Angell, Scott Edward (2008-05). Genomic and metagenomic approaches to natural product chemistry. Doctoral Dissertation. Thesis uri icon

abstract

  • For many years, natural products have been a primary source of new molecules for the treatment of disease, and microorganisms have been a prolific source of these molecules. Recent studies have indicated, however, that many biosynthetic pathways are present in organisms for which no natural product can be associated, and only a small fraction of the microbial life present in the environment can be grown in culture. This indicates that if methods could be developed for the isolation of these pathways and production of their target molecules in heterologous hosts, great numbers of potentially valuable compounds might be discovered. In these investigations, large insert libraries of two microorganisms were constructed, one a bacterial artificial chromosome (BAC) library, the other a fosmid library, and two large insert fosmid libraries were constructed with DNA isolated from marine environmental samples. A mathematical formula was derived to estimate probabilities of cloning intact biosynthetic pathways with large insert genomic libraries and tested with a computer simulation. This indicated that even large pathways could be cloned intact in large insert libraries, provided there was an adequate size difference between the target pathway and the library inserts, and there was a concomitant increase in the size of the library with the targeting of these larger pathways. In addition, an investigation into a mixed marine culture sample lead to the identification of an unusual relationship between two bacteria for which extended co-culture leads to the production of pyocyanin. However, no useful biosynthetic pathways were located within the genomic libraries. It is concluded that significant improvements would be required to make this approach feasible for larger scale investigations. It is further concluded, on the basis of recent developments in the field, including a reduction in the cost of sequencing, improvements in techniques of whole-genome shotgun sequencing, and the development of recombination based cloning, that the employment of mass sequencing efforts and sequence-driven, recombinationbased cloning, might prove to be a more fruitful and efficient alternative to large-insert library construction for the isolation and expression of these pathways. A possible paradigm for the cloning of pathways on the basis of this technology is proposed.
  • For many years, natural products have been a primary source of new molecules for the
    treatment of disease, and microorganisms have been a prolific source of these molecules. Recent
    studies have indicated, however, that many biosynthetic pathways are present in organisms for
    which no natural product can be associated, and only a small fraction of the microbial life
    present in the environment can be grown in culture. This indicates that if methods could be
    developed for the isolation of these pathways and production of their target molecules in
    heterologous hosts, great numbers of potentially valuable compounds might be discovered.
    In these investigations, large insert libraries of two microorganisms were constructed,
    one a bacterial artificial chromosome (BAC) library, the other a fosmid library, and two large
    insert fosmid libraries were constructed with DNA isolated from marine environmental samples.
    A mathematical formula was derived to estimate probabilities of cloning intact biosynthetic
    pathways with large insert genomic libraries and tested with a computer simulation. This
    indicated that even large pathways could be cloned intact in large insert libraries, provided there
    was an adequate size difference between the target pathway and the library inserts, and there was
    a concomitant increase in the size of the library with the targeting of these larger pathways. In
    addition, an investigation into a mixed marine culture sample lead to the identification of an
    unusual relationship between two bacteria for which extended co-culture leads to the production
    of pyocyanin. However, no useful biosynthetic pathways were located within the genomic
    libraries.
    It is concluded that significant improvements would be required to make this approach
    feasible for larger scale investigations. It is further concluded, on the basis of recent
    developments in the field, including a reduction in the cost of sequencing, improvements in techniques of whole-genome shotgun sequencing, and the development of recombination based
    cloning, that the employment of mass sequencing efforts and sequence-driven, recombinationbased
    cloning, might prove to be a more fruitful and efficient alternative to large-insert library
    construction for the isolation and expression of these pathways. A possible paradigm for the
    cloning of pathways on the basis of this technology is proposed.

publication date

  • May 2008