A study on microbial patents

2003/5/1
HARAKENZO WORLD PATENT & TRADEMARK
(Written by: Kiyooka)

Deposit of microorganisms

Among inventions related to biotechnology, if the microorganism is not easily available to a person skilled in the art, the microorganism must be deposited with the international depositary authority under the Budapest Treaty before filing, and the deposit must be certified. It is necessary to attach the written document to the application (Article 27-2 of the Patent Law Enforcement Regulations). In Japan, the National Institute of Advanced Industrial Science and Technology's Patent Organism Depositary is the country's only patent microorganism depository, and is also designated as an international depositary authority under the Budapest Treaty.

Therefore, for example, when an inventor applies for a patent as an invention for a novel microorganism screened from the natural environment, it is first necessary to deposit the microorganism with the Patent Organism Depositary Center and have it assigned a accession number.
When depositing microorganisms with the Patent Organism Deposit Center, submit documents such as a deposit application form. This deposit application form includes optional information on ``scientific properties and taxonomic position,'' but the Patent Organism Deposit Center requests that these items be included as much as possible in order to streamline deposit technology operations. (Reference 1, p. 7)

The above-mentioned scientific properties include the shape of the organism, physiological and biochemical properties, etc., and the taxonomic position includes the type of the deposited organism, the genus name, and the type of the organism. Therefore, when depositing microorganisms, it is highly preferable that the microorganisms be classified to the extent that their genus and species are clear.
The basic unit of biological classification is the "species", and the main ranks that make up the classification hierarchy are "species", "genus", "family", and "family". They are divided into orders, classes, and phylums (divisions in botany). Among these, the species and genus can be determined from the scientific name of the organism (Reference 2).

The scientific names of all organisms are expressed in two names, consisting of the genus name and the species name. For example, the scientific name for Escherichia coli is Escherichia coli, which refers to the coli species of the genus Escherichia. The scientific names of bacteria are defined by the International Code of Nomenclature of Bacteria. As a general rule, scientific names are written in italics, but if italics cannot be used (for example, when handwritten), they should be underlined.

By the way, it is very important to classify the microorganisms to be deposited to the extent that the genus and species are clear, not only for the convenience of the technical work involved in the deposit, but also for the reason of clarifying the patentability of inventions related to microorganisms. preferable. In inventions related to microorganisms, since the microorganism is an important feature of the invention, the scientific properties of the microorganism are generally known; however, in addition, the microorganism is novel. From the point of view of making a claim, it is highly desirable to clarify the genus and species.
For example, even if microorganisms are taxonomically determined to belong to the same genus or species, their properties differ depending on each individual (ie, "strain"). Therefore, even microorganisms that are taxonomically known need to be deposited (Reference 3, p. 27). Conversely, even if the genus/species of a microorganism is known, if the strain has novel scientific properties that correspond to the problem of the invention, the strain can be sufficiently novel. It seems possible.

As described above, when filing an application for an invention related to microorganisms, it is extremely important to classify the microorganisms and identify their scientific properties.

Classification of microorganisms 1: Classical method

Classification of living things began on the basis of physical similarities. The same applies to microorganisms; for example, attempts have been made to classify bacteria based on their appearance (shape), such as spherical, rod-shaped (rod-shaped or cylindrical), spiral-shaped, and the presence or absence of flagella.
However, most microorganisms are single-celled organisms with little change in appearance, so it is difficult to visually classify them. Therefore, for microorganisms, classification based on physiological and biochemical properties is more important than the above-mentioned appearance.

Furthermore, in the classification of bacteria, Gram staining is extremely important in addition to the above-mentioned physiological properties. Gram staining is a bacterial staining method invented in 1853 by Danish bacteriologist Christian Gram (1938-1884), in which bacteria are stained with gentian violet or crystal violet, decolorized with alcohol treatment, and then treated with fuchsin or safranin. Post-stain with etc. Bacteria that are not decolorized by alcohol treatment and remain stained purple are Gram-positive bacteria, and bacteria that are decolorized by alcohol treatment and stained red by post-staining are Gram-negative bacteria. Judgment of positive/negative by Gram staining is one of the major basis for classifying bacteria. Note that whether or not Gram staining is possible depends on the structure of the bacterial cell membrane, but the detailed mechanism is not clear.

According to the classification based on the above-mentioned physiological and biochemical properties and Gram staining, bacteria can be classified, for example, as follows (Reference 4, pp. 78-80).

(a) Photosynthetic bacteria
(b) Gram-negative heterotrophic aerobic bacteria
(c) Autotrophic chemosynthetic bacteria
(d) Gram-negative any anaerobic bacteria
(e) Gram-negative anaerobic bacteria
(f) Gram-positive bacteria that do not produce spores
(g) Gram-positive spore-forming bacteria
(h) Spirochete
(i) Rickettsia and chlamydia
(j) Mycoplasma
(k) Hyperthermic bacteria

Classification of microorganisms 2: Molecular biological methods

Furthermore, in recent years, due to advances in molecular biology, microorganisms have come to be classified using molecular biological methods in addition to the classical methods described above.
As a molecular biological classification method of microorganisms, for example, a method using the following classification index is known.

◎Classification index is nucleic acid
　○DNA probe
　○rRNA gene base sequence
　○RFLP (Restriction Fragment Length Polymorphism)
　○G+C content of DNA
　○DNA-DNA homology
◎Target molecules are bacterial cell components other than nucleic acids
　○Isoprenoid quinone
　○Bacteria fatty acid composition
　○Cell wall components
　○Enzyme electrophoresis pattern
　○ Electrophoresis pattern of whole bacterial protein

Among these, all methods other than the method using isoprenoid quinone as a target molecule can identify the species, and in particular, the electrophoretic patterns of RFLP, enzymes, and whole bacterial proteins can identify "strains" that are subordinate to the species. (Reference 5, p. 4, Figure 1.1).

Among the above, the mainstream classification index is the rRNA gene base sequence, particularly the 16S rRNA base sequence. 16S rRNA is an rRNA that constitutes the 30S subparticle of the basic ribosomal particle, and is possessed by almost all microorganisms, with small size differences between species and phylogenetically conserved, so it is used as an indicator. It is considered very meaningful.

Furthermore, in recent years, the entire genomes of various organisms have been sequenced, and the use of genome data is expected to significantly advance microbial classification technology.
For example, the complete genome sequences of microorganisms began with Hemophilas influenzae in 1995, Saccharomyces cerevisiae in 1996, E. coli in 1997, and Bacillus subtilis in XNUMX. However, these genome analyzes were usually carried out only at research institutions with large-scale facilities.

Furthermore, in the late 1990s, the introduction of the shotgun method as a sequencing method and the practical use of multi-capillary type DNA sequencers made it possible to downsize equipment, and Genome analysis has been completed or is in progress for amino acid-producing bacteria (Corynebacterium glutamicum), aspergillus (genus Aspergillus), etc. (Reference 6).

Classification of inventions related to microorganisms

Among the microorganisms existing in the natural world, it is thought that only a few have been artificially isolated and scientifically confirmed, so it is thought that there are many new and unknown microorganisms. From this point of view, it is judged to be meaningful to file a patent application for an invention related to microorganisms.
If we were to classify the inventions related to microorganisms mentioned above, they can be roughly divided into the following two types.

(1) Inventions that create new organisms using genetic recombination technology, etc.
Example: A gene for b trait obtained from organism B is introduced into bacterium A to obtain a new bacterium A' that expresses b trait.
(2) Invention of a new system using newly discovered microorganisms obtained from nature
Example: A system that efficiently and safely decomposes waste by using a new bacterium C obtained from soil at a certain location through screening.

In inventions of type (1), in most cases it is possible for a third party to reproducibly describe the process of creating the new microorganism (in the above example, "bacterium A'") in the specification. It is. Therefore, as long as the "original" microorganism ("Bacterium A" in the above example) is easily available, the microorganism does not necessarily have to be deposited.

On the other hand, in the type (2) of invention, the most important point of the new system is the newly discovered microorganism itself ("Bacterium C" in the above example). No matter how much the process of discovering a newly discovered microorganism is described in the specification, it does not mean that a third party will be able to obtain the microorganism. It becomes necessary to deposit the information before filing the application.
Furthermore, if the invention of type (2) above is further classified, it is possible to classify it into the following two subtypes.

(2-1) An invention in which metabolites produced by microorganisms are the key to establishing a system
Example: In a system that decomposes waste efficiently and safely, a special enzyme C produced by the bacteria C used is the key to decomposing the waste.

(2-2) Inventions in which microorganisms themselves are the key to establishing a system
Example: A transformation system constructed using a new bacterium D and a vector exclusively for this bacterium D.
In the invention of the type (2-2) above, the bacterium D itself is the most distinctive feature of the invention, but in the invention of the type (2-1) above, the bacterium C itself is not necessarily the most distinctive feature of the invention. do not have. In this case, if the special enzyme c produced by the bacterium C and the gene encoding it can be identified, it would be clearly advantageous to file an application as an invention whose main feature is a "substance" such as a gene or protein. In other words, for inventions of the type (2-1) above among inventions related to microorganisms, it is considered preferable to make genes and proteins the main subject of the invention as much as possible, and make the microorganism itself a dependent invention.

Classification and patentability of microorganisms

On the other hand, in the invention of type (2-1) above, if microorganisms are used as the main subject of the invention, the following problems are considered to occur.
In the first place, in the type of invention (2-1), for example, if the above-mentioned "system using bacterium C" is the main invention, there is a high possibility that similar microorganisms other than bacterium C will not be effectively covered by rights.

Furthermore, even if it is sufficient to secure the rights to just Bacteria C, making a "living form" such as a microorganism the most distinctive feature is different from making a "substance" such as a gene or protein the main feature. It may not be a fully valid right.
This is largely related to problems in classification and identification of microorganisms.
Compared to the classical classification method described in Section II above, the molecular biological classification method described in Section III may seem superior, but in reality, molecular biological classification methods are absolutely They are not standard indicators, and each has its own problems.

Specifically, for example, when looking at the technology that uses the 16S rRNA base sequence as a classification index, there is not necessarily a correlation between the homology of the 16S rRNA base sequence and the similarity of scientific properties. do not have. That is, even if a high degree of homology is observed in the above base sequences, there may be significant differences in appearance (shape), physiological and biochemical properties, and scientific properties such as Gram staining. Furthermore, it is said that there is no correlation between the base sequence of the 16S rRNA and DNA-DNA homology.

Therefore, even the classification index of 16S rRNA base sequence homology, which is currently the mainstream, cannot be a definitive index for identifying the genus or species. The same applies to the identification of "strains" which are lower than species.

As described above, it is extremely difficult to judge whether a newly discovered microorganism is truly new. Therefore, for example, in the example of the above-mentioned "system using bacterium C", enzyme c produced by bacterium C is not specifically specified, belongs to the same species as bacterium C, and has the ability to decompose waste. If the bacterium E having the following was previously known, the novelty of the bacterium C would be compromised. In this case, it is necessary to prove how novel and useful Bacteria C is as a "strain."

Furthermore, proteins such as enzymes are not always produced under the same conditions, and are largely dependent on the environment in which microorganisms grow. Therefore, even if the physiological and biochemical properties appear to be different, it cannot be concluded that this proves that they are different "strains." To take a more extreme example, molecular biological classification methods could conversely be used for materials that deny novelty.

For example, suppose that an application is filed for the invention of the above-mentioned "system using bacterium C" after specifying the base sequence of 16S rRNA of bacterium C. At this time, it is assumed that it is only known that bacteria C decomposes waste, and that it is not known that this decomposition is caused by enzyme c. Here, it is assumed that at the time of filing of this invention, a bacterium F, which is the same species but is apparently a "strain" that is clearly different from its physiological and biochemical properties, was known. After filing the application, research on this bacterium F revealed that the base sequence of the 16S rRNA of this bacterium F is homologous to that of bacterium C, and that it can produce enzyme c if induced under specific conditions. The patentability of the invention of the above-mentioned "system using bacterium C" will be greatly shaken.

On the other hand, if we say ``a specific enzyme and gene produced by bacteria C'' and ``a system that utilizes it'' rather than ``a system that uses bacteria C,'' the most distinctive feature of the invention is the gene and gene. It becomes a “substance” called protein. Therefore, the risk of uncertain factors being involved is much lower than when microorganisms are the most significant feature.

For example, in the above example, if the amino acid sequence and base sequence of enzyme c were identified before research on bacterium F progressed, even if bacterium F and bacterium C were the same "strain," the patent would be granted. It is possible to avoid a major impact on sexuality.

Therefore, if the invention is related to microorganisms and falls into the type (2-1) above, it is best to avoid using microorganisms as the main feature as much as possible. Of course, from the viewpoint of satisfying the enablement requirement, it goes without saying that it is preferable to deposit the bacterium C even if the invention involves a "specific enzyme and gene produced by the bacterium C." It seems necessary to reconsider whether these characteristics are actually microorganisms themselves.