氧化葡萄糖酸杆菌中硫辛酸合成模块对维生素C一步混菌发酵的影响

在由氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌构建的维生素C两菌一步发酵体系中,为了强化氧化葡萄糖酸杆菌对普通生酮古龙酸杆菌生长和产酸的促进作用,文中在氧化葡萄糖酸杆菌中构建硫辛酸合成功能模块。由含硫辛酸功能模块的氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌组成的两菌一步体系,能减轻普通生酮古龙酸杆菌单菌培养时的生长抑制,强化两菌的互作关系,使维生素C前体(2-酮基-L-古龙酸,2-KGA)的产量提高到73.34 g/L(对照组为59.09 g/L),醇酸转化率提高到86.0%。研究结果为进一步优化维生素C两菌一步发酵体系提供了新思路。     

巨大芽胞杆菌与普通生酮基古龙酸菌共培养过程中相互作用分子机制研究进展

混菌发酵法广泛应用于维生素C前体2-酮基-L-古龙酸的生产.为进一步改善工艺,多年来,科研人员一直致力于研究发酵过程中两菌相互作用的科学本质.目前,随着组学技术、高通量技术、生物信息学和生理学等多种技术与学科的迅速发展,为深入研究相互作用分子机制提供了新的方法和工具.通过蛋白质组学、代谢组学、比较基因组学、转录组学等多种组学数据的挖掘和分析,提供了系统中各层次之间的相互作用关系网络.在此基础上结合高通量的生理学验证分析,为诠释两菌相互作用分子机制和开发代谢工程改造策略奠定了基础.本文就近些年来在该研究方向的进展及其应用进行了简要归纳,并提出进一步研究的方向.     

氧化葡萄糖酸杆菌SCB329的培养及保存

氧化葡萄糖酸杆菌SCB329和苏云金芽孢杆菌SCB933是混合发酵产生维生素C前体2－KLG两株主要菌种,本文对氧化葡萄糖酸杆菌SCB329的纯培养,传代及纯小菌的保存及其对产酸的影响作了研究。     

维生素C第二步混菌发酵技术研究新进展

维生素C(简称维C)"两步发酵法"是我国科学家独创、目前唯一工业化应用的维C工业生产技术,其显著特征是第二步发酵为两种菌的混合发酵。综述了近年来我国在维C第二步发酵技术领域的最新研究进展。大量研究认为混菌发酵效率取决于产酸菌数量和催化酶(山梨糖脱氢酶)活性,而产酸菌的转化能力又依赖于伴生菌所释放的"伴生物质",并认为伴生菌的"伴生物质"应该有多种,除了大家所公认的蛋白类物质外,氨基酸、维生素、嘌呤类等小分子有机物也具有促进产酸菌生长和产酸的作用,都应归属于"伴生物质"。另外,列举了当前维C工业化生产中面临的技术难题,提出了解决思路和未来研究方向,以期对推动维C发酵技术进步和维C产业可持续发展提供参考。     

巨大芽孢杆菌B．m2980产孢对氧化葡萄糖酸杆菌产酸的影响

为确定维生素C二步发酵中巨大芽孢杆菌（伴生菌）芽孢形成对氧化葡萄酸杆菌（产酸菌）产酸的影响,本研究通过对巨大芽孢杆菌生长特性分析,选取培养12h（未形成芽孢）和36h（芽孢大量形成）巨大芽孢杆菌B．m2980,检测其胞外液、胞内液以及混合液对产酸菌生成2-酮基-L-古龙酸的影响。结果表明,在未开始形成芽孢时,伴生菌胞外液、胞内液及混合液对产酸菌的生长和产酸有较低的促进作用,其中胞内液的促进能力大于胞外液;在芽孢生成后,胞外液以及混合液对产酸菌生长和产酸的促进能力显著提高。     

混合培养中巨大芽孢杆菌对氧化葡萄糖酸杆菌的作用

为查明维生素Ｃ二步发酵混合培养中巨大芽孢杆菌与氧化葡萄糖酸杆菌间的关系,通过生长曲线测定、静息细胞实验及摇瓶发酵实验研究了巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产生２－酮基－Ｌ－古龙酸作用的影响;采用超滤分离、凝胶层析及聚丙烯酰胺凝胶电泳技术对巨大芽孢杆菌胞外液中具有促进氧化葡萄糖酸杆菌产酸作用的活性物质进行了分离和纯化。结果表明,大菌胞内液和胞外液均可促进小菌生长,大菌胞外液中具有该作用的组分分子     

离子注入氧化葡萄糖酸杆菌的诱变效应

本文运用离子注入对维生素C前体2—酮基—L—古龙酸的产生菌氧化葡萄糖酸杆菌的诱变效应进行了研究,确立了以8％甘油作保护剂,并就低能离子注入条件进行了参数设定。     

山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析

酮古龙酸杆菌Ketogulonigenium vulgare是维生素C二步混菌发酵过程中的产酸菌。山梨酮脱氢酶(L-sorbosone dehydrogenase,缩写为SNDH)作为维生素C直接前体2-酮基-L-古龙酸(2-KGA)合成的关键酶,其作用机制并不十分清楚。借助全基因组测序抽提2个山梨酮脱氢酶基因,分别位于基因组(缩写为sndhg)和质粒(缩写为sndhp)上。通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块,比较其对2-KGA产量的影响。研究发现sndhg过表达对菌株产酸影响不明显,sndhp过表达使菌株明显产生副产物。将sndhg和sndhp分别配合辅因子PQQ合成基因pqq A,分别构建sndhg-pqq A和sndhp-pqq A模块,得到的工程菌株产酸情况与之前的结果大致相同。将4株K.vulgare工程菌株分别与内生芽孢杆菌Bacillus endophyticus混合培养传代50 d后,分离菌株进行混菌发酵,其2-KGA的转化率分别提高了15.4%、179%、0.65%和125%。表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性,进而快速获得优良性状菌种的有效方法。     

基于组学技术的维生素C二步混菌发酵研究进展

二步发酵法是工业化生产维生素C (vitamin C, Vc)的主要方法,其中第二步由伴生菌与产酸菌(普通生酮基古龙酸菌)组合进行混菌发酵产生Vc前体2-酮基-l-古龙酸(2-keto-l-gluonic acid, 2-KLG)的机制,一直是科研人员研究的重要科学问题。通过高通量基因组学、转录组学、蛋白组学、代谢组学等组学技术揭示生物系统中各个组分相互作用关系已经成为主要的研究手段。本文对近年来利用组学技术解析Vc混菌发酵中两菌互作关系、解除发酵系统的氧化胁迫、伴生活性物质、产酸菌群体感应、外源添加物、基因工程改造产酸菌促进产2-KLG等方面的研究进行综述,并为进一步的探索和深入研究提供思路。     

维生素C前体2-酮基-L-古龙酸二步混菌发酵研究新进展

维生素C（Vc）二步混菌发酵是我国首创具有自主知识产权的唯一应用于工业化生产Vc的微生物转化方法,该方法利用混合菌发酵L-山梨糖生产Vc前体物质-2-酮基-L-古龙酸,再经化学转化合成Vc;具有简化工艺,减少污染,降低能耗等优点。本文主要从产酸菌代谢关键酶、伴生菌胞外物质、组学以及外源添加物等方面综述Vc二步混菌发酵的最新研究进展,并提出进一步研究和探索的方向。     

Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS

Vibrio cholerae can be differentiated into epidemic and non-epidemic strains by sorbitol fermentation speed, but little research has been done on its mechanisms. In this study, we investigated differential protein expression of the two strains in response to sorbitol metabolism. V. cholerae strains were cultured in media with and without sorbitol, respectively. Proteins were separated by 2-DE, and those that showed different expression in the two media were identified by MALDI-TOF MS. Fifteen proteins in epidemic strains and 11 proteins in non-epidemic strains showed a different expression in sorbitol medium. Among them, 4 proteins were common to epidemic and non-epidemic strains. Gene sequence analysis showed that some mutations occurred in these proteins between the two strains. Potential functions of these proteins included sugar uptake, amino acid uptake, electron transport, sulfate and thiosulfate transport.     

基因打靶及其应用

用活细胞染色体DNA可与外源性DNA同源序列发生同源重组的性质,达到定点修饰改造染色体某基因的目的,此法称基因打靶．基因的同源重组是较普遍的生物现象,其分子机理尚未阐明,但活细胞内确有一酶系可使DNA的同源序列在细胞内发生重组,这一事实已无可争辨．此事实为基因打靶的理论基础．基因打靶技术操作的关键是建立一含筛选基因的重组载体,并有效地把它转入细胞核内．基因打靶命中的细胞可稳定遗传．基因打靶在改造生物品种,一些复杂生命现象（如发育的分子机制等）及临床理论研究均有广阔的前景．     

Nucleotide sequence changes between Streptococcus pneumoniae R6 and D39 strains determined by an oligonucleotide hybridization DNA sequencing technology

The Gram-positive pathogen Streptococcus pneumoniae, which can be responsible for serious cases of pneumonia and meningitis, has been intensely studied for almost 100 years. Many of the key experiments have been performed in two strains; the non-pathogenic S. pneumoniae R6 and its pathogenic progenitor, S. pneumoniae D39. Whereas the genomic sequence of the R6 strain has been published, there is relatively little genomic information available on D39. Since R6 was derived from D39, we wished to explore the utility of a new technology, Comparative Genome Sequencing, which uses a set of custom oligonucleotide arrays to compare DNA sequences between similar strains. We report here the nucleotide polymorphisms identified between the R6 strain and D39 based on an R6 sequencing array. During the process, we were also able to confirm all of the high confidence changes reported by the oligonucleotide array chip by sequencing the region in the genome around the changes identified with the genome hybridization chip. We also discuss the potential impact of some of the amino acid changes found between these two widely used strains of pneumococci.     

Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy

Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains.     

The long and winding road from the research laboratory to industrial applications of lactic acid bacteria

Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.     

我国食用菌遗传学的发展及展望

食用菌遗传学是食用菌学科体系的重要分支之一,40年来我国食用菌遗传学研究紧密围绕为育种服务、最新分子生物学技术的应用和生产实践中的科学问题的解决等主题开展了众多科学活动。为了促进食用菌遗传学研究的系统性和全面性,本文梳理出9个方面的研究主题,主要包括食用菌种质资源调查和地方品种研究、食用菌农艺性状控制基因定位和分子辅助育种技术研究、食用菌杂交育种的遗传学规律研究、食用菌菌种的遗传稳定性研究和变异风险监控、栽培基质分解利用和储存转运的分子机制、食用菌应对环境因素变化的分子机制、子实体发育的分子调控机制、食用菌次级代谢产物生物合成的分子机制以及食用菌鲜品采摘后代谢生理的分子机制等,目前这些研究主题有些正在成为研究热点,有些在研究的系统性上还有待完善,有些还缺少足够的关注兴趣。希望食用菌遗传学在未来的发展中,能够把一些源头和底层的科学规律弄清楚,形成完整的知识体系和理论体系,为推动食用菌产业高质量发展提供科学基础。     

灰黄链霉菌的一个新亚种

在研究敦煌壁画微生物生态学的过程中,从257窟分离到一株链霉菌,编号为25706.该菌株在合成琼脂上气丝为黄白色,孢子丝柔曲或松螺旋,所产生的抗生素对革兰氏阳性细菌和革兰氏阴性细菌均有较强的抑制作用.经鉴定,该菌株为灰黄链霉菌的一个新亚种,命名为灰黄链霉菌敦煌亚种.     

利用功能基因组学方法研究酿酒酵母乙醇生物合成的调控机理

酿酒酵母在发酵生产乙醇的过程中存在的主要问题是前期高浓度底物葡萄糖的抑制和后期高浓度产物乙醇的抑制。功能基因组学技术的发展为从基因组水平上系统研究酿酒酵母乙醇生物合成的调控机理提供可能。本研究模拟工业发酵的条件,对酿酒酵母实验菌株BY4743为遗传背景的116个单基因缺失菌株进行了乙醇发酵试验,以发现基因和乙醇发酵的关系。结果表明乙醇对菌体得率系数高于平均值30%以上的基因缺失株有20株,其中高于50%以上基因缺失株有5株;低于平均值30%以上的基因缺失株有11株,其中低于45%以上的有5株。本研究为从整个酿酒酵母基因组水平上系统研究乙醇生物合成的调控机理建立了研究方法,提供了可行性验证。     

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway

Summary It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his₃ strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.Abbreviations IGP imidazole glycerol phosphate - InGP indole glycerol phosphate - ASase anthranilate synthetase - InGPase indole-3-glycerol phosphate synthetase - TSase tryptophan synthetase - Tris tris (hydroxymethyl)-aminomethane This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Divergence in replicated phylogenies: the evolution of partial post-mating prezygotic isolation in bean weevils

By tradition, speciation research has been focused on processes leading to either premating or post-zygotic reproductive isolation. The processes which generate isolation after mating but before zygote formation are less well understood. Here, we study divergence in characters which contribute to post-mating prezygotic isolation, such as egg production and remating rate. We propose that 'replicated' laboratory phylogenies with known histories can be used to yield insights into the processes of divergence. We performed a series of cross-matings between populations within two strains of the bean weevil Callosobruchus maculatus. Each strain has a unique and independent origin and both have been kept in the same set of laboratories during the last few decades. Our results show that divergence has occurred between laboratory populations within strains with regards to the effects that mating has on female reproductive behaviour, showing that the evolution of partial post-mating prezygotic isolation can be rapid. More importantly, the pattern of divergence across populations was distinct in the two strains, suggesting that coevolutionary trajectories are not determined by environmental factors but are to some extent arbitrary. We discuss the limitations of the novel empirical strategy employed here, and conclude that our results lend support to the hypothesis that post-mating sexual selection is capable of rapidly generating post-mating prezygotic isolation.