Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus

Abstract Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase (MNDH) in bacteria grown in batch or chemostat culture showed that MNDH was induced by methylamine and repressed when methanol or NH₄⁺ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase (MDH) was present in bacteria growtn on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enzyme is induced by methanol.     

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。     

银杏根系甲醇溶提物对离体培养条件下丛枝菌根真菌生长发育的研究

银杏根系甲醇溶提物对离体培养条件下丛枝菌根真菌生长发育的试验结果表明,银杏根系甲醇溶提物对离体条件下丛枝菌根真菌(Glonus mosseae,Gigaspora margarita)生长发育有明显的促进作用,能显著提高孢子萌发率,增加菌丝生长长度。与对照相比,浓度为20％～100％的甲醇溶提物均可显著促进丛枝菌根真菌的生长发育,80％甲醇溶提物效果最明显,银杏根系甲醇溶提物中含丰富的黄酮类化合物,溶提物中黄酮类物质的含量与甲醇洗脱剂的浓度有关,当浓度为80％时,黄酮含量最高,对银杏根系甲醇溶提物中黄酮类化合物含量与丛枝菌根真菌生长发育相关性分析发现,溶提物中黄酮类化合物含量与离体条件下丛枝菌根真菌孢子的生长发育表现为极显著的正相关关系,溶提物中黄酮含量越高,其对丛枝菌根真菌孢子的生长发育促进作用越大。     

Cryopreservation of Chlamydomonas reinhardtii: A cause of low viability at high cell density

Cryopreservation is a practical method for stabilizing the genetic content of living algae over long periods of time. Yet, Chlamydomonas reinhardtii, the algal species most often utilized in studies requiring genetically defined strains, is difficult to cryopreserve with a consistently high post-thaw viability. Work described here demonstrates that C. reinhardtii retains high viability only when cryopreserved at a low cell density. Low viability at high cell density was caused by the release of an injurious substance into the culture medium. Rapid freezing and thawing under non-cryoprotective conditions released large amounts of the injurious substance. Heat denaturation of cells prevented the release of the injurious substance, but heating did not inactivate it after it was released. Even when concentrated, the injurious substance was non-toxic to cells under normal culture conditions. Reduced viability of cells cryopreserved in the presence of the injurious substance could not be attributed to changes in the tonicity of the medium. A mutant strain of C. reinhardtii (cw10) with a greatly diminished cell wall did not release a substance that reduced the post-thaw viability of wild-type or cw10 cryopreserved cells. Cryopreservation of cw10 cells was achieved with approximately the same post-thaw viability irrespective to the cell concentration at the time of freezing. Acid treatment of the injurious substance was able to partially diminish its injurious effect on cells during cryopreservation. We propose that diminished viability of C. reinhardtii cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component.     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.