精氨酸缺陷型菌株发酵生产反式-4-羟脯氨酸

为了获得高产反式-4-羟脯氨酸的菌株,基于大肠杆菌的代谢网络模型的指导,以大肠杆菌E.coli BL21(DE3)Δput A为出发菌株,通过基因敲除技术成功敲除arg B基因,阻断L-脯氨酸合成的前体物L-谷氨酸的分支代谢途径,增加L-脯氨酸合成的代谢流,构建了精氨酸缺陷型菌株E.coli BL21(DE3)Δput AΔarg B。同时转入表达质粒p UC19-pro B2A-Ptrp2-hyp,该质粒含有突变基因pro B2,该突变基因所编码的谷氨酸激酶受L-脯氨酸的反馈抑制作用显著降低。摇瓶发酵结果表明,在外源添加600 mg/L L-精氨酸时,该重组菌株产反式-4-羟脯氨酸的量达到312.67 mg/L,较菌株E.coli BL21(DE3)Δput A/p UC19-pro B2A-Ptrp2-hyp提高了25.29%。     

putA与vgb基因对反式-4-羟基-L-脯氨酸产量的影响

反式-4-羟基-L-脯氨酸是在自然界中分布最广泛的一种羟脯氨酸,在医药、化工、食品和美容业等领域有广泛应用。为了在生物转化法生产反式-4-羟基-L-脯氨酸的过程中减少菌体对底物脯氨酸的降解,利用Red/ET同源重组系统敲除putA基因,并比较野生型菌株与缺失型菌株在不同培养基中的反式-4-羟基-L-脯氨酸产量,再引入vgb基因,考察该基因对反式-4-羟基-L-脯氨酸产量的影响。结果表明:putA基因的缺失能阻止菌体降解脯氨酸;碳源充足的情况下,GC培养基更有利于反式-4-羟基-L-脯氨酸的生产;putA基因缺失型菌株能将被消耗的脯氨酸全部转化为反式-4-羟基-L-脯氨酸;vgb基因的存在能显著提高反式-4-羟基-L-脯氨酸的产量。     

无外源脯氨酸发酵产反式-4-羟脯氨酸重组大肠杆菌的构建及发酵优化

为实现无外源脯氨酸的条件下直接从葡萄糖转化生成反式-4-羟脯氨酸,本实验采用定点突变和基因共表达的方法构建重组大肠杆菌:首先对L-脯氨酸生物合成途径中的关键酶谷氨酸激酶进行定点突变E143A、K145A,以增强L-脯氨酸生物合成能力;然后引入脯氨酸4-羟化酶基因,通过两个基因的共表达可以实现从葡萄糖到反式-4-羟脯氨酸的连续转化,而不再需要添加外源L-脯氨酸。得到的L-脯氨酸生物合成能力增强的菌株在摇瓶阶段L-脯氨酸的产量可达到1.4 g/L;pro BA2与hyp双基因共表达菌株的反式-4-羟脯氨酸产量为98.9 mg/L,比原始菌株提高了一倍,经发酵优化后得到培养基为:葡萄糖10 g/L,胰蛋白胨15 g/L,硫酸亚铁3 mmol/L,硫酸镁1 g/L,磷酸氢二钾3 g/L,氯化钙0.015 g/L,在这个培养条件下反式-4-羟脯氨酸的产量为220.0 mg/L,比优化前提高1.2倍。      

产顺式-4-羟脯氨酸枯草芽孢杆菌工程菌的构建及发酵优化

顺式-4-羟脯氨酸（CHOP）是一种重要的手性结构物质,可广泛用于药物以及香料制造。本研究为开发CHOP的生物合成方法,将来源于中华根瘤菌（Sinorhizobium meliloti）的L-脯氨酸-4-羟化酶在枯草芽孢杆菌WB800N中实现了成功表达,构建获得了能够以L-脯氨酸为底物合成CHOP的工程菌株,进而对该菌株的发酵条件以及发酵组分进行了优化研究。结果表明:工程菌株的最适发酵温度为25℃,最适诱导剂浓度为1 mmol/L,最适发酵p H为5;在发酵体系中加入5 mmol/L硫酸亚铁和2.5 g/L L-脯氨酸时最有利于合成CHOP。在最优发酵体系下发酵60 h后,顺式-4-羟脯氨酸的产量可以达到120.2 mg/L。本文成功的构建了羟脯氨酸生产菌株枯草杆菌WB800 p HT-P4H,而研究结果为枯草杆菌工程菌发酵生产顺式-4-羟铺氨酸提供了基础。      

以甘油为碳源发酵高产反式-4-羟脯氨酸菌株的选育及营养优化

以低品级甘油为碳源进行微生物发酵合成反式-4-羟脯氨酸(Trans-4-hydroxy proline,Hyp)的探索。从实验室构建的重组菌E.coli BL21(DE3)/p UC19-ptrp2-Hyp出发,通过易错PCR随机突变和常压室温等离子体复合诱变处理,利用单菌落琼脂块和氨基酸显色相结合高通量筛选出1株以甘油为唯一碳源的Hyp高产菌P71。与葡萄糖培养基相比,该菌株更适合在甘油上生长并转化L-脯氨酸合成Hyp,发酵20 h产Hyp高达1.20g/L,比生长在葡萄糖培养基上高70%以上;比其出发菌株在葡萄糖培养基上产量提高了1倍以上。通过培养基成分系统优化,发现胰蛋白胨、Fe SO4和L-脯氨酸是3大主要影响因素,最适加量分别为7.01 g/L、11.51 g/L和1.41 mmol/L;在该条件下突变菌株摇瓶发酵12 h产Hyp达1.61 g/L,比优化前提高了50%。     

产3-羟脯氨酸重组菌的构建及发酵优化

顺式-3-羟基-L-脯氨酸(顺式-3-羟脯氨酸)可用于合成多种抗癌药物,具有重要的商业价值,目前大多通过添加IPTG来诱导表达脯氨酸-3-羟化酶,采用两步法生物合成顺式-3-羟脯氨酸。作者通过目的基因优化设计,引入强启动子色氨酸串联启动子(Ptrp2)来避免异源表达时的诱导剂使用,构建重组质粒p ES-Ptrp2-P3H,成功构建了重组大肠杆菌BL21(DE3)/p ET21a-Ptrp2-P3H,优化后的脯氨酸-3-羟化酶基因(P3H)改变了168个碱基,GC含量由原来的64.83%降低到49.31%。该菌在初步优化培养基(葡萄糖1 g/d L,甘油0.125 g/d L,胰蛋白胨1.6 g/d L,(NH_4)_2SO_40.5 g/d L,K_2HPO_40.1 g/d L,NaCl 0.2 g/d L,FeSO_41 mmol/L,MgSO_40.5 g/d L,CaCl_20.015 g/d L,脯氨酸10 g/L,pH 7.5)上能一步法原位合成顺式-3-羟脯氨酸,摇瓶发酵24 h,产量0.8 g/L,比优化前提高一倍以上,为进一步开展顺-3-羟脯氨酸产业化提供了依据。     

sucA缺失型大肠杆菌菌株的构建与发酵条件优化

反式-4-羟脯氨酸在多领域具有重要应用价值。为了进一步提高反式-4-羟脯氨酸的产量,在已构建好的E. coli BL21(DE3)△putA的基础上,敲除基因sucA的同时插入密码子优化后的反式-4-羟化酶基因(hyp),并将构建好的质粒pUC19-ptrp2-hyp-vgb导入该基因敲除菌株中。之后在摇瓶水平上,单因素优化发酵条件与发酵培养基的组分,并通过正交实验进一步确定敲除菌株的培养条件。结果表明:与含有同样质粒的原菌和两种单敲除菌E. coli BL21(DE3)△sucA、E. coli BL21(DE3)△putA相比,E. coli BL21(DE3)△putA△sucA双敲除菌具有最高的全细胞酶活,大约是原菌的2.6倍;在摇瓶水平上,发酵条件优化后,以100 mmol/L的L-脯氨酸为底物进行发酵,12 h后可得到1.08 g/L的羟脯氨酸,是优化前的3.87倍。     

代谢工程改造大肠杆菌合成反式-4-羟基-L-脯氨酸

通过比对筛选,从Micromonospora sp. CNB394中获得高活性的L-脯氨酸-4-羟基化酶,随后利用规律成簇的间隔短回文重复-associated protein 9基因编辑方法,通过强化L-脯氨酸合成途径和引入高活性的L-脯氨酸-4-羟基化酶,构建1株以葡萄糖为碳源合成反式-4-羟基-L-脯氨酸的大肠杆菌基因工程菌HYP15。摇瓶发酵30 h,工程菌的反式-4-羟基-L-脯氨酸产量达到13.6 g/L。5 L发酵罐分批补料发酵40 h,反式-4-羟基-L-脯氨酸产量达到48.6 g/L,糖酸转化率和平均生产强度分别达到21.6%和1.22 g/(L·h),具有良好的工业应用前景。     

1株产顺式-4-羟基-L-脯氨酸基因工程菌的构建及发酵初步优化

顺式-4-羟基-L-脯氨酸是一种可用于合成多种药物和香料的手性结构物质。通过对L-脯氨酸顺式-4-羟化酶基因的优化设计,并引入色氨酸串联启动子,构建出1株能表达L-脯氨酸顺式-4-羟化酶的重组大肠杆菌JM109/p EHC4,从而可以将游离的L-脯氨酸转化为顺式-4-羟基-L-脯氨酸。对该菌株进行摇瓶发酵优化,得出优选培养基为(g/L):葡萄糖10,甘油10,蛋白胨10,NaCl 6,FeSO_4·7H_2O 0.278,L-抗坏血酸0.528,(NH_4)_2SO_45,K_2HPO41,MgSO_40.2,CaCl_20.015,L-脯氨酸4。在该条件下发酵12 h,顺式-4-羟基-L-脯氨酸的产量达到657.08mg/L,比优化前提高了3.76倍;在24 h时产量达到1 582.75 mg/L。研究结果为顺式-4-羟基-L-脯氨酸的微生物转化法生产提供了基础。     

密码子优化及透明颤菌血红蛋白共表达提高耐热脂肪酶在毕赤酵母的表达

根据毕赤酵母密码子的偏好性,通过在线软件对Thermomyces lanuginosus脂肪酶基因（tll）和透明颤菌血红蛋白基因（vhb）进行密码子优化。优化后的脂肪酶基因（tll-opt）和透明颤菌血红蛋白基因（vhb-opt）中碱基GC含量分别由原来的46%提高到49%和42%提高到51%,碱基A、T、C、G均匀分布,减少了AT和GC富集区,利于tll-opt和vhb-opt基因在毕赤酵母X33中的表达。将tll和tll-opt连接到表达载体pPICZαA并转入毕赤酵母X33中。摇瓶培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为7 U/mL和16 U/mL。50 L发酵罐培养条件下,含有tll和tll-opt重组工程菌的最大酶活力分别为201 U/mL和430 U/mL。为了进一步提高含tll-opt重组工程菌的表达酶活性,将vhb-opt转入该重组工程菌得到工程菌VHb＋（含有tll-opt和vhb-opt）。重组工程菌VHb＋在摇瓶培养条件下最大酶活力为23 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.39 倍和3.28 倍。重组工程菌VHb＋在50 L发酵罐瓶培养条件下最大酶活力为610 U/mL,分别是优化后基因和原始基因最大表达酶活力的1.41 倍和3.03 倍。     

CRISPRi/ddCpf1促进大肠杆菌中丙二酰辅酶A的积累

丙二酰辅酶A是多种有价值化合物（包括食品、保健品等）合成的重要前体物质,其合成已成为大肠杆菌中生产目标代谢物的潜在瓶颈。为解决其生物合成的瓶颈问题,作者通过CRISPR干扰（CRISPR interference,CRISPRi） 促进丙二酰辅酶A的积累。首先,构建了丙二酰辅酶A的生物传感器,实现了其快速、可视化的胞内测量。随后,构建了CRISPRi/ddCpf1系统,实现了基因转录的抑制,并尝试用ddCpf1（催化失活的Cpf1）和RNA聚合酶抑制蛋白Gp2融合表达（CRISPRi/ddCpf1-Gp2）。结果表明,Gp2虽然有一定的转录抑制效果,但是严重影响了生长。最后,利用CRISPRi/ddCpf1系统进行两组双靶点基因抑制,分别靶向乙醛脱氢酶和3-氧代酰基-酰基载体蛋白合酶II基因,以及3-氧代酰基-酰基载体蛋白合酶I和琥珀酰辅酶A合成酶基因,均实现了丙二酰辅酶A浓度的提高。     

The prevalence of Listeria, Salmonella, Escherichia coli and E. coli O157:H7 on bison carcasses during processing

Bison meat is a relatively new, emerging meat species gaining increased popularity in the US and European meat markets, but little is known of its microflora or pathogens that may be present. This study was carried out to determine the incidence of the foodborne pathogens Listeria, Salmonella, Escherichia coli/E. coli O157:H7 on slaughtered bison and to evaluate the bison slaughter process. Bison carcass sampling was carried out at monthly intervals over a period of 1 year at a Bison processing facility in the Midwestern United States. A total of 355 Bison carcasses were sampled by surface swabbing the carcasses at five points on the production line: pre-dehiding, post-evisceration, post-USDA inspection, post-washing and 24 h chilled carcass. Overall, the prevalence of Listeria spp., Salmonella spp., E. coli and E. coli O157:H7 was 18.3%, 3.94%, 38.3% and 1.13%, respectively. The prevalence of Listeria spp. at each sampling point tested was 42.24%, 18.1%, 6.03%, 1.72% and 3.77% while the prevalence of E. coli at each sampling point was: 88.79%, 73.28%, 52.59%, 56.89% and 11.3%, respectively. The data obtained suggests that current antimicrobial intervention strategies used at the plant are relatively effective in reducing Listeria and E. coli contamination on bison carcasses to some extent, however further study is required to determine the influence of current slaughter practices on carcass contamination. The data reported in this study to the authors’ knowledge is some of the first information reporting on the bacteriological status of Bison, and provides some useful baseline information for future research.     

Microtiter plate-based assay for screening antimicrobial activity of melanoidins against E. coli and S. aureus

A rapid plate reader based method examining the antimicrobial activity of both model and food melanoidins (coffee, beer, sweet wine) is described. Antimicrobial activity against Escherichia coli and Staphylococcus aureus is evaluated as area under the growth curve compared to a control. Method was settled for an aqueous melanoidin concentration of 2 mg/ml inoculated to 10⁶ cfu/ml culture. All tested model and food melanoidins exerted antimicrobial activity in some extent, but inhibition was significantly higher over S. aureus (Gram-positive) than E. coli (Gram-negative). Antimicrobial activity can be further quantified by expressing it as OTEV (oxytetracyclin equivalent value, μg/l) which could serve to compare the results obtained within different laboratories, methodologies and/or compounds. Results indicate that both strains have different sensitivity against the presence of melanoidins and probably different mechanism of inhibition. Procedure can be used for a rapid screening of the potential antimicrobial properties of melanoidins, and subsequently to Maillard reaction products as well, against pathogenic strains in order to isolated substances with biological activity.     

Understanding E. coli internalization in lettuce leaves for optimization of irradiation treatment

Irradiation penetrates food tissues and effectively reduces the number of food microorganisms in fresh produce, but the efficacy of the process against internalized bacteria is unknown. The objective of this study was to understand the mechanisms of pathogen colonization of plants relative to lettuce leaf structures so that radiation treatment of fresh leafy vegetables can be optimized. Leaves of iceberg, Boston, green leaf, and red leaf lettuces were cut into pieces, submerged in a cocktail mixture of two isolates of Escherichia coli (Rifampicin resistant), and subjected to a vacuum perfusion process to force the bacterial cells into the intercellular spaces in the leaves. Sixty bags containing 20 g of lettuce each were tested. The inoculated leaves were gamma irradiated (Lanthanum-140, 0.16 kGy/h) at 0.25–1.0-kGy (surface dose values), with increments of 0.25 kGy at 15 °C. Microbial analysis was performed right after irradiation, including non-irradiated leaf pieces (controls). A dose uniformity ratio (max/min dose) of 2.8 was set to confirm the effect of non-uniform dose distribution. Calculated D₁₀-values varied between 48 and 62% based on the dose distribution from the entrance dose. However, despite the subtle differences in composition and structure among the four lettuce varieties, the D₁₀-values were not significantly different. Irradiation up to 1.0-kGy resulted in 3–4-log reduction of internalized E. coli on the lettuce leaves. The SEM images suggest that the contamination sites of pathogens in leafy vegetables are mainly localized on crevices and into the stomata. This study shows that irradiation effectively reduces viable E. coli cells internalized in lettuce, and decontamination is not influenced by lettuce variety. Ionizing irradiation effectively reduced the population of internalized pathogen in a dose-dependent manner and could be used as an effective killing step to mitigate the risk of foodborne disease outbreaks.     

A survey of the incidence of E. coli O157 in the UK dairy industry

A survey of the incidence of Escherichia coli O157 in bovine faeces, pasteurised and unpasteurised dairy products, farm swabs and dairy factory environmental samples was undertaken. A single enrichment broth was employed, together with three detection methods, comprising a plating medium, a commercially available ELISA technique (Petrifilm) and an ELISA test under development (Organon-Teknika). For all detection methods, the efficiency of recovery, determined using artificially contaminated samples, was poor and this was attributed to the growth of competitors in the enrichment broth. Both the plating and the Organon-Teknika methods yielded a higher proportion of false positives than the Petrifilm method; however, the former two methods also detected a wider range of verotoxigenic E. coli than the Petrifilm method. Verotoxigenic E. coli were not detected in any of 1146 samples examined. The poor performance of the methodolgy may explain why the environmental incidence appears to be low although the incidence of infection by E. coli O157 is increasing.     

丝胶抗冻肽在大肠杆菌中的重组表达及其抗冻活性初探

为了高效制备抗冻肽,研究一种丝胶抗冻肽目的基因SerD在大肠杆菌BL21菌株中的重组表达,并分析表达产物的抗冻活性。首先合成SerD基因片段,经KpnI和XhoI双酶切后定向插入质粒载体Pet32a中,构建表达质粒Pet32a-SerD,然后电转化入大肠杆菌BL21(DE3),在IPTG诱导下进行目的基因表达,利用镍琼脂糖亲和层析对目的重组蛋白进行纯化,并对其进行抗冻活性分析。结果表明,最佳表达条件为重组菌在20℃诱导16 h;通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis,SDS-PAGE)和蛋白质免疫印迹试验(Western-Blot)鉴定重组蛋白表达成功且His-SerD融合蛋白表达的分子量在25~35 kDa之间;胞内表达His-SerD融合蛋白的大肠杆菌BL21-SerD复苏后的生长活性明显高于PBL空载菌;添加His-SerD融合蛋白可明显降低溶液中冰晶颗粒大小,具有较好的重结晶抑制效果。本文通过基因工程方法在大肠杆菌中成功构建丝胶肽抗冻肽的重组表达系统,并最终获得具有抗冷冻胁迫保护作用的His-SerD融合蛋白。     

Modelling the variability of lag times and the first generation times of single cells of E. coli

A mathematical model combining deterministic and stochastic elements describes the growth and division of single cells. Its deterministic part is based on the model of Baranyi and Roberts [International Journal of Food Microbiology 23 (1994) 277] modelling the gradual adjustment of the cells to a new environment. The stochastic part assumes a random threshold size for the division of a single cell, which accounts for the variability of the individual generation times. Experimental results of the first division times of thousands of single cells using a microscopic flow system could be reproduced with this model, and it has the potential to be used to study the effects of different stress and environmental factors on the distribution of the lag and generation times of individual cells.     

基于dsDNA-CuNCs的荧光ELISA检测牛奶中的E.coli O157:H7

目的:建立了一种灵敏检测牛奶中大肠杆菌O157:H7的新型荧光酶联免疫吸附方法。方法:以碱性磷酸酶水解焦磷酸盐-Cu²⁺配位复合物,释放出Cu²⁺形成铜纳米簇作为信号报告探针,通过对关键因素Cu²⁺浓度、焦磷酸盐浓度、DNA模板的浓度和长度进行优化。结果:在最优条件下,大肠杆菌O157:H7的菌数为5×10⁴～1×10⁸ CFU/mL时,与荧光信号值有较好的线性关系(R²=0.980 8),最低检出限为2.4×10⁴ CFU/mL。结论:该方法成功地应用于低脂牛奶和脱脂牛奶样本中大肠杆菌O157:H7的测定,平均回收率为92.2%～108.5%,相对标准偏差为4.9%～10.4%。     

Effects of supercritical carbon dioxide treatment against generic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and E. coli O157:H7 in marinades and marinated pork

This study was conducted to evaluate the effects of supercritical carbon dioxide (SC-CO₂) treatment on soy sauce and hot-pepper paste marinades, as well as in marinated pork products, for the inhibition of generic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and E. coli O157:H7. SC-CO₂ was more effective at destroying foodborne pathogens when it was applied to the marinades than the marinated products. SC-CO₂ treatment at 14 MPa and 45 °C for 40 min resulted in a greater reduction in soy sauce (2.52–3.47 log CFU/cm²) than in hot-pepper paste marinade (2.12–2.72 log CFU/cm²). In the case of the marinated pork, when SC-CO₂ was applied at 14 MPa and 45 °C for 40 min, the reduction levels of L. monocytogenes were 2.49 and 1.92 log CFU/cm² in soy sauce and hot-pepper paste marinated pork, respectively. The results should be useful in the meat industry to help increase microbial safety and assure the microbial stability of marinades and marinated products.     

代谢工程改造大肠杆菌合成酪醇

酪醇是一种天然存在于橄榄油、酒及绿茶中的酚类化合物。由于酪醇具有抗炎症和抗氧化的生理活性,因此广泛应用于医药、化工等工业领域。传统的酪醇生产方法是化学合成法,但是这些方法存在着工艺复杂、得率低和环境污染等问题。另外,植物中较低的酪醇含量也限制了酪醇的分离纯化工艺研究。因此微生物发酵法生产酪醇研究越来越受到重视。通过在大肠杆菌内异源表达酿酒酵母中的丙酮酸脱羧酶基因ARO10,成功构建了合成酪醇的重组大肠杆菌。通过敲除预苯酸脱水酶编码基因pheA和苯乙醛脱氢酶编码基因feaB,提高了酪醇的合成能力。在最适的培养条件下,过表达ARO10基因的重组菌利用10 g/L葡萄糖作为碳源,发酵48 h酪醇产量可达4.15 mmol/L。研究发现,发酵培养基中外源添加酪氨酸能够提高重组大肠杆菌的酪醇合成能力。同时,研究还发现在大肠杆菌细胞中存在能够催化酪氨酸合成酪醇的前体物质4-羟基苯丙酮酸的酶。为工业水平微生物发酵法合成酪醇提供了思路。