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Metagenomic approach to study the polyhydroxyalkanoate genes in hydrocarbon contaminated site.

机译:运用元基因组学方法研究烃污染位点中的聚羟基链烷酸酯基因。

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摘要

Plastics being xenobiotic are recalcitrant to microbial degradation. Plastic production also involves a number of harmful chemicals which pose environmental as well as human health risks. There is an obvious need to minimize the generation of plastic waste and to search for newer technologies that can play a vital role in mitigating these problems.;Research has been going on for quiet some time with the idea of developing an alternate environment friendly product. Many biodegradable plastic materials have been exploited such as: 1) PHAs (polyhydroxyalkanoates), 2) polylactides, 3) aliphatic polyesters, 4) polysaccharides, 5) co-polymers and /or blends of above. Of these, PHAs have gained more and more importance the world over due to their structural diversity and close analogy to plastics. The other advantage which PHAs have is that they are biodegradable and environmental friendly. Many microorganisms have the ability to degrade these macromolecules enzymatically.;PHAs are polyesters of various hydroxyalkanoates that are synthesized by many grampositive and gram-negative bacteria. It was in the last century (1926) when Lemoigne reported the formation of poly(3-hydroxbutyrate) (PHB) inside bacteria for the first time. Since then research in this field has developed considerably with the discovery of many more different PHAs from at least 75 different genera. This polymer is accumulated intracellularly to levels as high as 90% of the cell dry weight under conditions of nutrient stress and acts as a carbon and energy reserve.;Research over the years has focused on the use of alternative substrates, novel extraction methods, genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive. Enormous advances in molecular analysis of PHA biosynthesis genes and PHA production have been well documented. Many strategies such as homologous or heterologous gene probes, short consensus oligonucleotide hybridization or PCR techniques have been employed for identifying PHA synthase genes and other genes involved in PHA biosynthesis. In Escherichia coli, a previous non-PHA producer, many strategies such as pathway engineering has been developed to set up microbial production by recombinant DNA technology. The sequence analysis of 89 complete and 34 partially sequenced genomes was done in an attempt to identify domains of PHA genes using RPSBLAST. Computer simulations of PHA granule formation in vivo are being done to help design strategies to optimize the fermentation process and achieve higher yields of PHA.;Although PHA have been commercially developed and marketed, there are several important factors influencing the large scale commercial production. Efforts are being devoted with a renewed interest to increase PHA yield and productivity. A search is on to identify organisms capable of PHA production at higher levels.;Conventional methods for searching microbes employ ineffective laboratory method with the result that a major portion of the bacteria remain largely untapped, unknown, and uncharacterized due to lack of proper culture conditions. On the other hand, Metagenomics represents a powerful tool to access the abundant microbial diversity of native environmental samples. It helps to effectively characterize the genetic diversity present in any samples regardless of the availability of laboratory culturing techniques.;The term “metagenome” was first coined by Handelsman, 1998, as: The genomes of the total microbiota found in nature. Metagenomics, instead of removing microbes from their environment to isolate them, just directly isolates genomic DNA. This DNA is then cloned and environmental genetic libraries are constructed. Relevant clones from the libraries are sequenced or screened for the expression of gene products. Nucleotide-sequence based screening is highly efficient in comparison to enzyme-activity based screening. The success of metagenomic approach has already been demonstrated in identifying novel products ranging from small-sized genes conferring enzymatic activities such as lipases, amylases. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community.;The application of metagenomics to PHA will facilitate the trapping of those microbes that might have a better potential to produce PHA but are being missed out due to lack of proper culture techniques. This study is aimed at using metagenomics approach for bioprospecting of the PHA synthase gene in the environmental sample. Hydrocarbon contaminated sites are rich in carbon and have nutrient imbalance, providing excellent conditions for microbes to accumulate large amounts of PHA. And therefore are appropriate sources of sampling for our study. (Abstract shortened by UMI.).
机译:作为异源生物的塑料难以抵抗微生物降解。塑料生产还涉及许多有害化学物质,对环境和人类健康构成威胁。显然需要最大程度地减少塑料废料的产生,并寻求在减轻这些问题中起着至关重要作用的新技术。研究已经悄悄进行了一段时间,以开发另一种环保产品。已经开发了许多可生物降解的塑料材料,例如:1)PHA(聚羟基链烷酸酯),2)聚丙交酯,3)脂族聚酯,4)多糖,5)上述共聚物和/或共混物。其中,由于其结构的多样性和与塑料的相似性,PHA在世界范围内变得越来越重要。 PHA的另一个优点是它们可生物降解且对环境友好。许多微生物具有酶促降解这些大分子的能力。PHA是由许多革兰氏阳性和革兰氏阴性细菌合成的各种羟基链烷酸酯的聚酯。在上个世纪(1926年),Lemoigne首次报道了细菌内部聚(3-羟丁酸)(PHB)的形成。从那以后,随着至少75个不同属的更多不同PHA的发现,该领域的研究有了长足发展。在营养胁迫下,这种聚合物可在细胞内积累至细胞干重的90%,并具有碳和能量储备的功能。多年来的研究集中在使用替代性底物,新颖的提取方法,遗传上增强物种和混合文化,以期使PHA在商业上更具吸引力。 PHA生物合成基因和PHA产生的分子分析已取得了巨大的进步。已经采用了许多策略,例如同源或异源基因探针,短共有寡核苷酸杂交或PCR技术来鉴定PHA合酶基因和参与PHA生物合成的其他基因。在以前的非PHA生产者大肠杆菌中,已经开发了许多策略(例如途径工程)来通过重组DNA技术建立微生物生产。尝试对89个完整和34个部分测序的基因组进行序列分析,以尝试使用RPSBLAST识别PHA基因的结构域。体内PHA颗粒形成的计算机模拟正在进行中,以帮助设计策略优化发酵过程并实现更高的PHA产量。尽管PHA已商业化开发并投放市场,但仍有一些重要因素影响大规模商业化生产。人们正在重新努力提高PHA的产量和生产率。正在进行搜索以鉴定能够生产更高水平PHA的生物体;传统的微生物搜索方法采用的实验室方法无效,结果由于缺乏适当的培养条件,大部分细菌仍处于未开发状态,未知状态和未表征状态。另一方面,元基因组学是访问本地环境样品丰富的微生物多样性的强大工具。无论实验室培养技术的可用性如何,它都可以有效地表征任何样品中存在的遗传多样性。;“元基因组”一词最早由Handelsman,1998年提出,其含义是:自然界中发现的总微生物群的基因组。元基因组学并没有直接从基因组DNA中分离出来,而是从环境中去除微生物来分离它们。然后克隆该DNA,并构建环境遗传文库。对来自文库的相关克隆进行测序或筛选以表达基因产物。与基于酶活性的筛选相比,基于核苷酸序列的筛选非常高效。宏基因组学方法的成功已经在鉴定新颖的产品中得到了证明,这些产品包括赋予酶促活性的小尺寸基因,例如脂肪酶,淀粉酶。多个基因组的重组提供了有关社区内能量和营养循环,基因组结构,基因功能,种群遗传学和微异质性以及未培养社区成员之间横向基因转移的见解。宏基因组学在PHA中的应用将有助于捕获那些可能具有更好的生产PHA潜力但由于缺乏适当的培养技术而被遗漏的微生物。这项研究旨在使用宏基因组学方法对环境样品中的PHA合酶基因进行生物勘探。碳氢化合物污染的地点富含碳,并且营养失衡,为微生物积累大量PHA提供了极好的条件。因此是我们研究的适当抽样来源。 (摘要由UMI缩短。)。

著录项

  • 作者

    Cheema, Simrita.;

  • 作者单位

    TERI University.;

  • 授予单位 TERI University.;
  • 学科 Biology Molecular.;Biology Genetics.;Environmental Sciences.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 180 p.
  • 总页数 180
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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