WO2025018332A1 - 5-aminolevulinic acid-producing bacterium, method for producing same, and method for producing 5-aminolevulinic acid using same - Google Patents

Abstract

The present invention addresses the problem of: producing a 5-aminolevulinic acid high-producing strain, which is a strain suitable with respect to safety in the industrial production of 5-aminolevulinic acid, by utilizing a technology that is a recombinant DNA technology but does not leave a foreign gene in a finally produced strain, particularly a self-cloning technology; and producing 5-aminolevulinic acid using the produced strain.　The problem is solved by producing a strain of a photosynthetic bacterium, and culturing the produced strain, the strain belonging to the class α-Proteobacteria and having, upstream of a gene (hemT) encoding HemT that is a 5-aminolevulinic acid synthesis enzyme, an expression regulation region including an rrnB promoter, wherein the expression regulation region is originated from a photosynthetic bacterium that is the same type of the strain and is capable of inducing the expression of hemT, and the induction causes the increase in 5-aminolevulinic acid productivity compared with a wild-type strain thereof.

Description

5-aminolevulinic acid producing bacteria, method for producing same and method for producing 5-aminolevulinic acid using same

The present invention relates to the creation of a self-cloning strain of α-proteobacteria using a self-cloning technique, and a method for producing 5-aminolevulinic acid using the created strain.

5-aminolevulinic acid (ALA) is a compound that is widely present in the biosphere as a metabolic intermediate in the pigment biosynthesis pathway that synthesizes tetrapyrrole compounds (vitamin B12, heme, chlorophyll, etc.) and plays an important role in living organisms. 5-aminolevulinic acid is also a natural compound that exhibits excellent effects as a herbicide, insecticide, plant growth regulator, and plant photosynthesis enhancer (Patent Documents 1 and 2). Furthermore, 5-aminolevulinic acid is used as a raw material for pharmaceuticals, supplements, cosmetics, feed, fertilizer, and more, and supports people's lives in various ways.

It is known that 5-aminolevulinic acid is biosynthesized in vivo from glycine and succinyl CoA by 5-aminolevulinic acid synthase, or from glutamic acid via glutamyl tRNA (Patent Document 3).

The usefulness of 5-aminolevulinic acid is widely known, but one problem with its practical use is the high production costs. Chemical synthesis methods have been considered for some time, but no fully satisfactory method has yet been developed.

Meanwhile, methods for producing 5-aminolevulinic acid using microorganisms have also been investigated. For example, a method using the genera Propionibacterium, Methanobacterium, or Methanosarcina has been proposed (Patent Document 4). However, the production of 5-aminolevulinic acid by fermentation was insufficient in terms of production volume, and was not industrially satisfactory.

　A method using photosynthetic bacteria of the genus Rhodobacter is also known, and it has been reported that this method produces a larger amount of 5-aminolevulinic acid than the method using the above-mentioned microorganisms (Patent Document 5, etc.). Furthermore, a method has been carried out in which bacteria of the genus Rhodobacter are mutagenized, a strain with high 5-aminolevulinic acid productivity is selected, and the selected strain is subjected to repeated mutagen treatment and selection to obtain a mutant strain with higher 5-aminolevulinic acid production, which is then used (Non-Patent Documents 1 and 2).

As another approach to enabling microorganisms to produce high levels of 5-aminolevulinic acid, microbial strains have been selected or created using recombinant gene technology. For example, a 5-aminolevulinic acid synthase gene derived from photosynthetic bacteria is introduced into Escherichia coli to create a transformant (Non-Patent Document 3, Patent Document 6), and a 5-aminolevulinic acid synthase gene derived from photosynthetic bacteria is introduced into photosynthetic bacteria to create a transformant (Patent Document 7), etc. are known.

In terms of production costs and productivity, in the past, the selection or creation of strains with high 5-aminolevulinic acid production has been carried out mainly by (1) drug-induced mutation or (2) recombinant gene technology in order to industrially produce 5-aminolevulinic acid. However, in the case of (1), it is necessary to repeat the introduction of mutations and selection multiple times before obtaining the desired highly productive strain, and further, careful analysis of the properties of the selected strain and the mutated genes is required to ensure safety. In addition, in the case of (2), it is considered necessary to comprehensively analyze the properties of the strains created to ensure safety, because in many cases, the transformants contain foreign genes. Therefore, in either case, it has taken a considerable amount of time and effort to achieve industrial production, and it has often been accompanied by difficulties.

Japanese Unexamined Patent Publication No. 61-502814 Japanese Unexamined Patent Publication No. 2-138201 JP 2013-208074 A Japanese Patent Application Publication No. 5-184376 Japanese Patent Application Publication No. 5-184376 JP 2005-333907 A Japanese Patent Application Publication No. 9-173071

Nishikawa, S., et al. (1999) Rhodobacter sphaeroides mutants which accumulate 5-aminolevulinic acid under aerobic and dark conditions., J. Biosci. Bioeng., vol. 87, pp. 798-804. Kamiyama et al. (2000) Production of 5-aminolevulinic acid by a mutant strain of a photosynthetic bacterium. -Monograph-, Journal of the Japan of Biological Technology, vol. 78, no. 2, pp. 48-55. Xie, L. et al., (2003) Appl. Microbiol. Biotechnol., vol. 63, pp. 267-273.

When using drug-induced mutant strains or transformants with foreign genes introduced for the industrial production of 5-aminolevulinic acid, as mentioned above, there is a problem that considerable effort and cost are required to ensure safety, and that difficulties are often involved. The present invention aims to avoid or alleviate this problem.

The inventors of this case therefore attempted to create a strain with high 5-aminolevulinic acid productivity that is suitable from the standpoint of safety for industrial production of 5-aminolevulinic acid by using a technique that, although it is a recombinant gene technique, does not leave foreign genes in the final strain produced, particularly a self-cloning technique. As a result, they unexpectedly discovered that the above problems could be solved by combining a specific expression control region (promoter, etc.) and the 5-aminolevulinic acid synthase gene (ALAS gene), both of which are derived from the same genome, and thus completed the present invention.

In some aspects, the present invention provides the following [1] to [20].

[1] A photosynthetic bacterial strain belonging to the class α-proteobacteria, which has an expression control region including a ribosomal RNA operon promoter (PrrnB) upstream of a gene (hemT) encoding 5-aminolevulinic acid synthase HemT, wherein the expression control region is derived from the same species of photosynthetic bacteria as the strain and induces expression of hemT, and the induction results in increased productivity of 5-aminolevulinic acid compared to a wild-type strain.
[2] The strain according to [1] above, into which an expression control region containing PrrnB has been inserted by homologous recombination technology and which is substantially free of foreign gene sequences derived from heterologous species.
[3] The strain according to [1] or [2] above, wherein the photosynthetic bacterium belonging to the class α-proteobacteria is a bacterium of the genus Rhodobacter, Rhodospirillum, Rhodopseudomonas, Chromatium, Ectothiorhodospira, Chlorobium, Prosthecochloris, Chloroflexus, Chloronema, or Helicobacterium.
[4] The strain according to any one of [1] to [3] above, wherein the photosynthetic bacterium belonging to the α-proteobacteria class is a bacterium of the genus Rhodobacter.
[5] The strain according to [4] above, wherein the species of the bacterium of the genus Rhodobacter is Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter sulfidophillus, Rhodobacter adriaticus, or Rhodobacter veldkampii.
[6] The strain according to any one of [1] to [5] above, wherein hemT contains any one of the following base sequences:
(i) the base sequence of SEQ ID NO: 1;
(ii) a base sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2;
(iii) a base sequence which has a sequence identity of 90% or more with the base sequence of (i) or (ii) above and encodes a protein having 5-aminolevulinic acid synthase activity, and (iv) a base sequence in which 1 to 120 bases have been deleted, substituted, added or inserted relative to the base sequence of (i) or (ii) above and which encodes a protein having 5-aminolevulinic acid synthase activity.
[7] The strain according to any one of [1] to [5] above, wherein hemT is derived from the genome of any of Rhodobacter sphaeroides strains 2.4.1, H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, ATH 2.4.9 (ATCC 17029), IFO 12203, CH10, or KD131, and the photosynthetic bacterial species is Rhodobacter sphaeroides.
[8] The strain according to any one of [1] to [7] above, wherein PrrnB contains any one of the following base sequences:
(i) the base sequence of SEQ ID NO: 3;
(ii) the base sequence of SEQ ID NO: 4;
(iii) the base sequence of SEQ ID NO:5;
(iv) a functional fragment of PrrnB, comprising a base sequence having a 5'-end at any one of positions 1 to 158 of SEQ ID NO: 5 and a 3'-end at any one of positions 188 to 195 of SEQ ID NO: 5, and having an activity of inducing the expression of hemT;
(v) a base sequence having 90% or more sequence identity with the base sequence of SEQ ID NO: 4 or 5 and having hemT expression-inducing activity, and (vi) a base sequence in which 1 to 19 bases have been deleted, substituted, or added to the base sequence of SEQ ID NO: 4 or 5 and having hemT expression-inducing activity.
[9] The strain according to any one of [1] to [7] above, wherein PrrnB is derived from the genome of any of Rhodobacter sphaeroides strains 2.4.1, H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, ATH 2.4.3 (ATCC17025), ATH 2.4.9 (ATCC 17029), IFO 12203, CH10, KD131, or HJ, and the photosynthetic bacterial species is Rhodobacter sphaeroides.
[10] The strain according to any one of [1] to [9] above, wherein the inserted PrrnB is located upstream of the coding region of the hemT gene and within 10,000 bases upstream of the start codon of the hemT gene, preferably within 1,000 bases, more preferably within 100 bases, and even more preferably adjacent to the transcription start site of the hemT gene.

[11] A method for producing 5-aminolevulinic acid, comprising culturing any one of the strains according to [1] to [10] in a medium, producing and accumulating 5-aminolevulinic acid in the strain and the medium, and collecting 5-aminolevulinic acid from the culture.
[12] The method according to [11] above, wherein the medium is a liquid medium and the culture is performed by shaking culture.
[13] The method according to [10] or [11], wherein the culture is carried out under microaerobic or anaerobic conditions under oxygen restriction.
[14] The method according to [10] or [11], wherein the culture is carried out under aerobic conditions.

[15] A method for producing a photosynthetic bacterial strain belonging to the class α-proteobacteria, which has an expression control region including a ribosomal RNA operon promoter (PrrnB) upstream of a gene (hemT) encoding 5-aminolevulinic acid synthase HemT, wherein the expression control region is derived from the same species of photosynthetic bacteria as the strain and induces expression of hemT, such induction resulting in increased productivity of 5-aminolevulinic acid compared to a wild-type strain, the method for producing the strain comprising the step of functionally linking PrrnB to hemT.
[16] A method for producing any one of the strains according to [1] to [10] above, comprising a step of functionally binding PrrnB to hemT.

[17] A method for producing a photosynthetic bacterium strain with high productivity of 5-aminolevulinic acid, comprising the step of inserting, into a wild-type strain of the photosynthetic bacterium, an expression control region containing a ribosomal RNA operon promoter (PrrnB) derived from the same species upstream of a gene (hemT) encoding 5-aminolevulinic acid synthase HemT in the genome of the wild-type strain by homologous recombination technology, wherein the inserted PrrnB induces expression of hemT, and the induction results in increased productivity of 5-aminolevulinic acid compared to the wild-type strain, and the photosynthetic bacterium is a species belonging to the class α-proteobacteria.
[18] The method of [17] above, which uses a plasmid vector for homologous recombination, wherein the plasmid vector contains an expression control region containing PrrnB, and homologous regions consisting of sequences homologous to the upstream and downstream regions of the genome adjacent to the target region of homologous recombination are arranged on both sides of the expression control region containing PrrnB.
[19] The method according to [17] or [18], wherein the sequence of the homologous region is a base sequence of 20 bases or more, 100 bases or more, 500 bases or more, or 1,000 bases or more.
[20] The method according to any one of [17] to [19] above, wherein the region in the plasmid vector containing an expression control region including PrrnB, which is sandwiched between two homologous regions, is substantially free of foreign genes derived from a biological species that is heterologous to a species of photosynthetic bacteria belonging to the class α-proteobacteria.

The present invention provides a method for producing a photosynthetic bacterial strain by using a self-cloning technique, in which the expression of HemT, a 5-aminolevulinic acid synthase, is induced by a homologous or endogenous ribosomal RNA operon promoter (PrrnB), thereby increasing the productivity of 5-aminolevulinic acid without using a heterologous gene, and the produced strain is substantially free of heterologous nucleic acid sequences.
The strain of the present invention produced by the self-cloning technique is unlikely to cause unexpected side effects due to genetic recombination, and is therefore a boon for industries requiring high safety for products, such as the pharmaceutical, food and beverage industries, as well as for consumers. For example, the European Regulation on the Protection of Human Health and the Environment (Directorate-General XI, Commission of the European Communities, 1992) stipulates that self-cloning used for genetic recombination of non-pathogenic microorganisms should be exempt from regulatory oversight. Thus, the strain of the present invention has an advantage over drug-induced mutants and transformants into which foreign genes have been introduced in terms of regulatory treatment, and is advantageous in the industrial production of 5-aminolevulinic acid.

This is a schematic diagram of a procedure for preparing a promoter-inserted strain of photosynthetic bacteria by self-cloning technology. More specifically, FIG. 1 shows a procedure for inserting the rrnB promoter (PrrnB) or rsp_7571 promoter (Prsp_7571) derived from the photosynthetic bacterium Rhodobacter sphaeroides 2.4.1 strain upstream of the hemA gene in the genome of the same strain by homologous recombination. In the vector used for homologous recombination, Up (genomic sequence upstream of the hemA locus) and hemA are arranged on both sides of the promoter as homologous regions adjacent to PrrnB or Prsp_7571. Outside the homologous region, a gentamicin resistance gene (Gm ^r ) is arranged as a positive selection marker, and a levansucrase gene (sacB) is arranged as a negative selection marker. The finally prepared strain in which PrrnB is inserted upstream of the hemA gene is called the BA strain, and the strain in which Prsp_7571 is inserted is called the 7A strain. The genomic gene configuration around the ALAS gene (hemA or hemT) in the strain constructed by inserting a promoter by the self-cloning technique is shown. (A) shows the gene configuration of the region of chromosome 1 where the hemA gene is present in the genome of R. sphaeroides. (B) shows the genes of the region of chromosome 2 where the hemT gene is present in the genome of R. sphaeroides. In the figure, the white arrow indicates the coding sequence of the gene, and the black arrow indicates the transcription start point and transcription direction. The thick black line indicates the inserted promoter sequence (PrrnB or Prsp_7571). WT indicates a rifampicin-resistant strain derived from R. sphaeroides strain 2.4.1 (wild strain). BA indicates a strain in which PrrnB is inserted upstream of hemA. BAT indicates a strain in which PrrnB is inserted upstream of hemA and upstream of hemT. 7A indicates a strain in which Prsp_7571 is inserted upstream of hemA. 7AT indicates a strain in which Prsp_7571 was inserted upstream of hemA and upstream of hemT, BT indicates a strain in which PrrnB was inserted upstream of hemT, and 7T indicates a strain in which Prsp_7571 was inserted upstream of hemT. The results of quantification by RT-PCR of the expression level of the ALAS gene (hemA or hemT) in strains constructed by inserting a promoter using the self-cloning technique are shown. The upper row shows the expression level of the hemA gene as a relative value to the expression level of the WT strain under aerobic conditions, which is set to 1. The lower row shows the expression level of the hemT gene as a relative value to the expression level of the WT strain under aerobic conditions, which is set to 1. The white bars show the results when the strains were cultured under aerobic conditions, and the gray bars show the results when the strains were cultured under microaerobic conditions. The names of the WT and the six strains created by the self-cloning technique are the same as in Figure 2. The figure shows the time course of 5-aminolevulinic acid productivity in the strain constructed by inserting a promoter using the self-cloning technique. The vertical axis shows the 5-aminolevulinic acid (ALA) concentration (mM), and the horizontal axis shows the culture time (h). The names of the WT and the six strains constructed by the self-cloning technique are the same as those in Figure 2.

Below, several embodiments of the present invention are described in detail. However, the present invention is not limited to the following embodiments.

The photosynthetic bacterial strain according to one embodiment of the present invention is a photosynthetic bacterial strain belonging to the α-proteobacteria class in which an expression control region including a ribosomal RNA operon promoter (PrrnB) is located upstream of the gene (hemT) that encodes the 5-aminolevulinic acid synthase HemT, and the expression control region is derived from the same species of photosynthetic bacteria as the strain and induces the expression of hemT, which leads to increased productivity of 5-aminolevulinic acid compared to the wild-type strain.

First, photosynthetic bacteria will be described. Photosynthetic bacteria are bacteria that belong to the α-proteobacteria class in biological classification. Photosynthetic bacteria may be any bacteria that grow by photosynthetic inorganic or organic nutrition using light energy. Examples of photosynthetic bacteria include bacteria belonging to the genera Rhodobacter, Rhodospirillum, Rhodopseudomonas, Chromatium, Ectothiorhodospira, Chlorobium, Prosthecochloris, Chloroflexus, Chloronema, and Helicobacterium, with bacteria belonging to the genus Rhodobacter being preferred.

Bacteria belonging to the genus Rhodobacter include bacteria belonging to Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter sulfidophillus, Rhodobacter adriaticus, Rhodobacter veldkampii, etc., with preferred examples being bacteria belonging to Rhodobacter sphaeroides.

The classification system of photosynthetic bacteria is still in flux, with successive divisions and reorganizations taking place. In recent years, some species of the genus Rhodobacter have been transferred to three new genera: Cereibacter, Fuscovulum, and Phaeovulum. As a result, Rhodobacter sphaeroides and Rhodobacter veldkampii, two bacteria belonging to the genus Rhodobacter, are now classified as Cereibacter sphaeroides and Phaeovulum veldkampii, respectively. However, these species remain classified in the order Rhodobacter, and are still widely recognized as species of the genus Rhodobacter. Therefore, in this specification, Cereibacter sphaeroides and Phaeovulum veldkampii are treated as synonyms with Rhodobacter sphaeroides and Rhodobacter veldkampii, respectively, and as bacterial species belonging to the genus Rhodobacter.

Next, 5-aminolevulinic acid synthase (ALAS) will be described. There are two known pathways for the biosynthesis of 5-aminolevulinic acid, the C4 pathway and the C5 pathway. ALAS (EC 2.3.1.37) possessed by photosynthetic bacteria has the enzyme activity to catalyze the reaction of producing 5-aminolevulinic acid from glycine and succinyl-CoA in the C4 pathway (also called the Shemin pathway). Since the basic gene structures of photosynthetic bacteria, Rhodobacter genus and Rhodobacter sphaeroides are substantially identical or highly similar to each other, it is believed that the same effects can be obtained in the present invention regardless of the ALAS gene derived from any bacterium, as long as it has an ALAS gene that is homologous in sequence and/or function.

Specific examples of ALAS include HemA and HemT of Rhodobacter sphaeroides. HemA and HemT have in common the fact that they catalyze the reaction that produces 5-aminolevulinic acid from succinyl-CoA, but each is under different expression control. The hemA gene that codes for the former is located on the first chromosome and is induced to be expressed under anaerobic conditions, whereas the hemT gene that codes for the latter is located on the second chromosome and has been reported to be a silent gene in the 2.4.1 strain of Rhodobacter sphaeroides. As described in detail in the examples below, self-cloning strains in which the expression control regions of these genes were replaced with those of other genes by homologous recombination technology were produced and tested. When the rrnB promoter (PrrnB) was placed upstream of the hemT gene, high productivity of 5-aminolevulinic acid was observed, whereas when it was placed upstream of the hemA gene, no improvement in productivity of 5-aminolevulinic acid was observed. Therefore, the strain of the present invention is a photosynthetic bacterium strain that has a gene (hemT) encoding HemT under the control of PrrnB and expresses ALAS, and is highly productive of 5-aminolevulinic acid. Here, the hemT gene is preferably a gene derived from the same species as the photosynthetic bacterium or an endogenous gene.

Next, we will explain the hemT gene, whose expression is induced by the rrnB promoter (PrrnB) derived from the genome of the same species and introduced by homologous recombination technology in the photosynthetic bacterial strain of the present invention. The hemT gene may be of any sequence as long as it is derived from the genome of the photosynthetic bacteria and encodes a polypeptide having 5-aminolevulinic acid synthase activity, and examples of such hemT genes include the following: hemT genes derived from the genome of Rhodobacter sphaeroides strains 2.4.1, H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, ATH 2.4.9 (ATCC 17029), IFO 12203, CH10, or KD131. When inducing the expression of these hemT genes, self-cloning technology is used, and therefore the strain of the present invention is a strain of Rhodobacter sphaeroides, which is the same species as the biological species from which the hemT gene is derived. Here, the strain of the present invention only needs to be the same species as the biological species from which the hemT gene is derived, and does not necessarily need to be the same strain as the strain from which the hemT gene is derived.

A specific example of a hemT gene is a nucleic acid containing a nucleotide sequence encoding a protein having the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. Here, the nucleotide sequence of SEQ ID NO: 1 corresponds to the full length of the coding region of the hemT gene contained in the genome of Rhodobacter sphaeroides strain 2.4.1, and SEQ ID NO: 2 corresponds to the amino acid sequence encoded by the hemT gene. Also suitable for use in the present invention are nucleotide sequences which have 90% or more (preferably 92% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more) sequence identity with a nucleotide sequence encoding a protein having the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 and which encode a protein having 5-aminolevulinic acid synthase activity, and nucleotide sequences which have 1 to 120 (preferably 1 to 60, more preferably 1 to 40, even more preferably 1 to 20, and most preferably 1 to 10) bases deleted, substituted, added, or inserted from a nucleotide sequence encoding a protein having the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 and which encode a protein having 5-aminolevulinic acid synthase activity. When a BLAST search was performed on the base sequence of SEQ ID NO:1, the hemT genes derived from the genomes of the H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, CH10, and KD131 strains of Rhodobacter sphaeroides all showed sequence identity of 91% or more.

Next, we will explain PrrnB derived from the genome of the same species that is introduced into the photosynthetic bacterial strain of the present invention by homologous recombination technology to induce expression of the hemT gene. PrrnB is a nucleic acid derived from the ribosomal RNA operon promoter (PrrnB) in the genome of the photosynthetic bacteria, and an expression control region containing said PrrnB is used in the present invention. The expression control region containing the rrnB promoter may be of any sequence as long as it has the activity of inducing expression of the hemT gene under its control in the strain of the present invention, and examples include expression control regions containing PrrnB in the genomes of the following strains: That is, the expression control region contains PrrnB derived from the genome of any of the following strains: Rhodobacter sphaeroides strains 2.4.1, H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, ATH 2.4.3 (ATCC17025), ATH 2.4.9 (ATCC 17029), IFO 12203, CH10, KD131, or HJ.

A specific example of PrrnB is a nucleic acid containing the base sequence of SEQ ID NO: 4 or 5. Here, the base sequence of SEQ ID NO: 4 or 5 corresponds to the rrnB promoter region having a length of 58 or 195 bases contained in the genome of the 2.4.1 strain of Rhodobacter sphaeroides, or a nucleic acid containing the same. In addition, a base sequence having a sequence identity of 90% or more (preferably 94% or more, more preferably 96% or more, even more preferably 98% or more, and most preferably 99% or more) with the base sequence of SEQ ID NO: 4 or 5 and having hemT expression-inducing activity, or a base sequence in which 1 to 19 bases (preferably 1 to 15, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 3) have been deleted, substituted, added, or inserted from the base sequence of SEQ ID NO: 4 or 5, and having hemT expression-inducing activity, are also preferably used in the present invention. When a BLAST search is performed on the base sequence of SEQ ID NO: 4 or 5, PrrnB derived from the genome of any of the Rhodobacter sphaeroides strains H2, DSM158, MBTLJ-20, MBTLJ-13, MBTLJ-8, AB24, AB25, AB27, AB29, CH10, KD131, and HJ strains all show a sequence identity of 96% or 94% or more.

Functional fragments of the rrnB promoter (PrrnB) are also used in the present invention as long as they have the activity of inducing the expression of hemT. According to Henry et al. (Proc. Natl. Acad. Sci. USA, (2020) vol. 117, pp. 29658-29668), PrrnB contains a 6-base sequence (TTGCGC) in the -35 region recognized by transcription factors and a 6-base sequence (TAGAAA) in the -10 region recognized by RNA polymerase, and a 29-base sequence (TTGCGCCCGGGGCCGTCTGCTCCTAGAAA; SEQ ID NO: 3) containing both sequences, or a 58-base sequence (TACGGAGCCCAAAAAATCCGCTTGCGCCCGGGGCCGTCTGCTCCTAGAAACCGCTTCA; SEQ ID NO: 4) containing both sequences is considered to be the minimum unit or promoter region that functions as a functional fragment of the rrnB promoter, and is a suitable functional fragment of PrrnB. Here, the base sequence of SEQ ID NO: 3 or 4 corresponds to the base sequence of positions 159 to 187 or positions 138 to 195 of the base sequence of SEQ ID NO: 5. Therefore, a nucleic acid having a 5'-end at any position from 1 to 158 of SEQ ID NO: 5 and a 3'-end at any position from 188 to 195, which includes the entire base sequence of SEQ ID NO: 3 or 4 and corresponds to a partial sequence of the base sequence of SEQ ID NO: 5, or a nucleic acid having a 5'-end at any position from 1 to 137 of SEQ ID NO: 3 and a 3'-end at position 194 or 195, is also preferably used in the present invention as a functional fragment or promoter region of PrrnB of the present invention. In addition, in each of the base sequences of SEQ ID NO: 4 and SEQ ID NO: 5, the adenine residue at the 3'-end serves as the transcription initiation site.
In the present invention, the expression-inducing activity of hemT literally means an activity to induce the expression of hemT, and although it is not necessarily limited to this, it means, for example, an activity to induce expression of hemT 5-fold, 10-fold, 20-fold or 30-fold, and particularly preferably 40-fold or more, compared to the expression of hemT in a wild-type or standard strain; and from another perspective, it means an activity that is equal to or greater than the expression-inducing activity of hemT by a nucleic acid having the base sequence of any one of SEQ ID NOs: 3, 4 or 5.

Next, the insertion site of the rrnB promoter (PrrnB) or a functional fragment thereof will be described.
The insertion position is not necessarily limited as long as PrrnB or a functional fragment thereof functions as a promoter and induces transcription of the hemT gene located downstream thereof. Usually, the insertion position is the coding region of the hemT gene, the transcription start site, or upstream of the ribosome binding sequence. An example of the insertion position is a position upstream of the coding region of the hemT gene, and within 10,000 bases upstream of the start codon of the hemT gene, preferably within 1,000 bases, more preferably within 100 bases, and even more preferably adjacent to the transcription start site of the hemT gene.
However, since the purpose is to induce expression of the hemT gene, it is required that there is no transcription termination sequence between the inserted PrrnB or its functional fragment and the start codon of the hemT gene, and if such a sequence exists, the sequence is modified to remove the transcription termination sequence. The inserted sequence containing the promoter region of PrrnB or its functional fragment may also contain other appropriate components such as a transcription initiation site (adenine base), a ribosome binding sequence, or other genes.

The photosynthetic bacterial strain according to one embodiment of the present invention is produced by a self-cloning technique and is substantially free of nucleic acid sequences derived from heterologous organisms, such as foreign genes. In self-cloning, organisms of the same species are used as the source of transforming DNA and the host for transformation. Thus, in the present invention, an insertion sequence containing PrrnB or a functional fragment thereof derived from a certain type of photosynthetic bacteria is used as the source and inserted into the host genome of the same type of photosynthetic bacteria. This insertion functionally links PrrnB or a functional fragment thereof derived from the same type of photosynthetic bacteria to the hemT gene, and expression of hemT is induced in the self-cloning strain by the expression-inducing activity of the rrnB promoter. The genome of the strain of the present invention produced by the self-cloning technique is completely free or substantially free of nucleic acid derived from organisms of a species different from the host photosynthetic bacteria. Here, "substantially free" means that only heterologous nucleic acid or artificial sequence nucleic acid with a very short sequence length of about 1 to 30 bases (preferably 20 bases or less, more preferably 10 bases or less, and even more preferably 6 bases or less) is allowed to exist, such as a polylinker used to facilitate in vitro cloning. Here, it is not intended to include heterologous nucleic acids, such as heterologous antibiotic resistance genes, that are used as selection markers.

Self-cloning is a technique well known to those skilled in the art (see Akada et al. (1999) J. Biosci. Bioeng., vol. 87, pp. 43-48, JP 2003-144164 A, etc.), and can be carried out appropriately by those skilled in the art by combining and applying general molecular biology experimental techniques.

One embodiment of the present invention is a method for producing a photosynthetic bacterial strain with high productivity of 5-aminolevulinic acid. The method includes a step of inserting an expression control region including a ribosomal RNA operon promoter (PrrnB) into a wild-type strain of photosynthetic bacteria upstream of a gene (hemT) encoding HemT, a 5-aminolevulinic acid synthase, in the genome of the wild-type strain by homologous recombination, where the inserted PrrnB induces expression of hemT, and the induction results in increased productivity of 5-aminolevulinic acid compared to the wild-type strain, and the photosynthetic bacteria is a species of bacteria belonging to the class α-Proteobacteria.

One embodiment of the method for producing a photosynthetic bacterium strain with high productivity of 5-aminolevulinic acid of the present invention is a method using a plasmid vector for homologous recombination. Here, the plasmid vector includes an expression control region containing PrrnB, and homologous regions consisting of sequences homologous to the upstream and downstream regions of the genome adjacent to the target region of homologous recombination are arranged on both sides of the expression control region containing PrrnB. The homologous region may have any sequence length as long as it can efficiently induce homologous recombination in a target site-specific manner, for example, a sequence length of 20 bases or more, 100 bases or more, 500 bases or more, or 1,000 bases or more. The portion located outside the homologous region from the expression control region containing PrrnB is not inserted into the host by homologous recombination, so it may contain a nucleic acid heterologous to the genome of the host. Examples of the heterologous nucleic acid include a positive selection marker (antibiotic resistance gene such as Gm ^R ) and a negative selection marker (sacB, etc.).

Another embodiment of the present invention is a method for producing 5-aminolevulinic acid, characterized in that a strain of the photosynthetic bacteria of the present invention is cultured in a medium, 5-aminolevulinic acid is produced and accumulated in the strain and the medium, and 5-aminolevulinic acid is collected from the culture. Cultivating photosynthetic bacteria for the purpose of producing 5-aminolevulinic acid and collecting the product 5-aminolevulinic acid are both well-known techniques, and a person skilled in the art can appropriately carry them out by referring to known literature. As long as 5-aminolevulinic acid can be produced, the medium may be a solid medium or a liquid medium, and the medium components may be any as long as they contain a substrate (glycine, etc.) related to the production of 5-aminolevulinic acid and are suitable for the growth of photosynthetic bacteria. As described in Patent Document 3, a medium containing a plant protein hydrolysate in addition to ordinary nutritional components such as yeast extract can also be used. A preferred example of the method for producing 5-aminolevulinic acid of the present invention is a method using a liquid medium and performing shaking culture. The oxygen conditions in the culture may be aerobic, microaerobic (oxygen-limited), or anaerobic as long as 5-aminolevulinic acid can be produced, but microaerobic conditions are preferred. This is because oxygen inhibits pigment synthesis in photosynthetic bacteria, particularly purple nonsulfur bacteria, and 5-aminolevulinic acid synthase is said to be inactivated by oxygen. Microaerobic conditions can be achieved by artificially suppressing oxygen supply, but they can also be achieved naturally during continuous shaking culture in an environment with insufficient ventilation. If light irradiation is not performed during culture, the photosynthetic bacteria will be placed in a heterotrophic environment and will require oxygen for growth, so anaerobic conditions are not suitable. In order to increase the productivity of 5-aminolevulinic acid, the various environmental conditions related to the culture of the strain, such as medium components, oxygen supply conditions, and light irradiation amount, including combinations thereof, can be appropriately adjusted.

The culture temperature and the pH of the medium may be any temperature and pH as long as the photosynthetic bacterial strain of the present invention grows and produces 5-aminolevulinic acid. For example, the culture temperature is preferably 10 to 40°C, particularly 20 to 35°C, and the pH of the medium is preferably 3 to 9, particularly 6 to 8. If the pH changes during the production of 5-aminolevulinic acid, it is preferable to adjust the pH using an alkaline solution such as sodium hydroxide, ammonia, or potassium hydroxide, or an acid such as hydrochloric acid, sulfuric acid, or phosphoric acid.

The 5-aminolevulinic acid produced by the photosynthetic bacterial strain of the present invention through cultivation can be purified by standard methods. For example, it can be separated and purified as necessary by standard methods such as ion exchange, chromatography, and extraction.

The present invention will be described in detail below with reference to examples, but these are presented for illustrative purposes only and the present invention is not limited to these examples.

Materials and Methods
1. Strains, media, culture, plasmids, and primers Rhodobacter sphaeroides strains were grown aerobically in PYS medium (0.3% Bacto peptone, 0.3% Bacto yeast extract, 2 mM _CaCl2, and 2 mM _MgSO4 ) as the growth medium in a shaker at 30°C. E. coli strains were grown at 37°C in Luria-Bertani medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) as the growth medium.
The strains, plasmids and primers used in the following examples are shown below.

E. coli
JM109; Yanisch-Perron et al., Gene (1985) vol. 33, pp. 103-119.
JM109 λ pir; JM109 lysogenized with λ pir bacteriophage; Penfold and Pemberton, Gene (1992) vol. 118, pp. 145-146.
S17-1; F-, thi, pro, hsdR, [RP4-2 Tc::Mu Km::Tn7 (Tp Sm)]; Simon et al., Bio/Technology (1983) vol. 1, pp. 784-791.
S17-1; S17-1 lysogenized with λ pir bacteriophage; De Lorenzo, et al., J. Bacteriol. (1990) vol. 172, pp. 6568-6572.

Photosynthetic bacterium Rhodobacter sphaeroides
2.4.1; Wild strain; Sistrom, J. Gen. Microbiol., (1960) vol. 22, pp. 778-785.
WT; rifampicin-resistant strain derived from strain 2.4.1
BA; The rrnB promoter was inserted upstream of the hemA transcription start siteBT; The rrnB promoter was inserted upstream of the hemT transcription start siteBAT; The rrnB promoter was inserted upstream of both the hemA and hemT transcription start sites
7A inserted; The rsp_7571 promoter was upstream of the hemA start codon
7T; The rsp_7571 promoter was upstream of the hemT start codon
7AT inserted; The rsp_7571 promoter was upstream of both the hemA start codon and hemT start codon
The WT, BA, BT, BAT, 7A, 7T, and 7AT strains were newly constructed in the present invention.

Plasmids
pZJD29A; Suicide vector, sacB, Gm ^r ; Swem et al., EMBO J. (2003) vol. 22, pp. 4699-4708.
pZJD29A PrrnB hemA; Sequence to insert PrrnB upstream of the hemA transcription start site on pZJD29A.
pZJD29A PrrnB hemT; Sequence to insert PrrnB upstream of the hemT transcription start site on pZJD29A.
pZJD29A Prsp_7571 hemA; Sequence to insert Prsp_7571 upstream of the hemA coding sequence on pZJD29A.
pZJD29A Prsp_7571 hemT; Sequence to insert Prsp_7571 upstream of the hemT coding sequence on pZJD29A

Primers (for cloning)
MCShemAUP-F; sequence number 6 upstream of hemA transcription start site
PrrnBhemAUP-R; SEQ ID NO: 7; upstream of hemA transcription start site
PrrnBhemADW-F; SEQ ID NO:8; downstream of hemA transcription start site
MCShemADW-R; SEQ ID NO:9; downstream of hemA transcription start site
hemAUPPrrnB-F; SEQ ID NO: 10 Promoter of rrnB
hemADWPrrnB-R; SEQ ID NO:11; Promotor of rrnB
MCShemTUP-F; SEQ ID NO:12; upstream of hemT transcription start site
PrrnBhemTUP-R; SEQ ID NO: 13; upstream of hemT transcription start site
PrrnBhemTDW-F; SEQ ID NO:14; downstream of hemT transcription start site
MCShemTDW-R; SEQ ID NO: 15; downstream of hemT transcription start site
hemTUPPrrnB-F; SEQ ID NO: 16; Promoter of rrnB
hemTDWPrrnB-R; SEQ ID NO: 17; Promotor of rrnB
MCShemAUP-F2; SEQ ID NO:18; upstream of hemA start codon
P7571hemAUP-R; SEQ ID NO:19; upstream of hemA start codon
P7571hemADW-F; SEQ ID NO:20; downstream of hemA start codon
MCShemADW-R2; SEQ ID NO:21; downstream of hemA start codon
hemAUPP7571-F; SEQ ID NO:22; Promotor of rsp_7571
hemADWP7571-R; SEQ ID NO:23; Promotor of rsp_7571
MCShemTUP-F2; SEQ ID NO:24; upstream of hemT start codon
P7571hemTDW-F; SEQ ID NO:25; upstream of hemT start codon
P7571hemTUP-R; SEQ ID NO:26; downstream of hemT start codon
MCShemTDW-R2; SEQ ID NO:27; downstream of hemT start codon
hemTUPP7571-F; SEQ ID NO:28; Promotor of rsp_7571
hemTDWP7571-R; SEQ ID NO:29; Promotor of rsp_7571

2. Quantitative real-time PCR
RNA was extracted from logarithmically growing cells using RNeasy Mini Kit (QIAGEN). cDNA was synthesized from RNA by reverse transcription using PrimeScript RT reagent Kit (TaKaRa bio). cDNA was quantified by real-time PCR using TB Green Premix Ex Taq II (TaKaRa bio) with Thermal Cycler Dice Real Time System III (TaKaRa bio). Each cDNA sample was quantified three times as technical replicates. The primer sequences used for amplification are shown below. The cycle threshold (Ct) value of each sample was calculated by the 2nd derivative maximum method. The normalized ΔCt was calculated using the rpoZ gene encoding the ω subunit of RNA polymerase as an endogenous control for each cell. The ΔΔCt of each sample was calculated using the ΔCt of WT cultured under aerobic conditions as a calibrator.

The primers used for quantitative real-time PCR are shown below.
rpoZqRT-F; SEQ ID NO: 30; Gomelsky et al., Microbiology (2003) vol. 149, pp. 377-388.
rpoZqRT-R; SEQ ID NO: 31; Gomelsky et al., Microbiology (2003) vol. 149, pp. 377-388.
hemTqRT-F; SEQ ID NO: 32; newly designed in this application
hemTqRT-R; SEQ ID NO: 33; newly designed in this application
hemAqRT-F; SEQ ID NO: 34; newly designed in this application
hemAqRT-F; SEQ ID NO: 35; newly designed in this application

3. Detection of ALAS activity ALAS activity in crude cell extracts was measured according to the method of Yubisui et al. (Arch. Biochem. Biophys., (1972), 150, 77-85.). The culture medium was centrifuged at 8,000 × g for 5 min at 4°C, and the resulting cell pellet was suspended in ice-cold 50 mM Tris-HCl buffer (pH 7.5). The cell suspension was sonicated and centrifuged at 10,000 × g for 5 min at 4°C, and the protein concentration of the resulting supernatant was measured by Bradford protein assay. Enzyme solution (95% cell homogenate supernatant, 20 mM Tris-HCl (pH 7.5), 0.35 mM pyridoxal phosphate) and substrate solution (0.5 M glycine, 1 mM succinyl CoA) were incubated at 37°C for 5 min. The enzyme reaction was started by adding 25% of the substrate solution to the enzyme solution. After 5 minutes, the reaction was stopped by adding 10% trichloroacetic acid in an amount of 30% by volume relative to the reaction solution and cooling on ice.
The concentration of 5-aminolevulinic acid in each reaction solution was measured according to the method of Mauzerall et al. (J. Biol. Chem., (1956), 219, 435.). 5-aminolevulinic acid in each sample was boiled in 2 M acetate buffer (pH 4.6) at 100°C for 15 minutes and reacted with 1% acetylacetone. 175% volume of modified Ehrlich reagent (20 g/L p-dimethylaminobenzaldehyde, 12% perchloric acid, 60% glacial acetic acid) was added to the reaction solution, and the pyrrole compound formed was colorimetrically quantified by measuring the absorbance at 553 nm.

4. Fermentative Production of 5-Aminolevulinic Acid 5-Aminolevulinic acid was produced in various strains of Rhodobacter sphaeroides using the method of Nishikawa et al. (J. Biosci. Bioeng., (1999), 87, 798-804.). Each strain was cultured in 120 mL of GGY2 medium (50 mM glucose, 3.8 g/L sodium L-glutamate monohydrate, 2.0 g/L Bacto yeast extract, 1.13 g/L NaH2PO4·12H2O, 1.07 g _/ L _NaH2PO4 · _2H2O , 0.8 g/L ( _NH4 )2HPO4, 0.2 g _/ L MgSO4 _{· 7H2O} , 53 mg/L CaCl2· _2H2O , _{1.2 mg} /L MnSO4, 1.0 mg/L nicotinic _acid , 1.0 mg/ _L thiamine hydrochloride, and 0.01 _mg /L biotin) in a 300 mL baffled Erlenmeyer flask, and incubated at 30°C for 170 min. The strains were cultured in a dark, aerobic condition at 150 rpm for 48 h with shaking. Each culture was centrifuged at 5,000 × g for 5 min. A cell pellet (1.0 g wet weight) was suspended in 20 ml of fresh GGY2 medium containing substrate (60 mM glycine) and metabolic inhibitor (30 mM levulinic acid) and cultured in a test tube at 30°C with shaking at 150 rpm in the dark. Each strain was cultured in three independent test tubes. After 0, 12, 18, 24, 36, 42, and 48 h of culture, the 5-aminolevulinic acid concentration in each culture supernatant was measured as described above (detection of ALAS activity).

Example 1: Construction of a self-cloning strain in which the rrnB or rsp_7571 promoter is inserted upstream of the hemA or hemT gene of Rhodobacter sphaeroides (FIGS. 1 and 2)
(1)
In order to insert the rrnB promoter upstream of the transcription start site of hemA, a suicide vector pZJD29A PrrnB hemA was constructed. A 600 bp DNA fragment upstream and downstream from the transcription start site of hemA and the rrnB promoter were PCR amplified and inserted into pZJD29A. Similarly, pZJD29A PrrnB hemT was also constructed. These plasmids were conjugated to Rhodobacter sphaeroides using E. coli, and a single recombinant in which the plasmid was homologously recombined on the genome was obtained using the antibiotic resistance gene on the plasmid as a marker. These strains were selected on sucrose plates to obtain a double recombinant in which the sequence derived from the plasmid was removed from the genome by homologous recombination. The genome of the obtained strain was confirmed to confirm that the promoter was inserted. Thus, a Rhodobacter sphaeroides strain in which the rrnB promoter was inserted upstream of hemA, hemT, or both hemA and hemT was constructed.
More specifically, the 195 bp upstream of the transcription start site of rrnB was inserted upstream of the transcription start sites of hemA, hemT, or both hemA and hemT (Fig. 2). The strain in which PrrnB was inserted only in hemA was named BA, the strain in which PrrnB was inserted only in hemT was named BT, and the strain in which PrrnB was inserted in both hemA and hemT was named BAT.
The transcription initiation sites of rrnB, hemA, and hemT were determined by reference to Dryden et al. (J. Bacteriol., (1993), 175, 6392-6402.) and Neidle et al. (J. Bacteriol., (1993), 175, 2292-2303.). The rrnB promoter (PrrnB) is known as a constitutive promoter that exhibits the highest transcription activity among the promoters of ribosomal RNA operons (rrnA, rrnB, rrnC).

(2) A self-cloning strain of Rhodobacter sphaeroides in which the rsp_7571 promoter was inserted upstream of the transcription initiation site of the hemA or hemT gene was produced by the following procedure.
To insert the rsp_7571 promoter upstream of the hemA start codon, pZJD29A Prsp_7571 hemA was constructed. Two 600 bp DNA fragments, upstream and downstream of the hemA start codon, and the rsp_7571 promoter were amplified by PCR and inserted into pZJD29A. Similarly, pZJD29A Prsp_7571 hemT was also constructed. Using these plasmids, R. sphaeroides strains containing the rsp_7571 promoter were constructed.
More specifically, 498 bp upstream of the initiation codon of rsp_7571 was inserted upstream of the initiation codon of hemA and/or hemT (Fig. 2). The strain in which Prsp_7571 was inserted only into hemA was designated 7A, the strain in which Prsp_7571 was inserted only into hemT was designated 7T, and the strain in which Prsp_7571 was inserted into both hemA and hemT was designated 7AT.
The promoter of rsp_7571 (Prsp_7571) is known to be a highly active constitutive promoter in Rhodobacter sphaeroides strain 2.4.1.

Example 2 Confirmation of the Function of the Inserted PrrnB and Prsp_7571 Promoters To confirm whether the inserted PrrnB promoter was functional, the mRNA expression of hemA and hemT in WT, BA, BT, and BAT cultured under aerobic and microaerobic conditions was quantified (Figure 3).
In BA and BAT, where PrrnB was inserted upstream of hemA, the hemA mRNA levels increased 3.5-fold and 2.4-fold, respectively, under aerobic conditions. Under microaerobic conditions, the difference was narrowed because expression of WT hemA was induced, but the hemA expression levels in BA and BAT were still increased 1.3-fold compared to WT (Fig. 3, top).
In BT and BAT, where PrrnB was inserted upstream of hemT, the expression levels of hemT increased 24-fold and 41-fold, respectively, under aerobic conditions, and 37-fold and 46-fold, respectively, under microaerobic conditions (Fig. 3, bottom). These results indicate that insertion of PrrnB into the promoter region of the ALAS gene increases its expression.
On the other hand, no positive effect was observed due to the insertion of Prsp_7571 into the hemA and hemT promoters: the transcription levels of hemA and hemT did not increase in 7A, 7T, and 7AT compared to those of the WT under aerobic and semi-aerobic conditions (Fig. 3).
From the above results, it was confirmed that in the present invention, good results were obtained with the BT or BAT strain, which is a self-cloning strain in which the rrnB promoter is placed upstream of the hemT gene.

Example 3 Fermentative Production of 5-aminolevulinic Acid The productivity of 5-aminolevulinic acid of the constructed strains was evaluated using a medium supplemented with glycine, which is a substrate of 5-aminolevulinic acid, and levulinic acid, which is a metabolic inhibitor of 5-aminolevulinic acid.
After the cells were transferred to the production medium, the amount of 5-aminolevulinic acid accumulated increased (around 20 hours) and decreased (after 20 hours) in the BT and WT cultures (Fig. 4). In particular, the maximum concentration of 5-aminolevulinic acid in the BT culture was 9.2 mM, about 12-fold higher than that of the WT (0.78 mM). In the cultures of the other strains, BA, and BAT, 5-aminolevulinic acid accumulated at a lower level than that of the WT (Fig. 4).

Claims (3)

A photosynthetic bacterial strain belonging to the class α-proteobacteria that has an expression control region containing a ribosomal RNA operon promoter (PrrnB) upstream of the gene (hemT) that codes for the 5-aminolevulinic acid synthase HemT, the expression control region being derived from the same species of photosynthetic bacteria as the strain and inducing the expression of hemT, the induction resulting in increased productivity of 5-aminolevulinic acid compared to a wild-type strain. A method for producing 5-aminolevulinic acid, comprising culturing the strain according to claim 1 in a medium, producing and accumulating 5-aminolevulinic acid in the strain and the medium, and collecting 5-aminolevulinic acid from the culture. A method for producing a photosynthetic bacterial strain belonging to the α-proteobacteria class, which has an expression control region including a ribosomal RNA operon promoter (PrrnB) upstream of the gene (hemT) encoding the 5-aminolevulinic acid synthase HemT, said expression control region being derived from the same species of photosynthetic bacteria as said strain and inducing expression of hemT, said induction resulting in increased productivity of 5-aminolevulinic acid compared to a wild-type strain, said method for producing said strain, characterized in that it includes a step of functionally binding PrrnB to hemT.