Topic outline

  • Discovery and utilization of useful genes from aquatic metagenomes

  • Discovery and utilization of useful genes from aquatic metagenomes × SDGs

    •  SDGs14   

       So far, microorganisms have provided various benefits to human life, such as the production of useful substances and fermented foods. However, it is believed that more than 99% of microorganisms in the environment have not yet been isolated and cultured. In other words, uncultured microorganisms are considered to be a huge unused genetic resource. We are searching those microorganisms by metagenomic method, which directly obtains DNA without culturing them. Once genetic resources are lost, they can never be recovered. We are working to maintain biodiversity and search for unused genetic resources. Through this kind of research, I would like to contribute to SDG (14 LIFE BELOW WATER).

       The United Nations has designated the decade starting in 2021 as the "Decade of Ocean Science," with the aim of contributing to SDGs. Ocean science, as defined by the UN, includes the field of fisheries.


  • Abstract

     

    • More than thirty thousand compounds have been found so far in marine organisms, and many of them are useful as medicines.

      Many of these compounds are thought to be produced by microorganisms such as bacteria, but more than 99% of microorganisms cannot be cultured in an artificial environment yet.

      Therefore, we are conducting research to find unused genetic resources by using metagenomic methods to obtain the genomes of microorganisms without culturing them.

  • Purpose of the research

    • Fig. Environmental bacteria cultivated in an artificial environment

      Microorganisms have been utilized in many aspects such as pharmaceutical development, food processing, and industrial material development. However, it is believed that less than 1% of all microorganisms have been cultured so far, and it is expected that a vast amount of unused genetic resources remain. As a method to obtain genetic resources of uncultured bacteria, a metagenomic method has been developed to extract DNA directly from environmental samples without depending on culture.

      By applying metagenomic methods to various marine environmental samples and marine biological resources, we are developing methods to extract high-quality metagenomic DNA, to find biosynthetic genes for the production of useful substances, and to produce compounds using the obtained biosynthetic genes.

      In this article, we introduce the method of finding genetic resources by metagenomics and show partial results.

    • {Column} 
      Why can't many bacteria be isolated and cultured in an artificial environment?
      In some cases it is because they are dependent on other bacteria for the production of essential nutrients, but in many cases the reason is not clear.

  • Metagenomics: getting the genes of bacteria that cannot be cultured


    • Figs.
      (left) Metagenomics, DNA (genetic information) of environmental microorganisms
      (middle) Environmental microorganisms, leftward arrow: environmental microorganisms, rightward arrow: culture in artificial environment
      (right) Cultivation-dependent method, only a few culturable microorganisms (less than 1% of environmental microorganisms)

      Microorganisms are collected from environmental samples (seafloor sediments and seawater) and large host organisms (benthic invertebrates such as sponges and ascidians), and the cells of the microorganisms are crushed to obtain genomic DNA.

      Considering the purpose, it is desirable to obtain as long DNA fragments as possible and high quality DNA with few impurities.  However, the samples vary in their properties, making it difficult to use a standardized method.

  • Finding useful genes from metagenomic DNA


    • Figs.
      (left) Samples from the ocean (environmental microorganisms), rightward arrow: metagenomic extraction
      (middle) Metagenomic DNA, upper right arrow: method 1) Incorporating DNA into culturable bacteria, lower left arrow: method 2) Sequencing all DNA

      There are two major methods to find useful genes from the acquired metagenomic DNA.

      One is to randomly incorporate them into easily culturable bacteria (e.g., E. coli) and look for functional genes in them (1) random cloning), and the other is to determine the sequence of all DNA fragments and look for functional genes in the DNA information (2) full sequencing).

  • Method 1) random cloning


    • Figs.
      (left) Random metagenomic library (E. coli hosts), right arrow: capability test
      (upper-right) Pigment production: production of various pigments such as blue, red, yellow, purple, and fluorescent can be confirmed visually. some of them are luminescent clones that glow at night (they have acquired a luminescent gene)
      (middle-right) Siderophore production: We have added a pigment to the medium that turns yellow when siderophores (substances necessary for the acquisition of iron, an essential nutrient. siderophores' structure varies among bacteria)  are present
      (lower-right) Production of antibacterial substances: when a metagenomic clone produces an antimicrobial substance, the bacteria around the clone die and black circles appear.

       The obtained fragments of metagenomic DNA are randomly incorporated into a culturable host such as E. coli. Each of the colonies in the lower left photo contains a different metagenomic DNA fragment.

      Each colony is called a metagenomic clone, and the aggregate (the entire plate in this example) is called a metagenomic library.

      If the metagenome contains a gene that is required to produce some substance (such as a pigment), the clone will produce that substance ( in the case of a pigment, the colony will be colored).

    • {Column}
      In many cases, multiple genes are required to make a single compound.
      Fortunately, in bacteria, the necessary genes are often found in a continuous and coherent set (called a gene cluster).
       Therefore, it is possible to incorporate all the necessary genes together into a single clone.
      In the case of the biosynthetic gene cluster of antimicrobial substances, self-resistance genes are also included.

    • Advantages and disadvantages of method 1)

      Advantages

      Genes that have never been sequenced before can be obtained.
      No need for DNA sequencing costs.

      Disadvantages

      Metagenomic genes do not always work in E. coli.
      Difficult (but not impossible) when multiple genes are required.
      It takes time and effort.

  • Method 2) full sequencing


    • Fig. Homology search on database

      The sequenced metagenomic information is subjected to a database search.

      If the sequence is similar to that of a gene related to the intended function, then a similar function is expected.

      In addition, by searching for homology, you can obtain various information other than the intended function, such as what kind of organism the metagenomic DNA was derived from and what kind of compound it can produce.

    • Advantages and disadvantages of method 2)

      Advantages

      It requires little time and effort, and can process a huge amount of genetic information in a short period of time.
      Genes that do not function in E. coli can be found.

      Disadvantages

      Only genes that are similar to already identified genes can be found (completely new genes cannot be found).
      Since we only have data, we need to artificially synthesize the genes.

  • Examples of materials and genes obtained from marine metagenomes


    • Fig.
      (left) Vibrioferrin form sand and mud on a tidal flat
      (middle) Bisucaberin from deep-sea sediment
      (right) Agrobactin from sponges


      Examples of siderophores produced from marine metagenomes and their origin and composition of biosynthetic gene clusters.

      Metagenomic DNA is obtained from various marine environmental samples and organisms, and the genes are practically cloned for material production.

      Some of them are new types of genes or substances discovered for the first time in the ocean.