大肠杆菌利用废弃生物质高效生产丁二酸的代谢工程研究

利用木质纤维素类生产生物化学品及生物燃料是生物炼制的核心内容。丁二酸是一种平台化合物，广泛应用于医药、食品、餐饮等行业，同时，丁二酸还可用于生产一些重要的聚酯、烷烃等。目前，利用木质纤维素生产丁二酸有很多挑战，比如木质纤维素预处理成本控制、基因工程菌的构建、五碳糖和六碳糖的利用等等。在本论文中，我们利用Palmaria palmata、Strophanthus preussii、Cocos nucifera water（可可椰子水）以及 elephant grass stalk（大象草茎）四种生物质为原料，开展了生物合成丁二酸的研究。首先，我们利用红巨藻P. palmata进行了微生物合成丁二酸的研究。在工程菌E. coli KLPPP中分别敲除了乳酸脱氢酶A（ldhA）、丙酮酸甲酸裂解酶（pflB）、磷酸转乙酰激酶A（pta-ackA）、丙酮酸氧化酶B（poxB）等基因，并且过表达了磷酸烯醇式丙酮酸羧化酶。P. palmata 水解产物含有葡萄糖（12.57 &plusmn; 0.17 g/L）和半乳糖（18.03 &plusmn; 0.10 g/L）。经过72小时得双阶段发酵，重组菌利用半乳糖（1.20 &plusmn; 0.02 mol/mol）作为底物比利用葡萄糖（0.48 &plusmn; 0.03 mol/mol）得到的丁二酸摩尔产率更高。丁二酸的浓度及摩尔产率分别达到22.40 &plusmn; 0.12 g/L和1.13 &plusmn; 0.02 mol/mol（总糖）。这一结果表明，P. palmata生物质是一种新型的、具有替代性的生物基化学品的发酵原料。第二，对E. coli工程菌利用S. preussii发酵生产丁二酸的生物炼制过程进行了研究。我们对S. preussii的甲醇浸提预处理工艺进行了优化。S. preussii甲醇浸提物主要含有葡萄糖(9.00 &plusmn; 0.02 g/L)、半乳糖(4.00 &plusmn; 0.02 g/L)、木糖(6.00 &plusmn; 0.02 g/L)、阿拉伯糖(0.50 &plusmn; 0.02 g/L)。工程菌E. coli K3OS中过表达了吡啶核苷酸转氢酶基因（sthA），同时敲除ldhA, pflB, pta-ackA, poxB。该菌有效利用了S. preussii的甲醇浸提产物。在M9培养基中，经过72小时的双阶段发酵，丁二酸浓度达到14.39 &plusmn; 0.02 g/L，产率为1.10 &plusmn; 0.01 mol/mol（总糖）。这一水平达到利用S. preussii甲醇浸提物发酵产生丁二酸最大理论值的64%。第三，我们构建了工程菌E. coli M6PM，该菌过表达了Bacillus subtilis的丙酮酸羧化酶基因（pyc）基因，并对ldhA、pflB、pta-ackA、poxB、pgi、murein cluster C (mreC)进行了突变，下调了磷酸烯醇式丙酮酸羧化酶（ppc）基因的活性。我们利用该菌进行了可可椰子水混合糖发酵生产丁二酸的研究。C. nucifera water主要含有葡萄糖（5.00 &plusmn; 0.02 g/L）、果糖（6.10 &plusmn; 0.01 g/L）、蔗糖（6.70 &plusmn; 0.02 g/L）。经过72小时得双阶段发酵，丁二酸最终浓度达到11.78 &plusmn; 0.02 g/L，产率为1.23 &plusmn; 0.01 mol/mol（总糖）。这一水平达到利用C. nucifera water发酵产生丁二酸最大理论值的72%。这一研究也显示了可可椰子水作为底物生产生物化学品的重要性。第四，利用大香草茎水解物进行了丁二酸发酵。大香草茎水解物主要含有葡萄糖（11.60 &plusmn; 0.04 g/L）、木糖（27.22&plusmn; 0.04 g/L）、阿拉伯糖（0.65&plusmn; 0.04 g/L）。工程菌E. coli M6PM可有效利用大香草茎水解物发酵生产丁二酸。该工程菌对ldhA、pflB、pta-ackA、poxB、pgi、mreC等多基因进行了突变、下调了ppc基因、过表达了B. subtilis的pyc。经过72小时双阶段发酵，产生的丁二酸最终浓度达到30.03 &plusmn; 0.02 g/L，产率为1.09 mol/mol。这一研究说明了工程菌利用可再生的生物质作为原料发酵生产丁二酸得可能性。最后，这些研究结果揭示了生物质可以作为一种可替代原料来生产生物化学品。在生物炼制中木质纤维素生物质的利用将有望提高生物质在生产生物化学品的使用，从而改善综合生物炼制中丁二酸的生产。&nbsp;;AbstractProduction of biochemicals and biofuels from lignocellulosic biomass has the potential to enhance the overall economic view of the lignocellulosic biorefinery. Succinic acid is a platform chemical for the production of some important polyester, alkyl resins, and it is also widely used in the pharmaceutical, medicine, food and beverage industry. Production of succinic acid from lignocellulosic biomass is laced with challenges including, the cost of biomass pretreatment, genetic engineering and utilization of cost-effective medium. Here, we investigated the biosynthesis of succinic acid from Palmaria palmata, Strophanthus preussii, Cocos nucifera water and elephant grass stalk.Firstly, microbial production of succinic acid from Palmaria palmata was investigated for the first time. In engineered Escherichia coli KLPPP, lactate dehydrogenase A (ldhA), phosphotransacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB) and pyruvate oxidase B (poxB) genes were deleted while phosphoenolpyruvate carboxykinase was overexpressed. The recombinant exhibited higher molar yield of succinic acid on galactose (1.20 &plusmn; 0.02 mol/mol) than glucose (0.48 &plusmn; 0.03 mol/mol). The concentration and molar yield of succinic acid were 22.40 &plusmn; 0.12 g/L and 1.13 &plusmn; 0.02 mol/mol total sugars respectively, after 72 h dual-phase fermentation from P. palmata hydrolysate, which composed of glucose (12.57 &plusmn; 0.17 g/L) and galactose (18.03 &plusmn; 0.10 g/L). The results demonstrate that P. palmata red macroalgae biomass represents a novel and alternative feedstock for biochemical production.Secondly, a biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant E. coli. In this study, a novel biomass methanol extracts of Strophanthus preussii was used for the synthesis of succinic acid and optimization of the process parameters was carried out for pretreatment of biomass. Glucose (9.00 &plusmn; 0.02 g/L), galactose (4.00 &plusmn; 0.02 g/L), xylose (6.00 &plusmn; 0.02 g/L) and the arabinose (0.50 &plusmn; 0.02 g/L) were the major sugars present in methanol extracts of S. preussii. E. coli K3OS used methanol extracts of S. preussii effectively. E. coli K3OS with overexpression of soluble nucleotide pyridine transhydrogenase (sthA) and mutation of ldhA, pta-ackA, pflB and poxB, produced a final succinic acid concentration of 14.39 &plusmn; 0.02 g/L and yield of 1.10 &plusmn; 0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium. This value was 64 % of the maximum theoretical yield using methanol extracts of S. preussii.Thirdly, the genetically engineered strain with heterologous overexpression of pyc from Bacillus subtilis and ldhA, pflB, pta-ackA, poxB, pgi, murein cluster C (mreC) mutations and downregulation of phosphoenolpyruvate carboxylase (ppc) in E. coli were constructed and the mixed sugar fermentations was carried out with the best stains. C. nucifera water containing 5.00 &plusmn; 0.02 g/L glucose, 6.10 &plusmn; 0.01 g/L fructose and 6.70 &plusmn; 0.02 g/L sucrose was utilized for the production of succinic acid. Fermentation of C. nucifera water with E. coli M6PM produced a final succinic acid concentration of 11.78 &plusmn; 0.02 g/L and yield of 1.23 &plusmn; 0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium while modeled C. nucifera water was 0.38 &plusmn; 0.02 mol/mol total sugars. The novel substrate of C. nucifera water resulted in 72 % maximum theoretical yield of succinic acid. These investigations show the importance of C. nucifera water as a substrate for the production of biochemicals.Fourthly, efficient production of succinic acid from elephant grass stalk hydrolysate comprising of 11.60 &plusmn; 0.04 g/L glucose, 27.22 &plusmn; 0.04 g/L xylose and 0.65 &plusmn; 0.04 g/L arabinose was investigated. Fermentation of elephant grass stalk hydrolysate by engineered E. coli M6PM, involve the use of lactate dehydrogenase A, pyruvate formate lyase B, phosphotransacetylase-acetate kinase A, pyruvate oxidase B, phospho glucose isomerase, murein cluster genes mutations, down regulation of phosphoenolpyruvate carboxylase and overexpression of B. subtilis pyruvate carboxylase produced a final succinate concentration of 30.03 &plusmn; 0.02 g/L and a yield of 1.09 mol/mol during 72 h dual-phase fermentation. The high succinate yield from elephant grass stalk demonstrated possible application of renewable biomass as feedstock for the synthesis of succinic acid using recombinant E. coli.Conclusively, these results revealed that biomass represents an alternative feedstock for the production of biochemicals. The implementation of lignocellulosic biomass in biorefinery will enhance utilization of biomass for the production of biochemical and consequently improving succinic acid production in an integrated biorefinery.&nbsp;</p

Ameliorative role of diets fortified with Artocarpus altilis in a Drosophila melanogaster model of aluminum chloride-induced neurotoxicity

Abstract Backgrounds: Artocarpus altilis (breadfruit) belongs to the family Moraceae. Artocarpus altilis possesses antioxidative, anti-inflammatory, and anti-proliferative properties. Aluminum (Al) is extensively utilized for consumer products, cooking utensils, pharmaceuticals, and industries. Indication for the neurotoxicity of Al is investigated in various studies, notwithstanding the precise mechanisms of Al toxicity are yet to be fully elucidated, and, which requires novel therapy. In this study, we determined the ameliorative role of Artocarpus altilis on aluminum chloride-induced neurotoxicity in Drosophila melanogaster. Methods Varying concentration of the extract were used to formulate diets for 6 groups of flies. Group 1 contained basal diet, group 2 contained basal diet and aluminium chloride (AlCl3), group 3 contained basal diet + 0.1% unseeded breadfruit (UBF), group 4 contained basal diet + 1% unseeded breadfruit, group 5 and 6 contained basal diet + AlCl3 + 0.1% and 1% unseeded breadfruit. Assays such as acetylcholinesterase activity, malondialdehyde (MDA) concentration level, catalase activity, and superoxide dismutase (SOD) activity were carried out after 7 days of exposure respectively. Results The results showed low activity of acetylcholinesterase activity and MDA level and high catalase and SOD activity in the pretreated and post-treated flies with Artocarpus altilis compared to the normal and negative control respectively. Conclusions Taken together, Artocarpus altilis is a promising prophylactic, antiacetylcholinesterase, and antioxidant plant in the prevention, management and treatment of neurodegenerative diseases. Graphical Abstrac

Sercocephalous latifolius fruit attenuates aluminum chloride-induced oxidative stress and neurotoxicity in Drosophila melanogaster via Drn1 regulation of the IMD signaling pathway

Introduction: Alzheimer disease (AD) is a form of dementia that is a heterogeneous dysfunction of various pathophysiological features. Its morphological characteristics consist of tau aggregates and amyloid plaques. The need for novel therapy in the treatment of AD is required. Hence use of antioxidant, natural products are widely employed for the management and treatment of AD and other neurodegenerative diseases. However, there are still a lot of limitations. Sercocephalous latifolius fruit (SLF) is also known as the African peach and has numerous well-being-endorsing advantages. The Chinese name of SLF is Feizhou hong tao, while the Nigeria (Yoruba) name is Eso Gberesi. It can be grown all over the world. SLF has anti-neurodegenerative properties, however, there is a lack of evidence on their probable molecular mechanism for novel therapeutic properties. Investigations have been conducted on the SFL which shows its medicinal capability, but its ability to attenuate neurodegenerative diseases such as AD is yet to be fully elucidated, and the molecular signally pathways whichmay serve as a therapeutic target. Methods: The modulatory effect of SLF on aluminum chloride-induced neurotoxicity and oxidative stress in Drosophila melanogaster via Defense repressor-1 (Drn1) regulation of immune deficiency (Imd) signaling pathway which may be a useful therapeutic target. D. melanogaster was divided into nine groups. Group 1 served as the basal control as they were fed with a normal basal diet, group 2 served as the negative control as they were fed with Basal Diet and 0.5 ml of aluminum/100 ml distilled water, group 3 served as the positive control as they were fed with basal diet 0.5 ml aluminum chloride/100 ml distilled water and Garlic acid, group 4 and 5 were fed with basal diet and 0.1 and 1 % SLF respectively, group 6 and 7 were fed with basal diet, aluminum chloride and 0.1 and 1.0 % respectively, group 8 and 9 were fed with basal diet, garlic acid, aluminum chloride and 0.1 and 1.0 % SLF respectively. All the groups were left for seven days. The experiment was conducted for 3 months, and the fruit flies were changed every 5 days. Results: Superoxide dismutase, catalase, malondialdehyde, and nitric oxide enzymatic enzymes were assayed. However, the relative expression of Defense repressor-1 was also determined both in vitro and on aluminum-induced neurodegenerative D. melanogaster tissue. A significant increase was observed in the catalase when 0.1 % and 1 % were compared, it could be inferred that the extract proved effective at low concentrations, since its highest antioxidant potential was observed at low concentrations. The results revealed decrease in the activity of malondialdehyde, superoxide dismutase, and nitric oxide biological enzymes in the test groups. There was upregulation of Defense repressor -1 in groups 3, 4, 5, and 6, when compared with the positive and negative control, respectively. Discussion: The result provides evidence that SLF has the potential of upregulating defense repressor genes and modulating biological antioxidant enzymes, which could be further exploited to fully understand the underlying molecular mechanisms responsible for the attenuation of neurotoxicity, oxidative stress, and neurodegenerative diseases such as AD. The results suggest that SLF may be useful as a therapeutic target in AD and other neurodegenerative disease

Anti-proliferative, antioxidant effects of methanol extract of Calotropis procera leaf on lung cancer cells (H1299) and its ameliorative effect on expression of CD146 on blood cells

Abstract Background Calotropis procera leaf is one of the plants commonly utilized in phytomedicine in Nigeria. The present investigation explored the use of the extracts on cell viability and apoptosis respectively. In this study, the expression of the Cluster of differentiation 146 (CD146) in the blood of lung cancer patients on regulatory T cells (Tregs) was determined. The antioxidant and anti-proliferative effects of methanol extracts of Caloropis procera leaf on lung cancer cell H1299 were investigated. Methods From the flow cytometry, the expression of the CD146+ in the T cells were evaluated using the healthy patient, adenocarcinoma, squamous, and small cell lung cancer respectively. The apoptosis of granulocytes, monocytes, lymphocytes, CD4+, and Treg were determined by 7-amino-actinomycin D/Annexin V-Allophycocyanin (APC) staining during the resting stage and after 24 h respectively. Immunofluorescence was conducted. Cell viability assay, hydroxyl (OH), hydrogen peroxide (H2O2) and nitric oxide (NO) scavenging radicals were conducted. Reducing power and flavonoid content of Calotropis procera were investigated. The effect of the Calotropis procera at different concentrations at 24hrs was determined. Results From the flow cytometry, the expression of the CD146+ on the T cells includes 4.60 % in healthy patients, 10.10, 12.20, 9.80 % in adenocarcinoma, squamous and small cell lung cancer. The apoptosis of granulocytes, monocytes, lymphocytes, CD4+ and Treg were determined by 7-amino-actinomycin D/Annexin V-APC staining during the resting stage and after 24 h which indicate that apoptosis also occurred on Treg. Immunofluorescence shows the presence of CD146 in lung cancer patient’s tissues. The methanol extracts of Calotropis procera leaf have antioxidant and anti-proliferative effects. Methanol extract of Calotropis procera leaf reduced CD146 expression on blood cells at 24 h. Conclusion Increased CD146 expression in the Treg of lung cancer patients indicates that it may be a possible target for the treatment of lung cancer by utilizing potent immunotherapy or natural products such as methanol extract of Calotropis procera leaf which may ameliorate the expression of CD146. Calotropis procera has antioxidant, inhibitory capacity on H1299 lung cancer cells, and the ability to scavenge OH, H2O2, and NO radicals. Hence, this investigation strengthens the phyto-medicinal properties of Calotropis procera

Toxicological evaluation of the effect of phenol-contaminated water on the liver of albino rats

Phenol is a constituent of coal tar and is formed during the natural decomposition of organic materials. The effect of phenol-contaminated water on the liver of rats was investigated. Activities of some liver enzymes, alkaline phosphatase, acid phosphatase, alanine transaminase, aspartate transaminase, and gamma-glutamyl transpeptidase were determined alongside some serum indices of liver function such as serum bilirubin, albumin, globulin and serum enzymes. The total bilirubin of rats treated with phenol-contaminated water was observed to be 8.4 ± 0.8 g/l while that of control rats was 5.6 ± 0.5 g/l. Serum albumin of test rats was found to be 15 ± 2 g/l while that of control rats was 7 ± 3 g/l. Activity of all the enzymes studied reduced significantly in the liver of test rats compared with the control (P < 0.05). However, serum enzymes activity, with the exception of serum aspartate transaminase, of test rats increased significantly (P < 0.05) relative to that of test rats. It is viewed that phenol-contaminated water is hazardous to health as it may be responsible for the leakage of enzymes into the serum and may impair liver function as portrayed by reduced serum globulin and albumin

Succinate Production with Metabolically Engineered Escherichia coli Using Elephant Grass Stalk (Pennisetum purpureum) Hydrolysate as Carbon Source

Succinic acid is a spectacular chemical that can be used as the precursor of various industrial products including pharmaceuticals and biochemicals. The improvement of the succinic acid market depends on strains engineering that is capable of producing succinic acid at high yield and excellent growth rate which could utilize the wide range of carbon sources such as renewable biomass. Here we use counter selection using catAsacB for pathway design and strains developments. In this investigation, metabolically engineered Escherichia coli M6PM strain was constructed for the synthesis of succinic acid using elephant grass stalk (Pennisetum purpureum) as a carbon source. Elephant grass stalk hydrolysate was prepared which comprised of 11.60 +/- 0.04 g/L glucose, 27.22 +/- 0.04 g/L xylose and 0.65 +/- 0.04 g/L arabinose. Metabolically engineered E. coli M6PM was constructed and fermentation with pure sugars revealed that it could utilize xylose and glucose efficiently. E. coli M6PM produced a final succinate concentration of 30.03 +/- 0.02 g/L and a yield of 1.09 mol/mol during 72 h dual-phase fermentation using elephant grass stalk hydrolysate, which resulted in 64% maximum theoretical yield of succinic acid. The high succinate yield from elephant grass stalk demonstrated possible application of renewable biomass as feedstock for the synthesis of succinic acid using recombinant E. coli

Efficient degradation of rhodamine B using modified graphite felt gas diffusion electrode by electro-Fenton process

The electro-Fenton (EF) process treatment of 0.1-M (rhodamine B) RhB solution was studied with different graphite cathode materials, and graphite felt (GF) was selected as a promising material in further investigation. Then, the degradation performances of gas diffusion electrode (GDE) and graphite felt (GF) were compared, and GDE was confirmed to be more efficient in RhB removal. The operational parameters such as Fe2+ dosage and current density were optimized, and comparison among different modified methods-polytetrafluoroethylene-carbon black (PTFE-CB), polytetrafluoroethylene-carbon nanotube (PTFE-CNT), electrodeposition-CB, and electrodeposition-CNT-showed 98.49 % RhB removal by PTFE-CB-modified cathode in 0.05 M Na2SO4 at a current density of 50 A/m(2) and an air flow rate of 1 L/min after 20 min. Meanwhile, after cathode modified by PTFE-CB, the mineralization efficiency and mineralization current efficiency performed absolutely better than the pristine one. Cyclic voltammograms, SEM images, contact angles, and BET surface area were carried out to demonstrate stronger current responses and higher hydrophilicity of GF after modified. The value of biochemical oxygen demand/chemical oxygen demand (BOD5/COD) increased from 0.049 to 0.331 after 90-min treatment, suggesting the solution was biodegradable, and the modified cathode was confirmed to be stable after ten circle runs. Finally, a proposed degradation pathway of RhB was put forward

Efficient rhodamine B degradation using electro-fenton process with PbO2-coated titanium as the anode

To replace the high cost Pt anode, Ti based PbO2 electrode (PbO2-Ti) and Ti based SnO2 doped with Sb electrode (SnO2/Sb-Ti) were fabricated and compared for treating 0.1 mM Rhodamine B (RhB) in Electro-Fenton (EF) process. Scanning electron microscopy, energy dispersive X-ray, X-ray diffraction spectrometry were carried out to study the surface morphology, element composition, and substance composition of the anodes (PbO2-Ti and SnO2/Sb-Ti). Linear Sweep Voltammetry, cyclic voltammetry, Tafel, and electrochemical impedance spectroscopy were conducted to examine the electrochemical properties of the anodes (Pt, PbO2-Ti, SnO2/Sb-Ti, and Ti). These assessments showed that PbO2-Ti had the best performance. Then, the influence of varied parameters (pH, types of anodes, current density, and electrolyte) were explored. Almost complete decolorization (99.01%) was reached at pH 3, current density 50 A/m(2), 0.05 M Na2SO4 using PbO2-Ti as the anode after 20 min electrolysis. Under these conditions, 90.48% of total organic carbon was removed after 180 min. Finally, the durability of PbO2-Ti was evaluated via accelerated service life test and exhibited long service lifetime (similar to 4000 h). (c) 2018 American Institute of Chemical Engineers Environ Prog, 38: 189-197, 201