嗜盐嗜碱多能硫碱弧菌D306基因组编辑和脱硫途径工程研究

硫化氢通常存在于石油和天然气及其下游工艺中，由于其可形成酸雨，对环境的危害很大。多能硫碱弧菌 D306是一种化能自养型、GC含量高的革兰氏阴性嗜盐嗜碱细菌，对硫化氢有较强的脱硫作用，产物是单质硫和硫酸根。为了加强多能硫碱弧菌 D306对天然气的脱硫作用和单质硫的生成，本论文努力解决三个问题，即缺乏基因组编辑方法、硫代谢不明确和硫酸根的产生、 对工业设备和细胞的毒害作用。首先，利用已知的菌株信息，采用细胞死亡诱导 (cell death induced, CDI) 策略，开发出了编辑效率为8.2%的多能硫碱弧菌基因组编辑系统。建立了嗜盐嗜碱多能硫碱弧菌 D306降落PCR (touchdown PCR) 技术，利用不同表达水平的Cas9蛋白和sgRNA对基因组编辑系统进行优化，并通过CDI策略进行筛选，优化后的系统效率提高到41.2%。因此，CDI策略成功简化了多能硫碱弧菌CRISPR系统开发的工作流程，并可以应用于其他极端微生物。第二，我们在多能硫碱弧菌 D306中鉴定了约50个硫代谢相关蛋白，并推测了它们的定位，深化了对D306菌株硫代谢途径的认识。利用CRISPR系统敲除了产生硫酸根的四个关键基因，强化了单质硫的生成。与野生型菌株相比，HdrB基因的敲除使多能硫碱弧菌在以硫代硫酸钠和硫化钠为硫源时，硫酸盐的产量分别下降了20.8%和55.1%。这意味着HdrB的敲除可以防止菌株消耗硫作为能量来源，可以产生更多的单质硫。这些结果也被电子显微镜观察和菌株生长曲线所证实。第三，构建了严紧调控的诱导表达体系，设计、构建、检测和验证了Cas9蛋白、Ⅱ类CRISPR主效应蛋白等天然毒性蛋白的表达。采用补料分批策略提高了多能硫碱弧菌D306的生长速度和生物量，为提高生物脱硫效率奠定了基础。所构建的系统使用铁吸收调节剂调控基因转录水平，使蛋白表达量增加到对照组的27倍。利用该系统成功表达了一种剧毒蛋白，验证了系统的功能。第四，初步构建了具有通用性的多基因编辑载体系统。选择具有通用性的tRNA作为基因编辑靶标，在大肠杆菌中针对11种不同的tRNAs进行了多基因编辑研究，其中，1种tRNAs来源于多能硫碱弧菌D306，1种来源于水稻，9种来源于大肠杆菌。研究结果显示，所构建的多基因编辑载体对LeuW，ValU和LysV的编辑效率分别达到100%，66.7% 和33.3%。综上所述，我们首次在嗜盐嗜碱多能硫碱弧菌 D306中构建了一个基于CRISPR- cas9系统和细胞死亡诱导策略的敲除系统。该体系通过敲除硫酸盐产生途径的基因来提高脱硫效率。结果表明，该方法具有较好的应用价值。经过设计、构建、测试和验证的过程，开发了另一种利用铁抑制蛋白诱导蛋白表达的系统。最后，在大肠杆菌中进行了多重CRISPR的研究，为将来在嗜盐嗜碱多能硫碱弧菌 D306中的应用提供参考。;Hydrogen sulfide, normally found in oil and gas and their down streaming processes, represents a highly toxic environmental threat by forming acid rain. Thioalkalivibrio versutus D306, an autotrophic; high GC content and gram-negative polyextremophile, can strongly desulfurize natural gas and produce sulfur and the less favorable, sulfate. To strengthen the power of T. versutus D306 as a natural gas desulfurizing bacterium, this thesis aimed to solve three different obstacles for this objective, which are lack of genome editing method, unclear sulfur metabolism and unfavorable sulfate production which is both; harmful to industrial equipment and toxic to the cells. To develop efficient genome editing strategy for T. versutus D306 using CRISPR-cas9 system based upon known strain information, cell death induced (CDI) strategy was followed. The strategy used cell death induction, calculated as colony forming units, as an indicator for screening the best knockout plasmid without the need to check colonies using PCR.Firstly, a CRISPR-cas9 system with editing efficiency of 8.2% of the screened colonies was first developed. Further optimization of the system, using different expression levels of Cas9 protein and sgRNA, was screened again by the CDI strategy. The editing efficiency was increased to 41.2% using touchdown PCR technique developed especially for T. versutus D306. Therefore, CDI strategy showed a success in simplifying workflow for CRISPR system development in T. versutus D306 and can be applied to other autotrophs or polyextremophiles. Secondly, 50 sulfur related proteins were identified in T. versutus D306 and their localization were assumed, which gave us a clear picture for different sulfur metabolism steps in the strain. The CRISPR system was used to knockout four sulfate producing key genes to enhance the desulfurization process. HdrB protein knockout enabled us to decrease sulfate production by 20.8 % and 55.1 % for thiosulfate and sulfide grown T. versutus, respectively, as compared to the native strain, which means more sulfur, was produced. It also prevented the strain to consume sulfur as the strain energy substrate. These results were also confirmed by electron microscope and strain growth. The desulfurization process profitability was further improved by proving the ability of T. versutus D306 to produce nano sulfur (less than 50 nm) from sulfide as a substrate using XRD, electron microscope and grain size measurements.Thirdly, a tightly inducible expression system was built using fed-batch and Design, build, test and validate approach for expressing toxic proteins by nature like Cas9 protein, Class 2 CRISPR main effector protein. The fed batch strategy improved the growth of T. versutus D306 to facilitate expression system measurements. The final constructed system, using ferric uptake regulator, enabled the expression to be repressed near the control strain values and increased again to 27 times the control. The system successfully expressed a highly toxic protein as a validation step.Fourthly, a multi gene editing CRISPR-cas9 system was constructed. The universal tRNA was selected as the target of gene editing, and 13 different tRNAs, which were 1 native T. versutus D306, 1 rice native one and 11 E. coli ones, were studied in E. coli. The results showed that the editing efficiency of the constructed system for LeuW, ValU and LysV was 100%, 66.7% and 33.3% respectively.In conclusion, a first knockout system, based on CRISPR- cas9 system and cell- death induced strategy, was built in T. versutus D306. The system was used to improve productivity of desulfurization process by knocking out unfavorable sulfate production pathway genes. The nanometric characteristics of T. versutus sulfur was proved for more profitable bio desulfurization process. Another system for inducible protein expression was developed using ferric repression protein depending upon design, build, test and validate approach. Finally, multiplexing CRISPR was studied in E. coli for future application in T. versutus D306.&nbsp;</p

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

Improvement of D-lactic acid production at low pH through expressing acid-resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

BACKGROUND Blending d-lactic acid (d-LA) with l-lactic acid can significantly improve the thermostability of polylactic (PLA). Although microbial production of d-LA under acidic conditions is beneficial for the reduction of production costs, the yield is low due to the acidic toxicity of the source strains. Herein, an Issatchenkia orientalis glycosylphosphatidylinositol-anchored protein IoGas1, which is required for resistance to low pH and salt stress, was expressed in the YIP-J-C-D-A1 yeast strain. This strain was integrated with Escherichia coli d-lactate dehydrogenase gene and several attenuated key pathway genes, including pyruvate decarboxylases (PDC1, PDC6), JEN1 (a monocarboxylate transporter), d-lactate dehydrogenase1 (DLD1), l-lactate cytochrome-c oxidoreductase (CYB2) and alcohol dehydrogenase 1(ADH1). RESULTS The results revealed that the production of d-LA by the modified strain YIP-I-J-C-D-A1 was remarkably improved and reached 85.3 g L-1 d-LA, with a yield of 0.71 g g(-1) and a productivity of 1.20 g L h(-1) in batch-fed fermentation. The d-LA production of the YIP-I-J-C-D-A1 strain (CGMCC2.5785) was further improved by attenuating the ethanol and glycerol pathways. The resulting strain YIP-A15G12 (CGMCC2.5803) produced 92.0 g L-1 d-LA with a yield of 0.70 g g(-1) and a productivity of 1.21 g L h(-1) in batch-fed fermentation at a final pH of 3.58. CONCLUSION Taken together, the expression of the acid-resistant gene IoGAS1 in a modified yeast strain can significantly improve the efficiency of producing d-LA at low pH, which may prove beneficial for the industrial production of the biodegradable material, PLA

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

Degradation of Rhodamine B at Neutral pH Using Modified Sponge Iron as a Heterogeneous Electro-Fenton Catalyst

Sponge iron was used as heterogeneous electro-Fenton (EF) catalyst. Then, the catalytic activity and durability of sponge iron was greatly improved after modified by polytetrafluoroethylene (PTFE). Scanning electron microscopy, energy dispersive X-ray, X-ray diffraction spectrometry, and vibrating sample magnetometer were carried out to further understand the surface morphology, element composition, substance composition, and magnetic property, respectively. The PTFE modified sponge iron exhibited excellent mineralization performance for treatment of 20 mg/L Rhodamine B solution under the optimized conditions (catalyst dosage 0.5 g/L, current density 50 A/m(2)), achieving almost complete decolorization, 90.13% total organic carbon removal, and 93.24% Chemical Oxygen Demand removal after 120 min electrolysis. Moreover, the results confirmed that the modified sponge iron showed satisfactory durability after five times consecutive cycles, and could be a promising heterogeneous EF catalyst. (C) 2017 American Institute of Chemical Engineer

Effective degradation of rhodamine B by electro-Fenton process, using ferromagnetic nanoparticles loaded on modified graphite felt electrode as reusable catalyst: in neutral pH condition and without external aeration

Polytetrafluoroethylene/ferromagnetic nanoparticle/carbon black (PTFE/MNP/CB)-modified graphite felt (GF) was successfully applied as cathode for the mineralization of rhodamine B (RhB) in electro-Fenton (EF) process. The modified cathode showed high decolorization efficiency for RhB solution even in neutral pH condition and without external aeration, achieving nearly complete decolorization and 89.52 % total organic carbon (TOC) removal after 270-min oxidation with the MNP load 1.2 g at 50 A/m(2). Moreover, the operational parameters (current density, MNP load, initial pH, and airflow rate) were optimized. After that, adsorption isotherm was also conducted to compare the absorption quantity of CB and carbon nanotube (CNT). Then, the surface morphologies of MNPs were characterized by transmission electron microscope (TEM), energy-dispersive X-ray detector (EDX), and Fourier transform infrared spectroscopy (FTIR); and the modified cathode was characterized by SEM and contact angle. Finally, the stability and reusability of modified cathode were tested. Result uncovered that the PTFE/MNP/CB-modified cathode has the potential for industrial application and the solution after treatment was easily biodegradable

Enhanced growth-driven stepwise inducible expression system development in haloalkaliphilic desulfurizing Thioalkalivibrio versutus

Highly toxic and flammable H2S gas has become an environmental threat. Because of its ability to efficiently remove H2S by oxidation, Thioalkalivibrio versutus is gaining more attention. Haloalkaliphilic autotrophs, like the bio-desulfurizing T. versutus, grow weakly. Weak growth makes any trial for developing potent genetic tools required for genetic engineering far from achieved. In this study, the fed-batch strategy improved T. versutus growth by 1.6 fold in maximal growth rate, 9-fold in 0.D-600 values and about 3-fold in biomass and protein productions. The strategy also increased the favorable desulfurization product, sulfur, by 2.7 fold in percent yield and 1.5-fold in diameter. A tight iron-inducible expression system for T. versutus was successfully developed. The system was derived from fed-batch cultivation coupled with new design, build, test and validate (DPTV) approach. The inducible system was validated by toxin expression. Fed-batch cultivation coupled with DPTV approach could be applied to other autotrophs

Systematically redesigning and optimizing the expression of _D-lactate dehydrogenase efficiently produces high-optical-purity _D-lactic acid in<i> Saccharomyces cerevisiae </i>

(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. coli (D)-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 Pdc1 and 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

Enhanced Biodesulfurization with a Microbubble Strategy in an Airlift Bioreactor with Haloalkaliphilic Bacterium Thioalkalivibrio versutus D306

Biodesulfurization under haloalkaline conditions requires limiting oxygen and additional energy in the system to deliver high mixing quality control. This study considers biodesulfurization in an airlift bioreactor with uniform microbubbles generated by a fluidic oscillation aeration system to enhance the biological desulfurization process and its hydrodynamics. Fluidic oscillation aeration in an airlift bioreactor requires minimal energy input for microbubble generation. This aeration system produced 81.87% smaller average microbubble size than the direct aeration system in a bubble column bioreactor. The biodesulfurization phase achieved a yield of 94.94% biological sulfur, 84.91% biological sulfur selectivity, and 5.06% sulfur oxidation performance in the airlift bioreactor with the microbubble strategy. The biodesulfurization conditions of thiosulfate via Thioalkalivibrio versutus D306 are revealed in this study. The biodesulfurization conditions in the airlift bioreactor with the fluidic oscillation aeration system resulted in the complete conversion of thiosulfate with 27.64% less sulfate production and 10.34% more biological sulfur production than in the bubble column bioreactor. Therefore, pleasant hydrodynamics via an airlift bioreactor mechanism with microbubbles is favored for biodesulfurization under haloalkaline conditions

Enhanced Biodesulfurization with a Microbubble Strategy in an Airlift Bioreactor with Haloalkaliphilic Bacterium Thioalkalivibrio versutus D306

Biodesulfurization under haloalkaline conditions requires limiting oxygen and additional energy in the system to deliver high mixing quality control. This study considers biodesulfurization in an airlift bioreactor with uniform microbubbles generated by a fluidic oscillation aeration system to enhance the biological desulfurization process and its hydrodynamics. Fluidic oscillation aeration in an airlift bioreactor requires minimal energy input for microbubble generation. This aeration system produced 81.87% smaller average microbubble size than the direct aeration system in a bubble column bioreactor. The biodesulfurization phase achieved a yield of 94.94% biological sulfur, 84.91% biological sulfur selectivity, and 5.06% sulfur oxidation performance in the airlift bioreactor with the microbubble strategy. The biodesulfurization conditions of thiosulfate via Thioalkalivibrio versutus D306 are revealed in this study. The biodesulfurization conditions in the airlift bioreactor with the fluidic oscillation aeration system resulted in the complete conversion of thiosulfate with 27.64% less sulfate production and 10.34% more biological sulfur production than in the bubble column bioreactor. Therefore, pleasant hydrodynamics via an airlift bioreactor mechanism with microbubbles is favored for biodesulfurization under haloalkaline conditions