JPH04335894A - Production of insecticidal protein - Google Patents

Abstract

PURPOSE:To provide a process for stably producing a large amount of a specific insecticidal protein [CRY1A(b)] among insecticidal proteins having excellent insecticidal effect and produced by Bacillus thuringiensis. CONSTITUTION:An insecticidal protein is produced by culturing Bacillus thuringiensis containing essentially exclusively insecticidal protein gene cry1A(b) at <25 deg.C. An insecticidal protein having high insecticidal effect against larva of lepidoptera such as diamondback moth and low toxicity to silkworm can be stably produced in high purity in a mass.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a method for producing an insecticidal protein produced by Bacillus thuringiensis, which exhibits an insecticidal effect on the larvae of lepidopteran insects (such as the diamondback moth). This relates to a method for stably and efficiently producing insecticidal proteins, which are expression products, using bacterial cells that have only insecticidal protein genes as insecticidal protein genes, and is used in the agrochemical industry and agricultural field. be.

0002

[Prior Art] Various Bacillus thuringiensis (
The intracellular inclusion bodies formed during the sporulation stage of BT are called insecticidal proteins or crystal toxins, and are known to be toxic to the larvae of many insects and are widely used as insecticides. There is.

The insecticidal activity of insecticidal proteins differs depending on the subspecies of Bacillus thuringiensis that produces them, and when used as insecticides, they are used depending on their properties.

Bacillus thuringiensis subsp. kurstakii HD-1, which produces insecticidal proteins that have been widely used commercially as BT insecticides, has been shown to produce multiple insecticidal proteins with different genes through previous research. It has been shown that they are produced simultaneously during the culture process (Aronson A.
I. etal. :Microbiological
Review 50, 1, 1986). It is also known that these insecticidal spectra differ from each other (
HofteH. et al. :Microbiolog
ical Review 53, 242, 1989
).

Among the multiple insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD-1, Hofte H. et al.: Microb
iological Review 53,242
An insecticidal protein expressed from a gene encoded on a plasmid of about 44Md, named cryIA(b) type by Kondo et al.
tal. : Agric. Biol. Chem. ,5
1, 455, 1987), whose DNA base sequence was determined by Cry-1-2, which has high insecticidal activity against lepidopteran larvae such as the diamondback moth, but has low toxicity against silkworms. It has been known.

[0006] Therefore, instead of producing multiple insecticidal proteins in Bacillus thuringiensis cells as in the past, if only the cryIA(b) type insecticidal protein is selectively produced, it is possible to kill specific insect pests. Various attempts have been made to produce BT insecticides that are highly effective against silkworms and have little effect on silkworms, which are agriculturally useful.

[0007] That is, a strain that produces only the cryIA(b) type is treated to delete the plasmid from the wild-type parent strain (curing), or by conjugating (mating) a mutant strain lacking the plasmid with the parent strain. By performing a transfer treatment and further utilizing a spore-free mutant strain, it is possible to produce an insecticidal protein with less foreign substances such as spores.

[0008] A method for producing insecticidal proteins using Bacillus thuringiensis strains as described above includes Dalmage (
Dulmage H. T. :J. Invertebr
．． Pathol. 22, 273, 1971) is applied, and generally, when cultured for 2 to 5 days at 25 to 37°C in a medium containing sufficient carbon sources, nitrogen sources, minerals, vitamins, etc. has been done.

[0009]

[Problems to be Solved by the Invention] The above-mentioned range of suitability for culture conditions is a result of the bacterial growth ability of Bacillus thuringiensis, but the present inventors have determined that c
In addition to ryIA(b), cryIA named by Hofte et al.
(a), Bacillus thuringiensis subsp. kurstaki HD-1, which simultaneously produces cryIA (c) and cryIIA type insecticidal proteins, and cryIA (b), which is highly effective against diamondback moths and has low toxicity to silkworms. We found that when a Bacillus thuringiensis mutant strain that produces only the type of insecticidal protein was cultured under the same conditions, the amount of insecticidal protein produced by the mutant strain was extremely low.

[0010] The present inventors used a Bacillus thuringiensis mutant strain that produces only the insecticidal protein of the cryIA(b) type, which is highly effective against diamondback moths and has low toxicity against silkworms, and more efficiently. They also conducted various studies to find a method for producing insecticidal proteins in large quantities.

[0011]

[Means for Solving the Problems] The present inventors conducted a detailed study on culture conditions and found that under culture conditions at a temperature of 25 to 37°C, which is widely used as a method for producing cryIA with Bacillus thuringiensis strains, cryIA (b)
In Bacillus thuringiensis mutant strains that only produce insecticidal proteins of the type B. thuringiensis, the amount of insecticidal proteins produced is small, and rather at temperatures below 25°C, preferably between 15 and 23°C.
By culturing at a temperature of 10°C, a pure insecticidal protein consisting essentially of the cryIA(b) type can be produced to an extent comparable to the production per culture of the wild-type strain, which simultaneously produces multiple insecticidal proteins. The present invention was completed by discovering that it is possible to produce a synthetic protein.

Specifically, the present invention relates to a method for producing an insecticidal protein, which comprises culturing Bacillus thuringiensis carrying a cryIA(b) type gene as an insecticidal protein gene at a temperature below 25°C. , Bacillus thuringiensis carrying an insecticidal protein gene carried on an endogenous plasmid with a molecular weight of about 44Md of Bacillus thuringiensis subsp. Regarding the production method, Bacillus thuringiensis non-spore mutant strain BTK-MA312 (FERM P
10739) at a temperature below 25°C.

[0013] By culturing a Bacillus thuringiensis mutant strain that essentially possesses only the gene for the insecticidal protein of the cryIA(b) type at a temperature lower than the temperature conventionally considered to be the appropriate culture temperature, the insecticidal protein can be improved. B makes it possible to stably produce active ingredients in large amounts of protein, is highly effective against specific pests, and has little impact on silkworms.
This makes it possible to provide T-insecticides at low cost.

[0014]

[Action] A Bacillus thuringiensis mutant strain carrying only the cryIA(b) type insecticidal protein gene is
One of the reasons why the insecticidal protein can be stably produced in large quantities to produce its active ingredients by culturing at lower temperature conditions than conventional methods is that the insecticidal protein is structurally extremely unstable and cannot be cultured using normal culture. At a temperature of 25 to 37°C, the produced insecticidal protein is easily degraded by endogenous proteases, etc., but other details are unknown.

[0015]

[Example] The following is an example illustrating the procedure for carrying out the culture method of the present invention, and should not be considered as limiting. In other words, c
ryIA(b) type or as a plasmid carrying an insecticidal protein gene of about 44 Md.
It is obvious that if the strain is a Bacillus thuringiensis subsp. kurstaki derivative strain carrying an endogenous plasmid, the same results as the strain tested in the following Examples will be obtained.

〇BTK-MA312 (FERM P-
10739) BTK-MA312 is a mutant strain obtained by mutation treatment, plasmid removal treatment, and conjugation treatment of Bacillus thuringiensis subsp. It has been improved to produce only the cryIA(b) type encoded on the endogenous plasmid of are the same. Incidentally, BTK-MA312 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, located in Tsukuba City, Ibaraki Prefecture, on May 24, 1989, as Microbiological Research Institute No. 10739. The strain is inoculated and cultured, pre-cultured in a natural medium rich in nitrogen sources, carbon sources, minerals and vitamins. The culture temperature was 20°C. Furthermore, since the production amount of insecticidal proteins and bacterial cells largely depends on aeration and agitation conditions, cultivation should be carried out under sufficient aerobic conditions. The number of culture days is 3 to 5 days. After the cultivation is completed, a conventional centrifugation method, filtration method, etc. can be used to separate and collect the insecticidal protein-containing portion from the culture solution. In addition, the culture solution can be concentrated,
If you want to dry it into a powder, you can use the usual concentration method or drying method (
For example, a spray drying method may be used. These steps are
It can be easily scaled up to large fermentation equipment using techniques well known in the art. In the above-mentioned culture, whose main feature is that the culture temperature is 20° C., although the growth curve was different, the maximum amount of bacterial cells was the same as when cultured at a normal temperature. Furthermore, the yield of cryIA(b) type insecticidal protein per culture solution was stably produced and accumulated over both 3 and 5 days of culture. However, as a control, when culturing was carried out under the same medium conditions and at the usual culture temperature of 30°C, the insecticidal protein decreased from the second day onwards (see Figure 1). This result is based on cryIA(b)
This shows that the culture temperature conditions have a significant effect on the stable production culture method of this type of insecticidal protein.

[0017]

[Effects of the Invention] According to the present invention, it is possible to stably produce the active ingredients of a BT insecticide that is highly effective against specific pests and has little effect on silkworms, and at a relatively low cost. This will greatly contribute to the agrochemical industry and agricultural field.

[Brief explanation of the drawing]

[Fig. 1] Fig. 1 is a diagram showing the influence of culture temperature on the production amount of insecticidal protein.
This shows the results of 10% polyacrylamide gel electrophoresis of the culture solution after culturing at 30° C. or 30° C. for 3 days and 5 days in the presence of sodium dodecyl sulfate.

[Explanation of symbols]

The arrows in the figure indicate the location of cryIA(b) type insecticidal protein, and a indicates the location of 5μ of the culture solution at 30°C for 3 days.
l, b is 20°C, 5 μl of 3-day culture solution, c is 30°C,
5 μl of culture solution for 5 days, and d shows the results with 5 μl of culture solution for 5 days at 20°C.

Claims (3)

[Claims] [Claim 1] Cry as an insecticidal protein gene
A method for producing an insecticidal protein, which comprises culturing Bacillus thuringiensis carrying an IA(b) type gene at a temperature below 25°C. 2. The method for producing an insecticidal protein according to claim 1, wherein the insecticidal protein gene is carried on an endogenous plasmid of Bacillus thuringiensis subspecies kurstakii having a molecular weight of about 44 Md. Claim 3: Bacillus thuringiensis non-spore mutant strain BTK-MA312 (FERM P-10
739) at a temperature below 25°C.