JPWO2020100888A1 - Microorganisms belonging to the genus Cupriavidus - Google Patents

Abstract

According to the present invention, it is possible to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it. A microorganism belonging to the genus Cupriavidus into which a gene encoding α-amylase is linked to a secretory signal derived from a microorganism belonging to the genus Cupriavidus.

Description

The present invention relates to microorganisms belonging to the genus Cupriavidus.

Significance and importance of microbial production and microbial material production (eg, fermentation production, bioconversion, etc.) against the backdrop of growing awareness of environmental issues, food issues, health and safety; and increasing nature and nature orientation. The sex is getting higher and higher.

In the production of microorganisms and the production of substances by microorganisms, a carbon source suitably assimilated by microorganisms (for example, a carbon source for culturing and / or fermentation, etc.) is required. As a representative of the carbon source, the use of sugars (for example, starch), fats and oils (for example, vegetable fats and oils), fatty acids (for example, plant-derived fatty acids) and the like has been increasingly desired. Starch, which is a sugar raw material, is an inexpensive fermented raw material that can be produced from potatoes, corn, rice, cassava, and the like, and may be considered as one of the candidates for a suitable carbon source.

It is easy to use a carbon source having a structure that is easily catabolized and assimilated by microorganisms into cells, such as a low molecular weight sugar raw material such as glucose or sucrose, as a raw material. However, starch is a polymer to which a large number of glucoses are bound, and in particular, raw starch has a micelle structure (crystal structure) in which chains of amylose and amylopectin are regularly assembled by hydrogen bonds, and is decomposed by an enzyme. There is a problem that it is difficult to receive and cannot be assimilated by microorganisms as it is.

In order to decompose raw starch into low molecular weight sugar raw materials such as glucose, it is necessary to reduce the crystallinity by inputting energy such as heat and physical destruction, and usually heat gelatinization (solubilization, solubilization, etc.) Alternatively, it is also referred to as pregelatinization) (Patent Document 1). Then, after that, amylase, which is a degrading enzyme, is allowed to act to saccharify. However, such a method consumes energy in the gelatinization process. Therefore, it cannot be said that starch, especially raw starch, is effectively used industrially.

In order to reduce the energy consumption associated with the use of raw starch, it would be significant if the microorganisms responsible for substance production could secrete amylase capable of decomposing raw starch and directly produce substances using raw starch as a raw material.

Examples of hosts for substance production using microorganisms include microorganisms belonging to the genus Cupriavidus. In addition, examples of substance production using microorganisms belonging to the genus Cupriavidus include aliphatic polyesters such as polyhydroxyalkanoate (PHA), butanol, isopropanol and the like.

There is an example of producing acetone and butanol using Cupriavidus necator, which is a microorganism belonging to the genus Cupriavidus (Non-Patent Document 1). There is an example in which isopropanol was produced from fructose, which is a sugar raw material, using Cupriavidus necator (Non-Patent Document 2). In addition, an example in which polyhydroxyalkanoate (PHA) is produced from sucrose using Cupriavidus necator has been reported (Non-Patent Document 3). However, none of them use starch as a raw material.

Since Cupriavidus necator does not have a gene encoding α-amylase, it cannot express starch-degrading enzyme and starch cannot be suitably used. Therefore, an example of producing PHA using starch derived from saccharified potato has been reported. (Non-patent document 4).

Japanese Patent No. 4767128

Chakravarty J., et al., Appl. Microbiol. Biotechnol., 102 (12): 5021-5031 (2018) Marc J., et al., Metab. Eng., 42: 74-84 (2017) Arikawa H., et al, Appl. Microbiol, Biotechnol, 101 (20): 7497-7507 (2017) Haas R., et al., Biosci. Biotechnol. Biochem., 72 (1): 253-256 (2008) Matsui Y., et al., J. Appl. Glycosci., 54: 217-222 (2007) Shigechi H., et al., AEM., 70 (8): 5037-5040 (2004) Huong KH., et al., Int Biol Macromol., 116: 217-223 (2018) Syafig IM., Et al., Enzyme Microb Technol., 98: 1-8 (2017)

The present inventors first introduced α-amylase (Non-Patent Document 5) derived from Streptococcus bovis, which is reported to be capable of decomposing unsolubilized starch (also referred to as raw starch), into Cupriavidus necator H16 strain, and introduced the same. Starch decomposition activity was confirmed, but no decomposition activity was shown. As described above, it is not easy to suitably use starch, particularly raw starch, as a carbon source.

For example, in Non-Patent Document 6, a secretory signal sequence derived from Rhizopus is used to express α-amylase derived from Streptococcus bovis on the surface layer (a type of secretory expression) in yeast Saccharomyces cerevisiae. In order to secrete and express a protein, it is essential to introduce a gene sequence in which the secretory signal and the gene encoding the protein are linked. However, how the secretory protein and the secretory signal can be combined to produce starch in the target microorganism. It is difficult to find out if it exhibits degradative activity. It is said that secreting an enzyme extracellularly is more difficult than simply expressing a heterologous gene inside the cell. Further research is needed to industrially enable efficient substance production by microorganisms belonging to the genus Cupriavidus, which can produce aliphatic polyesters such as polyhydroxy alkanoate (PHA) using starch as a raw material. be.

An object of the present invention is to provide a microorganism belonging to the genus Cupriavidus, which can decompose starch and effectively assimilate it.

The present invention relates to a microorganism belonging to the genus Cupriavidus into which a gene encoding α-amylase is linked to a secretory signal derived from a microorganism belonging to the genus Cupriavidus.

In the microorganism, the α-amylase may be an α-amylase derived from a microorganism belonging to the genus Streptococcus.

Among the microorganisms, the microorganism belonging to the genus Cupriavidus may synthesize polyhydroxyalkanoates.

Among the microorganisms, the microorganism belonging to the genus Cupriavidus may be Cupriavidus necator.

The polyhydroxy alkanoate may be poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).

According to the present invention, it is possible to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it.

The microorganism of the present disclosure is a microorganism belonging to the genus Cupriavidus into which a gene encoding α-amylase is linked to a secretory signal derived from a microorganism belonging to the genus Cupriavidus.

In the microorganisms of the present disclosure, the present inventors have found a gene encoding a protein secreted extracellularly (secretory protein) from among genes possessed by a microorganism belonging to the genus Cupriavidus, and a signal present in the gene. A gene in which a similar sequence (secretory signal) and an α-amylase gene are linked is introduced into a microorganism belonging to the genus Cupriavidus. According to the microorganism, α-amylase, which is a starch-degrading enzyme having starch-degrading activity, can be secreted extracellularly, so that raw starch can be assimilated without pregelatinization and can be effectively used. The substance production using the microorganisms of the present disclosure is also suitable for industrial substance production.

The microorganism of the present disclosure is not limited as long as it is a microorganism belonging to the genus Cupriavidus, but it is preferable to synthesize polyhydroxyalkanoate, that is, to have a PHA synthesizing ability. Among the microorganisms belonging to the genus Cupriavidus, Cupriavidus necator is particularly preferable because it has a high PHA synthesizing ability.

Microorganisms belonging to the genus Cupriavidus having PHA synthesizing ability include not only wild strains that originally have a PHA synthase gene, but also mutant strains obtained by artificially mutating such wild strains and PHA synthesis. It may be a recombinant strain in which a foreign PHA synthase gene is introduced into a microorganism that does not originally have an enzyme gene by a genetic engineering technique.

When the microorganism belonging to the genus Cupriavidus having PHA synthesizing ability is a recombinant strain into which a foreign PHA synthase gene has been introduced by a genetic engineering method, the foreign PHA synthase gene is, for example, the Cupriavidus necator H16 strain. The PHA synthase gene encoding the amino acid sequence shown in SEQ ID NO: 2 possessed, or the PHA encoding a polypeptide having 85% or more sequence identity to the amino acid sequence and having PHA synthesizing activity. Synthetic enzyme gene; and PHA synthase gene encoding the amino acid sequence set forth in SEQ ID NO: 3 possessed by Aeromonas caviae, or PHA synthase gene having 85% or more sequence identity with respect to the amino acid sequence and PHA synthetic activity. Examples thereof include a PHA synthase gene encoding a polypeptide having the above, but the present invention is not limited to these, and other PHA synthase genes can also be preferably used. The sequence identity is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more. Among these, a PHA synthase gene having a synthetic activity for poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (sometimes referred to as PHBH) as PHA is preferable. PHBH may be synthesized by a transformant in which a PHA synthase gene derived from Aeromonas caviae has been introduced into Cupriavidus necator.

The gene introduced into the microorganism of the present disclosure encodes α-amylase linked with a secretory signal derived from a microorganism belonging to the genus Cupriavidus.

The secretory signal is a DNA sequence encoding a secretory signal peptide. The secretory signal peptide is a signal peptide required to secrete a protein synthesized intracellularly into the intracellular membrane or extracellularly.

The secretory signal is not particularly limited as long as it is a secretory signal derived from a microorganism belonging to the genus Cupriavidus. For example, secretory signals derived from microorganisms belonging to the genus Cupriavidus such as Cupriavidus necator, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus respiraculi, Cupriavidus taiwanensis and Cupriavidus metallidurans can be used.

The α-amylase is not restricted as long as it is an α-amylase having starch-degrading activity, and the above-mentioned gene introduced into the microorganism of the present disclosure is restricted as long as it encodes such an α-amylase. is not it.

Genes encoding α-amylase include, for example, genes encoding α-amylase derived from microorganisms of the genera Bacillus, Streptococcus, Panibacillus, and Rhyzopus. Among these, a gene encoding α-amylase derived from a microorganism of the genus Streptococcus is preferable. This is because the microorganisms of the present disclosure can suitably decompose raw starch.

Examples of genes encoding α-amylase derived from microorganisms belonging to the genus Bacillus include genes encoding α-amylase derived from microorganisms such as Bacillus stearothermophilus and Bacillus amyloliquefaciens.

Examples of the gene encoding α-amylase derived from a microorganism belonging to the genus Streptococcus include a gene encoding α-amylase derived from a microorganism such as Streptococcus bovis.

Examples of the gene encoding α-amylase derived from a microorganism belonging to the genus Paenibacillus include a gene encoding α-amylase derived from a microorganism such as Paenibacillus sp.

The "linkage" between a secretory signal from a microorganism belonging to the genus Cupriavidus and a gene encoding α-amylase may be a direct binding between the secretory signal and the portion encoding α-amylase, and the secretory signal peptide and α-amylase. It may be an indirect bond containing one or more bases with the encoding moiety.

As a method for introducing a gene encoding α-amylase linked with a secretory signal derived from a microorganism belonging to the genus Cupriavidus, a method for introducing into a microorganism belonging to the genus Cupriavidus using a plasmid vector, a method for introducing into genomic DNA, or the like is used. be able to. The introduction into a microorganism belonging to the genus Cupriavidus using a plasmid vector can be carried out by any commonly used method, for example, an electric introduction method, a calcium method or the like. Introduction to genomic DNA can be performed using any of the commonly used methods, such as homologous recombination, transposon-based methods, and phage-based methods.

The plasmid vector may be prepared by ligating a DNA fragment having a base sequence encoding α-amylase linked with a secretory signal derived from a microorganism belonging to the genus Cupriavidus to a plasmid vector such as pCUP2.

The culture conditions of the microorganism belonging to the genus Cupriavidus of the present disclosure may be any conditions as long as the microorganism can grow. For example, the temperature condition is 20 ° C. or higher and 50 ° C. or lower, and the culture time is 10 hours or more and 100 hours. It may be as follows.

In addition, the substance accumulated in the microorganism belonging to the genus Cupriavidus can be recovered by a known method. When the accumulated substance is PHA, it can be recovered by, for example, the following method. After culturing the microorganisms belonging to the genus Cupriavidus, the cells are separated from the culture solution by a centrifuge or the like, and the cells are washed with methanol or the like and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. The bacterial cell component is removed from the solution of the organic solvent containing PHA by filtration or the like, and a poor solvent such as hexane is added to the filtrate to precipitate PHA. Further, the supernatant liquid may be removed by filtration or centrifugation and dried to recover PHA.

According to the microorganism of the present disclosure, α-amylase having starch-degrading activity can be secreted extracellularly, so that starch can be decomposed and effectively assimilated.

Further, if the microorganism of the present disclosure belongs to the genus Cupriavidus that synthesizes PHA, PHA can be synthesized directly from starch, so that it is expected that PHA production can be performed at low energy and low cost.

Currently, non-petroleum-derived plastics are attracting attention due to growing awareness of the environment. Among non-petroleum plastics, especially PHA is a thermoplastic polyester produced and accumulated in cells by microorganisms, which is biodegradable and is incorporated into the natural carbon cycle process to the ecosystem. It is expected that the adverse effects of PHA will be small, and the practical application of PHA is eagerly desired. Therefore, PHA is one of the good examples of substances produced using the microorganisms of the present disclosure.

The microorganism belonging to the genus Cupriavidus of the present disclosure can be used for the production of alcohols such as ethanol, butanol and propanol; acids such as lactic acid, acetic acid, amino acids and nucleic acids, lipids and fats and oils in addition to PHA. .. For example, production examples of PHA using Cupriavidus malaysiensis (Non-Patent Document 7) and Cupriavidus sp. USMAA1020 (Non-Patent Document 8) have been reported.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Overall genetic manipulation can be performed according to the description of Molecular Cloning (Cold Spring Harbor Laboratory Press (1989 or 2001)). In addition, the enzyme used for genetic manipulation, the cloning host, etc. can be purchased from a supplier on the market and used according to the description thereof. The enzyme is not particularly limited as long as it can be used for genetic manipulation.

The KNK005ΔphaZ1,2,6 / nagEG793C, dR strain (hereinafter referred to as “KNK005dZG strain”) used in the following production examples, examples, and comparative examples is derived from Aeromonas caviae on the chromosome of the Cupriavidus necator H16 strain. The PHA synthase gene (the gene encoding the PHA synthase having the amino acid sequence shown in SEQ ID NO: 3) was introduced, and the phAZ1,2,6 gene, which is a PHA degrading enzyme gene on the chromosome, was deleted, and it was related to sugar uptake. Among the nag operon genes, G, which is the 793th base of the nagE structure gene, is replaced with C, and the nagR gene is a transformed microorganism in which the start codon to the end codon are deleted. This transformed microorganism can be produced according to the method described in International Publication No. 2017/104722.

(Production Example 1) Preparation of pCUP2-trc-amyA1535 / KNK005dZG strain First, a plasmid vector was prepared. The manufacturing method is as follows. A base encoding the amino acid sequence (SEQ ID NO: 1) of the trc promoter and the secretory α-amylase (hereinafter referred to as “amyA1535”) derived from the Streptococcus bovis 148 strain (NRIC 1535) by PCR using synthetic oligo DNA. A DNA fragment having a sequence (SEQ ID NO: 4) was obtained. The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI. This DNA fragment was ligated with a plasmid vector pCUP2 described in WO 2007/049716 cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-amyA1535. The secretory α-amylase is an α-amylase having a secretory signal peptide.

Next, the plasmid vector pCUP2-trc-amyA1535 was introduced into the KNK005dZG strain to obtain a transformant pCUP2-trc-amyA1535 / KNK005dZG strain.

The plasmid vector was introduced into cells by electrical introduction as follows. A gene pulsar manufactured by Bio-Rad was used as the gene transfer device, and a gap 0.2 cm also manufactured by Bio-rad was used as the cuvette. 400 μl of competent cells and 20 μl of expression vector were injected into the cuvette and set in a pulse device, and an electric pulse was applied under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance value of 800 Ω. After the pulse, the bacterial solution in the cuvette was cultured with shaking in Nutrient Broth medium (manufactured by DIFCO) at 30 ° C. for 3 hours, and cultured on a selection plate (Nutrient Agar medium (manufactured by DIFCO), kanamycin 100 mg / L) at 30 ° C. 2 After culturing for one day, the grown transformant pCUP2-trc-amyA1535 / KNK005dZG strain was obtained.

(Production Example 2) Preparation of pCUP2-trc-SPflicamyA1535 / KNK005dZG strain First, a plasmid vector was prepared. The production was carried out as follows. A DNA fragment having a base sequence encoding amyA1535 (SPflicamyA1535) in which the trc promoter and the secretory signal peptide were replaced with the secretory signal peptide derived from the cupriavidus necator H16 strain flic gene by PCR using synthetic oligo DNA (SEQ ID NO: 5). Got The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI. This DNA fragment was ligated with a plasmid vector pCUP2 described in WO 2007/049716 cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-SPflicamyA1535.

Next, the plasmid vector pCUP2-trc-SPflicamyA1535 was introduced into the KNK005dZG strain in the same manner as in Production Example 1 to obtain a transformant pCUP2-trc-SPflicamyA1535 / KNK005dZG strain.

(Production Example 3) Preparation of pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain First, a plasmid vector was prepared. The production was carried out as follows. A DNA fragment having a base sequence encoding amyA1535 (SPplcN4amyA1535) in which the trc promoter and the secretory signal peptide were replaced with the secretory signal peptide derived from the cupriavidus necator H16 strain plcN4 gene by PCR using synthetic oligo DNA (SEQ ID NO: 6). Got The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI. This DNA fragment was ligated with a plasmid vector pCUP2 described in WO 2007/049716 cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-SPplcN4amyA1535.

Next, the plasmid vector pCUP2-trc-SPplcN4amyA1535 was introduced into the KNK005dZG strain in the same manner as in Production Example 1 to obtain a transformant pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain.

(Comparative Example 1) Starch resolution test using KNK005dZG strain The composition of the seed medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast-extract, 0.9 w / v%. It was _{Na 2 HPO 4 · 12H 2 O} , 0.15w / v% KH 2 PO 4.

The composition of the starch-containing plate medium was prepared by adding 1 w / v% cornstarch to a nutrient agar medium (Difco).

A glycerol stock (50 μL) of the KNK005dZG strain was inoculated into a seed medium (5 mL) and cultured with shaking at a culture temperature of 30 ° C. for 24 hours, and the obtained culture solution was used as a seed mother.

The starch resolution test was performed as follows. 10 μl of the seed culture medium adjusted to OD600 = 2.0 using sterile water was spotted on a starch-containing plate medium (1 w / v% cornstarch on nutrient agar medium (Difco)) and grown at 30 ° C. for 3 days. .. It was determined whether or not a clear zone (also referred to as halo) due to the decomposition of starch was observed around the grown colony. Further, the larger the diameter of the clear zone, the higher and better the starch resolution.

As a result, in this comparative example, no clear zone was observed, and the KNK005dZG strain did not show starch resolution. The results are shown in Table 1.

(Comparative Example 2) Starch resolution test using pCUP2-trc-amyA1535 / KNK005dZG strain The composition of the seed medium and the starch-containing plate medium was the same as that described in Comparative Example 1. The pCUP2-trc-amyA1535 / KNK005dZG strain prepared in Production Example 1 was cultured and subjected to a starch resolution test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed medium and the starch-containing plate medium so as to have a final concentration of 100 μg / ml.

In this comparative example, no clear zone was observed, and the pCUP2-trc-amyA1535 / KNK005dZG strain showed no starch resolution. The results are shown in Table 1.

(Example 1) Starch resolution test using pCUP2-trc-SPflicamyA1535 / KNK005dZG strain The compositions of the seed medium and the starch-containing plate medium were the same as those described in Comparative Example 1. The pCUP2-trc-SPflicamyA1535 / KNK005dZG strain prepared in Production Example 2 was cultured and subjected to a starch resolution test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed medium and the starch-containing plate medium so as to have a final concentration of 100 μg / ml.

In this example, a clear zone was observed and the pCUP2-trc-SPflicamyA1535 / KNK005dZG strain showed starch resolution. The diameter of the clear zone was measured. The results are shown in Table 1.

(Example 2) Starch resolution test using pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain The composition of the seed medium and the starch-containing plate medium was the same as that described in Comparative Example 1. The pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain prepared in Production Example 3 was cultured and subjected to a starch resolution test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed medium and the starch-containing plate medium so as to have a final concentration of 100 μg / ml.

In this example, a clear zone was observed and the pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain showed starch resolution. The diameter of the clear zone was measured. The results are shown in Table 1.

It was shown that the microorganism of the example into which the gene encoding α-amylase linked with the secretory signal derived from the microorganism belonging to the genus Cupriavidus was able to decompose the starch and thus can effectively assimilate the starch.

Claims (5)

A microorganism belonging to the genus Cupriavidus into which a gene encoding α-amylase is linked to a secretory signal derived from a microorganism belonging to the genus Cupriavidus. The microorganism according to claim 1, wherein the α-amylase is an α-amylase derived from a microorganism belonging to the genus Streptococcus. The microorganism according to claim 1 or 2, wherein the microorganism belonging to the genus Cupriavidus synthesizes polyhydroxyalkanoate. The microorganism according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus necator. The microorganism according to claim 3 or 4, wherein the polyhydroxy alkanoate is poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).