Development of Novel and High-throughput Technology for Discovery of Gene Resources from Variety of Microorganisms in Soil
NEDO, Research and Development for an Intellectual Foundation
(January 12, 2005)
- Background and Project Objective
A huge amount of biological resources exist on Earth. Among the variety of such resources, microorganisms that can be utilized by human beings are the most valuable. To date, many useful microorganisms have been isolated from various environments around the world. However, such useful microorganisms are only a tiny part of the microorganism population present on Earth. In the past, it was possible to utilize the useful properties of only isolated and cultivable microorganisms. However, since 99% of the microorganism population has not been cultivated, the functions possessed by most microorganisms remain to be utilized.
In particular, soil is one of the richest environments for biological resources, and many microorganisms capable of producing antibiotics have been isolated. However, only a limited amount of gene resources from soil have been utilized because of difficulty in soil sample handling and the low concentration of microorganisms living in soil. Therefore, the main objective of this project is the development and establishment of novel and high-throughput technology that can be used to discover and utilize gene resources from uncultivable microorganisms in soil. In particular, hot environments are believed to contain microorganisms possessing thermostable proteins that are also stable in other extreme conditions. Because of this stability, enzymes isolated from thermophilic microorganisms are expected to be useful for industry. For this reason, a high temperature environment was selected as the target of this project.
This research was started in 2002 as a three-year project with Precision System Science Co. Ltd (PSS) as the primary research contractor. The project is a collaborative endeavor involving the industrial, governmental and academic sectors. Other project participants include the Faculty of Engineering of Tokyo University of Agriculture and Engineering, the Faculty of Engineering of Tokushima University, the Faculty of Agriculture of Kyushu University, and the National Institute of Advanced Industrial Science and Technology.
- Project Plan
The first research goal of the project was to develop a simple and compact instrument using magnetic micro-beads that collects entire microorganisms from the soil and then extracts gene information from microorganism DNA or RNA molecules. At the same time, it was planned to construct an automated instrument for extracting a large amount of DNA from a large volume of soil sample solution recovered and concentrated by the simple and compact instrument. By using these two elemental instruments together as a new system, it was expected that the collection of whole microorganisms or nucleic acids present in sample soil would be realized. Furthermore, it was planned to identify useful gene resources from soil samples obtained by utilizing this system.
For the simple and compact instrument to collect living microorganisms in a soil environment, newly developed magnetic micro-beads were used. For the large-scale DNA extraction instrument, Magtration® technology, which is proprietary to PSS, was utilized. In contrast with the conventional method in which a magnet used for separation of magnetic micro-beads is attached at the bottom of a reaction tube, Magtration® technology was developed as a unique technology for the separation of magnetic micro-beads within a plastic tip having a capillary section to which the magnet is attached. In addition, it is important that Magtration® technology can be applied to DNA extraction as well as separation and purification of proteins or microorganisms when magnetic micro-beads with different types of surface coating are used. Also, Magtration® technology can minimize the risk of cross-contamination between samples that was unavoidable with the conventional method as separation is performed within a plastic tip. A notable example of the application of this technology is that the Japan Red Cross Society uses it for screening virus contamination in donated blood.
The work described below for the discovery and characterization of novel important genes from a soil environment has been carried out through the collaboration of many scientists at the universities and national institutes participating in the project. Search areas include metabolic enzymes, DNA synthesis or repair genes with a correlation for cancer, and membrane proteins correlating with signal transduction or transportation. Novel genes isolated from these areas will be applied for the discovery of new medicines.
- Project Achievements (Interim Results)
Firstly, the simple and compact instrument, named the Portable Sampling Instrument (Figure 1), was designed and constructed as a sampling instrument to recover valuable microorganisms as a concentrated solution from a large amount of soil. With this instrument, the microorganisms present in approximately 1 kg of soil can be selected by binding to specific magnetic micro-beads and then be recovered as a solution of a few tens of ml.
During the development of the Portable Sampling Instrument, a new type of magnetic micro-beads (Table 1) that can bind to microorganisms in soil for efficient recovery was also developed. Since many organisms with different surface structures are present in soil, it is difficult for one type of magnetic micro-beads to be capable of binding to whole microorganisms. Thus, a combination of various magnetic micro-beads with different types of surface coating was planned for recovery of different microorganisms. This improvement made it possible to recover various microorganism species (e.g. gram-positive and gram-negative bacteria, eukaryotes) and even unknown microorganisms. In an evaluation test in which a cultured microorganism was used as a model, it was found that over 80% of the microorganisms present were recovered using a model solution with the Portable Sampling Instrument through combined use with the newly developed magnetic micro-beads.
Secondly, a new instrument designated Magtration® System 8Lx, (Figure 2) was designed and constructed to extract DNA or RNA from a large volume of solution containing microorganisms recovered from soil by the Portable Sampling Instrument. For development of the Magtration® System 8Lx, it was important to select and optimize reagents and protocols for efficient extraction of DNA. During reagent selection, an evaluation step was performed at a sampling site using an instrument called the SX-6GC. This instrument is a major commercial product of PSS, and with a weight of approximately 26 kg it can be handled by one person. Fortunately, it was found that DNA was efficiently recovered from microorganisms in soil by combined treatment of physical damage and use of various enzymes for cell lysis. Moreover, it was shown that this protocol could prepare DNA with less damage than that prepared by the currently available soil DNA extraction kit.
Thirdly, as an evaluation of the newly developed Portable Sampling Instrument, Magtration® System 8Lx and magnetic micro-bead system, it was considered whether some genes similar to genes characterized as being useful were collected from soil obtained from a high temperature environment. The site selected for the evaluation was a geothermal area in a hot spring where the temperature of venting water was over 90ºC and no artificial modifications were performed. The reasons why such a geothermal area was selected are as follows: (1) we wanted to confirm if many different types of hot springs could be utilized for different types of gene resources, (2) it was expected that thermostable proteins were present in such a thermal environment, and (3) the genes recovered from a thermal environment should not be toxic to humans because of different growth temperatures. In fact, many useful enzymes and genes have already been identified in microorganisms living in high temperature environments and some of these are being utilized in industry.
From phylogenetic analyses of microorganisms living in thermal locations using prepared DNA molecules, different types and amounts of microorganisms were detected according to the properties of the sample locations. The results of phylogenetic analyses also indicated evidence that some unknown and uncultivable Archaea are alive in the soil obtained from a geothermal environment (Fig. 3).
As a preliminary result for identification of useful genes from uncultivable microorganisms living in a high temperature soil environment, DNA fragments containing nucleotide sequences similar to DNA polymerase (a DNA amplifying enzyme), DERA, alanine dehydrogenase, chitinase and DPM synthetase (a membrane enzyme for synthesis of polysaccharide) were found to be amplified by PCR reaction. Since only a few microorganisms can be cultivable from such an environment, it is considered that immense gene resources are stored in geothermal soil environments and that they await identification by humans. It can be said that the discovery of unknown genes in soil will greatly contribute to the discovery of numerous useful resources.
Also, a system that includes a new PSS micro-array method named the Bio-Strand System was designed and constructed for easy detection of microorganisms existing in a soil environment. The Bio-Strand System was developed to analyze gene content or the rate of gene expression based on the concepts of easy handling and a low operating cost. Compared to a conventional DNA chip, it was found that the results obtained with the Bio-Strand System are more reliable.
- Future Work
We will continue to make great efforts to improve the developed system, including the two instruments, reagents and new type magnetic micro-beads, for more efficient recovery of microorganisms and DNA molecules from a high temperature environment. Also, we will continue to analyze the details of PCR fragments, which encode a part of useful enzymes, using amplified DNA prepared from high temperature environment soil. We believe it will be possible to develop new antibiotics, agricultural chemicals and biochemical reagents for commercialization by utilizing important information obtained from the environment in the near future. Finally, we will endeavor to improve fully automated instruments for effective amplification by PCR as well as techniques for DNA cloning and the recovery of DNA. A method to recover DNA, and some pathogenic microorganisms, from a soil environment will also be established.
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