代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先,构建了l-半胱氨酸合成酶基因cysK强化表达菌株,杆菌肽效价相比于对照菌株提高了9.47%。接着,为提高l-半胱氨酸合成前体供给,对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化,杆菌肽产量分别提高了7.23%和8.52%。随后,结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸,强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种,在整合表达了基因cysK基础上,利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子,得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP)),杆菌肽效价达到910.02 U/mL,相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后,通过3 L发酵罐小试实验,进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明,强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略,为杆菌肽工业生产提供了一株具有良好应用前景的菌株。     

Targeted nanoparticles for precise cancer therapy

<正>Cancer has become one of the biggest challenges in the development of modern medical science. In particular, many problems, such as low efficiency, severe side effects, metastasis, and tumor invasion, challenge the development of precise cancer therapy. Continuous advancements in medicine, however, have allowed clear differentiation between tumors and normal tissues that can be exploited for cancer     

Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.     

A fundamental regulatory role of formate on thuringiensin production by resting cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> YBT-032

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.     

Adsorption of insecticidal toxin from Bacillus thuringiensis subsp. Kurstaki by some Chinese soils: effects of organic acid ligands addition

We have investigated the effects of low molecular weight organic acid ligands on the adsorption of the insecticidal toxin from Bacillus thuringiensis (Bt) by the colloidal (<2 μm particle-size) fraction of some soils. The desorption of the bound toxin by NaCl and phosphate buffer has also been measured. The soils used were a red soil (Ultisol), a latosol (Oxisol), a yellow brown soil (Alfisol) and a yellow cinnamon soil (Alfisol) from central and southern China. The adsorption isotherms were all of the L-type, and the data fitted the Langmuir equation (R² > 0.97). When present at low concentrations, organic acids (acetate, oxalate, citrate) had an inhibitory effect on toxin adsorption. Uptake, however, was promoted when the organic acid concentration exceeded 10 mM. The toxin was very strongly bound by the soils but the soil-toxin interaction weakened in the presence of organic acids. A small portion of the toxin was adsorbed by electrostatic and ligand exchange interactions. The addition of organic acids appeared to enhance these interactions. Responsible Editor: Thomas B. Kinraide     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Synthesis, spectral and crystallographic characterization of manganese(II) coordination polymers based on aliphatic dicarboxylate and flexible bis(imidazolyl) derivatives

Two structurally related flexible imidazolyl ligands, bis(N-imidazolyl)methane (L₁) and 1,4-bis(N-imidazolyl)butane (L₂) reacted with Mn(II) salts of aliphatic dicarboxylic acids resulted in the formation of a number of novel metal-organic coordination architectures. All complexes have been structurally characterized by X-ray diffraction analysis. The different coordination modes of dicarboxylate anions due to their chain length, rigidity and diimidazolyl functionality lead to a range of different coordination structures. The coordination polymers exhibit 1D single chain, 2D sheet and 3D network structures. The aliphatic dicarboxylates can adopt chelating μ₂, bridging μ₂, and chelating-bridging μ₃ coordination modes, or act as uncoordinated counter anions. The central metal ions are coordinated in N₂O₄ and N₄O₂ fashions depending on the ancillary ligands. The topology of [Mn(male)(L₁)(H₂O)₂] (1, male = maleate) gives rise to singly bridged 1D chains, whereas compound [Mn(mal)(L₁)(H₂O)] · H₂O (2, mal = malonate) exhibits 2D sheets in which the metal centers are bridged by both imidazolyl ligands and dicarboxylates. Compounds [Mn(L₁)₂(H₂O)₂](suc) · 6H₂O (3, suc = succinate) and [Mn(L₁)₂(H₂O)₂](fum) · 6H₂O (4, fum = fumarate) show doubly bridged 1D chains, and the dicarboxylate groups are not coordinated but form 2D corrugated sheets with water molecules intercalated between the cationic layers. Compound [Mn(suc)(L₂)(H₂O)₂] (5, suc = succinate) was built from very flexible succinate and 1,4-bis(N-imidazolyl)butane which yielded three-dimensional interpenetrate networks, both succinate anion and the imidazolyl ligand act as bidentate bridging.     

意愿价值评估法应用于农业生态补偿研究进展

农业生态补偿是支持和保护农业发展的重要政策激励手段,有关补偿标准的估算及计量方法研究始终是学术界讨论的热点。意愿价值评估法(CVM)是国际社会进行非市场价值评估最广泛采用的陈述偏好方法。由于在农业生态补偿领域应用时间不长,农业产业的弱质性及农民认知水平的差距,导致国内CVM的实证研究面临许多问题和困惑。阐明CVM方法的内涵与理论基础,全面系统地梳理国内外CVM在农业补偿领域的研究进展。以激励农户采纳农业清洁生产技术补偿意愿问卷调查为例,深入探讨CVM应用于农业生态补偿政策评估中存在的假想特性偏差、研究手段偏差及调查实施偏差等。提出通过合理的技术途径和对策措施规避可能的偏差,包括:明确计量模型适用范围、开展效度与信度检验、选择两种尺度评估及处理调查过程偏差的方法等,为破解CVM应用于国内农业生态补偿政策制定中的问题提供方法和思路借鉴。研究指出存在的不足与局限性,以期未来不断完善和拓展。