ATR of S. albulus was preliminarily proposed. Notably, it is indicated that the biosynthesis of ε-PL is also a response mechanism for S. albulus to combat acid stress. These results provide new insights into the ATR of S. albulus and will contribute to the production of ε-PL via adaptive evolution or metabolic engineering.

Hydrogen Gas Treatment Improves the Neurological Outcome After Traumatic Brain Injury Via Increasing miR-21 Expression.

Hydrogen gas (H2) exerts a beneficial effect against traumatic brain injury (TBI). microRNA-21 (miR-21) is one of the most highly expressed members of small non-coding microRNA family in mammalian cells. miR-21 can improve the neurological outcome after TBI. In the present study, we investigated whether H2 treatment could improve the neurological outcome after TBI via increasing miR-21 expression. TBI was induced by controlled cortical impact in rats. H2 treatment was given by exposure to 2% H2 from 30 min to 5 h after TBI operation. Here, we found that H2 treatment significantly increased the expression of miR-21 in brain from 6 h to 3 d after TBI. The level of miR-21 expression in brain was significantly decreased after intracerebroventricular infusion of miR-21 antagomir in TBI-challenged rats with or without H2 treatment. Moreover, we found that H2 treatment conferred a better neurological outcome after TBI by improving neurological dysfunction, alleviating brain edema as well as decreasing lesion volume and blood-brain barrier permeability, which were significantly prevented by miR-21 antagomir. Furthermore, intracerebroventricular infusion of miR-21 agomir increased the level of miR-21 expression and decreased the lesion volume after TBI. In addition, H2 treatment decreased the levels of oxidative products (malondialdehyde and 8-iso-prostaglandin F2α) and increased the activities of endogenous antioxidant enzymes (superoxide dismutase and catalase) in brain after TBI, which were prevented by miR-21 antagomir. Taken together, these data indicate that H2 treatment improves the neurological outcome after TBI via increasing miR-21 expression.

Role of bone marrow-derived progenitor cells in cuff-induced vascular injury in mice.

OBJECTIVE: Arterial injury results in vascular remodeling associated with proliferation and migration of smooth muscle cells (SMCs) and the development of intimal hyperplasia, which is a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions. However, the origin of SMCs and other cells in the development of vascular remodeling is not yet fully understood. METHODS AND RESULTS: We utilized a cuff-induced vascular injury model after transplantation of the bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. We found that macrophages were major cells recruited to the adventitia of the vascular injury lesion along with SMCs and endothelial cells (ECs). While investigating whether those cells are derived from the donor, we found that most of the macrophages were GFP-positive, and some of the SMCs and ECs were also GFP-positive. Administration of the anti-c-fms antibody resulted in a marked decrease in macrophages and a relative increase of SMCs, while administration of antibodies against the platelet-derived growth factor receptor-beta caused a prominent decrease in SMCs and a relative increase in macrophages. CONCLUSIONS: The current study indicates that BM-derived cells play an important role in vascular injury, and that differentiation of macrophages and SMCs might be dependent on each other.

Expression of the novel Snai-related zinc-finger transcription factor gene Smuc during mouse development.

The Snai-related proteins are zinc-finger transcription factors that play important roles in cell-fate determination. We previously cloned a novel Snai-related gene known as snail-related transcription factor of muscle cells (Smuc) or, more recently, as snail homologue 3 (Snai3). In the present study, we investigated the functional roles of Smuc using in situ hybridization analysis at various stages of mouse development. Smuc was not detected until 12.5 days post-coitus (dpc). Its expression was observed in the skeletal muscles and thymus at 13.5 and 15.5 dpc, respectively, and these remained the major sites of Smuc expression until postnatal day 2. No Smuc expression was observed in the heart, large vessels, lungs, liver, kidney or brain. These results indicate that Smuc might be involved in the morphogenesis of the skeletal muscles and thymus at a relatively late stage of mouse development.

Improved propionic acid production with metabolically engineered Propionibacterium jensenii by an oxidoreduction potential-shift control strategy.

In this study, a three-stage oxidoreduction potential (ORP) control strategy was developed to improve propionic acid (PA) production using engineered Propionibacterium jensenii ATCC 4868 (pZGX04-gldA) in a 3-L bioreactor. Specifically, ORP was controlled at -200mV from 0 to 36h, -300mV from 36 to 156h, and -400mV after 156h. The PA titer increased from 21.38 to 27.31g/L. The effects of ORP regulation on key intracellular metabolites were studied, demonstrating that ORP can both regulate NADH/NAD(+) ratio and the activities of some enzymes involved in electron transport and redistribute metabolic flux. We integrated the ORP control strategy with a fed-batch culture method and increased PA production to 39.53g/L. This new ORP control strategy may be useful in the optimization of other anaerobic processes.

Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy.

Propionic acid (PA) production with metabolically engineered Propionibacterium jensenii (pZGX04-gldA) was improved by integrating fed-batch culture with a two-stage pH control strategy in a 3-L fermenter. The following two-stage pH control strategy was used: the pH was controlled at 5.9 for 0-36 h and shifted to 6.5 after 36 h. The PA titer was increased to 21.43 g/L. On the basis of pH control, the influence of fed-batch culture on PA production was further investigated and the maximum PA production (34.62 g/L) was obtained when glycerol was fed at a constant rate of 3.33 mL/h from 60 to 132 h with an initial glycerol concentration of 25 g/L. Crude glycerol was then used to produce PA using the optimized strategies, and maximal PA production reached 37.26 g/L. The strategies may be useful for the production of PA by other propionibacteria species.

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells.

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells.

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.

Association of GSTM1, CYP1A1 and CYP2E1 genetic polymorphisms with susceptibility to lung adenocarcinoma: a case-control study in Chinese population.

A case-control study of 164 lung adenocarcinoma (AC) patients with 181 age- and gender-matched healthy controls was conducted in order to assess any associations between glutathione-S-transferase M1 (GSTM1), cytochrome P4501A1 (CYP1A1) and cytochrome P4502E1 (CYP2E1) polymorphisms and susceptibility to lung AC in Chinese. The presence of CYP2E1 variant allele was significantly less frequent in cases than in controls, while the distribution of GSTM1 null genotype and variant CYP1A1 Msp1 allele did not vary between cases and controls. After adjustment for age, gender, smoking and all other genotypes, the CYP2E1 Rsa1 variant allele was significantly associated with decreased risk of lung AC [odds ratio 0.534 (95% confidence interval, 0.340-0.837)]. Furthermore, 3.0-fold increased risk was found in individuals with combined GSTM1 null genotype and CYP2E1 Rsa1 wild type versus those with combined GSTM1 non-null type and CYP2E1 variant allele. Our results suggest that CYP2E1 Rsa1 variant allele is associated with a decreased risk of lung AC, and combined GSTM1 null genotype and CYP2E1 Rsa1 wild type has a promoting effect on susceptibility to lung AC.