Method for producing 6-hydroxyhexanoic acid

Abstract

To provide a method for producing 6-hydroxyhexanoic acid (6-HHA) with increased production efficiency through a bioprocess.SOLUTION: A method for producing 6-hydroxyhexanoic acid comprises culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically controlled conditions, where the bacterium has a carboxylic acid reductase and an aldehyde reductase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing 6-hydroxyhexanoic acid. [Background technology]

[0002] Traditionally, petrochemical processes have been the main method for industrially producing useful chemical substances. However, petrochemical processes have had problems such as the difficulty of carrying out reactions at room temperature and pressure, and the large amount of CO2 emissions.

[0003] In recent years, bioprocessing has been attracting attention as a way to solve these problems, and various technologies using microorganisms have been developed to sustainably produce chemicals from renewable biomass. For example, Non-Patent Document 1 describes a method for producing major C2 to C6 platform chemicals using genetically engineered Corynebacterium glutamicum. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Baritugo KA et al., “Metabolic engineering of Corynebacteriumglutamicum for fermentative production of chemicals in biorefinery.” Appl Microbiol Biotechnol. 2018 May;102(9):3915-3937 Summary of the Invention [Problem to be solved by the invention]

[0005] However, it cannot be said that sufficient research has been carried out into methods for producing 6-hydroxyhexanoic acid by bioprocesses.

[0006] An object of the present invention is to provide a method for producing 6-hydroxyhexanoic acid (6-HHA) using a bioprocess with improved production efficiency. [Means for solving the problem]

[0007] The present inventors cultured Shewanella oneidensis, which possesses a specific enzyme, under aerobic conditions in a medium containing glucose and adipic acid but no nitrogen source. They found that the concentration of 6-HHA in the culture supernatant increased compared to when the same bacterium was cultured under the same conditions, except that the medium contained a nitrogen source. They also found that the pH of the medium was significantly lower than that at the start of the culture.

[0008] The present inventors also found that when Shewanella oneidensis, which possesses a specific enzyme, was cultured under anaerobic conditions in a medium containing glucose and adipic acid, the concentration of 6-HHA in the culture supernatant increased compared to when the same bacterium was cultured under the same conditions except for aerobic conditions. The present invention is based on these novel findings.

[0009] The present invention includes, for example, the following inventions. [1] The method comprises culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically inhibited conditions, The method for producing 6-hydroxyhexanoic acid, wherein the bacterium has a carboxylic acid reductase and an aldehyde reductase. [2] The method according to [1], wherein the metabolic inhibition conditions include one or more conditions selected from the group consisting of anaerobic conditions, conditions in which the medium is a nutrient-limited medium, and conditions in which the pH of the medium is 3 or higher and 5.5 or lower. [3] The method according to [1] or [2], wherein the medium does not contain a nitrogen source. [4] The method according to any one of [1] to [3], wherein the bacterium further has glucose kinase and sugar permease. [5] The method comprises culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically inhibited conditions, The method for improving the production efficiency of 6-hydroxyhexanoic acid, wherein the bacterium has a carboxylic acid reductase and an aldehyde reductase. [6] The method according to [5], wherein the metabolic inhibition conditions include one or more conditions selected from the group consisting of anaerobic conditions, conditions in which the medium is a nutrient-limited medium, and conditions in which the pH of the medium is 3 or higher and 5.5 or lower. [7] The method according to [5] or [6], wherein the medium does not contain a nitrogen source. [8] The method according to any one of [5] to [7], wherein the bacterium further has glucose kinase and sugar permease. [9] A bacterium belonging to the genus Shewanella, A bacterium having carboxylic acid reductase and aldehyde reductase.

[10] The bacterium according to [9], further comprising glucose kinase and a sugar permease.

[11] The bacterium according to [9] or

[10] , wherein the bacterium belonging to the genus Shewanella is Shewanella oneidensis. [Effects of the Invention]

[0010] According to the present invention, a method for producing 6-hydroxyhexanoic acid by a bioprocess with improved production efficiency can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the concentration of 1,6-hexanediol (1,6-HDO) (A) and the concentration of 6-HHA (B) in the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of Test Example 1. [Figure 2] 1 is a graph showing the concentration of adipic acid (A) and the concentration of glucose (B) in the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of Test Example 1. [Figure 3] 1 is a graph showing the pH of the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the non-growth conditions (A) or growth conditions (B) of Test Example 1. [Figure 4] 1 is a graph showing the concentration of 1,6-HDO (A) and the concentrations of glucose and 6-HHA (B) in the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of Test Example 2. [Figure 5] 1 is a graph showing the glucose concentration (A) and the concentrations of fumaric acid, succinic acid, and acetic acid (B) in the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of Test Example 2. [Figure 6] This is a graph showing the changes in the concentrations of 1,6-HDO, 6-HHA, and adipic acid (A), and the concentrations of glucose, lactic acid, and acetic acid (B) in the culture supernatant when the modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of the Reference Example. [Figure 7] Graph (A) shows the change in OD600 when a modified strain of Shewanella oneidensis prepared in the Examples was cultured under the conditions of the Reference Example, and graph (B) shows the change in pH of the culture supernatant of the above modified strain. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0013] [Method for producing 6-HHA] The method for producing 6-hydroxyhexanoic acid according to this embodiment (hereinafter also referred to as the "production method according to this embodiment") includes a step of culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically inhibited conditions (culturing step). Here, the bacterium has a carboxylic acid reductase and an aldehyde reductase.

[0014] The bacteria cultured in the culturing step possess carboxylic acid reductase and aldehyde reductase, thereby establishing a pathway within the bacteria that produces 6-HHA from adipic acid via adipic acid semialdehyde. Further reactions of 6-HHA can convert it to 1,6-hexanediol (1,6-HDO) via 6-hydroxyhexanal. In the culturing step, the bacteria are cultured in a medium containing adipic acid under metabolically inhibited conditions, thereby suppressing the conversion to 1,6-HDO and enabling efficient production of 6-HHA.

[0015] The bacterium to be cultured in the culture step is not particularly limited as long as it is a bacterium belonging to the genus Shewanella that has carboxylic acid reductase and aldehyde reductase, such as Shewanella oneidensis, Shewanella loihica, Shewanella putrefaciens, and Shewanella algae, with Shewanella oneidensis being preferred and the Shewanella oneidensis MR-1 strain (wild-type strain) being more preferred.

[0016] The carboxylic acid reductases and aldehyde reductases possessed by the bacteria may be endogenous or exogenous, and the bacteria may have multiple carboxylic acid reductases and / or multiple aldehyde reductases from different microbial species.

[0017] Carboxylic acid reductase (CAR) is an enzyme that reduces carboxylic acids to produce aldehydes. CARs can be derived from any source, including Mycobacterium avium, Mycobacterium marinum, Nocardia iowensis, Segniliparus rugosus, and Kutzneria albida.

[0018] The carboxylic acid reductase may be a variant of the carboxylic acid reductase as long as it has the above-mentioned activity. The variant of the carboxylic acid reductase may be, for example, a carboxylic acid reductase in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the wild-type carboxylic acid reductase derived from the microorganism, and which has at least the activity of reducing a carboxylic acid to produce an aldehyde. Furthermore, the variant of the carboxylic acid reductase may be, for example, a carboxylic acid reductase that exhibits 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, 96% or more, and more preferably 97% or more, 98% or more, or 99% or more sequence identity in amino acid sequence to the wild-type carboxylic acid reductase derived from the microorganism, and which has at least the activity of reducing a carboxylic acid to produce an aldehyde.

[0019] A specific example of the above-mentioned modified carboxylic acid reductase may be a protein encoded by a nucleic acid comprising the base sequence shown in SEQ ID NO: 1. The protein is a carboxylic acid reductase (MavCAR) derived from Mycobacterium avium, in which the asparagine residue at position 355 has been substituted with an arginine residue.

[0020] Aldehyde reductase is an enzyme that reduces aldehydes to produce alcohols. The source of the aldehyde reductase is not particularly limited, and examples include Escherichia coli, Pseudomonas putida, and Shewanella oneidensis. A specific example of the aldehyde reductase may be a protein encoded by a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 2. The protein is the aldehyde reductase YahK derived from Escherichia coli.

[0021] The aldehyde reductase may be a variant of the aldehyde reductase as long as it has the above-mentioned activity. The variant of the aldehyde reductase may be, for example, an aldehyde reductase in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of the wild-type aldehyde reductase derived from the microorganism, and which has at least the activity of reducing an aldehyde to produce an alcohol. Furthermore, the variant of the aldehyde reductase may be, for example, an aldehyde reductase that exhibits 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, or 96% or more, more preferably 97% or more, 98% or more, or 99% or more sequence identity in amino acid sequence to the wild-type aldehyde reductase derived from the microorganism, and which has at least the activity of reducing an aldehyde to produce an alcohol.

[0022] Bacteria belonging to the genus Shewanella that have carboxylic acid reductase and aldehyde reductase can be obtained by introducing genes that express these enzymes into the above bacteria and transforming them.

[0023] Bacterial transformation can be performed by any method known in the art, including, for example, a method of introducing an expression vector into which genes expressing carboxylic acid reductase and aldehyde reductase are inserted, or a method of integrating genes expressing carboxylic acid reductase and aldehyde reductase into the genome of a bacterium.

[0024] The gene to be introduced into the bacterium may be, for example, one whose codons have been optimized according to the type of bacterium (host) to be transformed.

[0025] In addition to the carboxylic acid reductase and aldehyde reductase, the bacterium may further possess modifying enzymes for these proteins. The modifying enzymes are enzymes that have the activity of modifying proteins synthesized within the bacterium, such as phosphorylation, glycosylation, methylation, and phosphopantetheinylation. For example, the stability and activity of carboxylic acid reductase are improved by undergoing phosphopantetheinylation modification by phosphopantetheinyl transferase (PPTase). In this way, when the bacterium further possesses the modifying enzymes for the above proteins, the production of 6-HHA from adipic acid becomes more efficient.

[0026] A specific example of a phosphopantetheinyl transferase (PPTase) is a protein encoded by the base sequence shown in SEQ ID NO: 3. This protein is a phosphopantetheinyl transferase (Sfp) derived from Bacillus subtilis.

[0027] The bacterium may also further have glucose kinase and sugar permease.

[0028] Glucose kinase (glk) is an enzyme that promotes the phosphorylation of glucose to glucose-6-phosphate. The source of glucose kinase is not particularly limited, and examples include Escherichia coli, Pseudomonas aeruginosa, and Corynebacterium glutamicum. A specific example of glucose kinase may be a protein encoded by a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 4. This protein is glucose kinase derived from Escherichia coli.

[0029] The glucose kinase may be any one that has the above-mentioned activity and may be a variant of the glucose kinase. The variant of glucose kinase may be, for example, a glucose kinase in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the wild-type glucose kinase derived from the microorganism, and which has at least the activity of promoting the phosphorylation of glucose to glucose-6-phosphate. The variant of glucose kinase may also be, for example, a glucose kinase that has an amino acid sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, 96% or more, and more preferably 97% or more, 98% or more, or 99% or more to the wild-type glucose kinase derived from the microorganism, and which has at least the activity of promoting the phosphorylation of glucose to glucose-6-phosphate.

[0030] Sugar permeases (also known as hexose permeases) are enzymes that transport glucose from the outside of bacterial cells into the cells. Glucose transport from the outside of cells into the cells can be active or passive. The origin of sugar permeases is not particularly limited, and examples include Escherichia coli, Pseudomonas aeruginosa, and Corynebacterium glutamicum. A specific example of a sugar permease is a protein encoded by a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 5. This protein is galactose permease (galP) derived from Escherichia coli.

[0031] The sugar permease may be any modified form of the sugar permease as long as it has the above-mentioned activity. The modified enzyme may be, for example, an enzyme in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of the wild-type sugar permease derived from the microorganism, and which has at least the activity of transporting glucose from the outside of the cell into the cell. Furthermore, the modified enzyme may be, for example, an enzyme that has an amino acid sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, or 96% or more, more preferably 97% or more, 98% or more, or 99% or more to the wild-type sugar permease derived from the microorganism, and which has at least the activity of transporting glucose from the outside of the cell into the cell.

[0032] The bacterium may have multiple glucose kinases and / or sugar permeases from different microbial species. Bacteria belonging to the genus Shewanella that further have glucose kinases and / or sugar permeases can be obtained by introducing genes that express these enzymes into the bacterium and transforming it. The transformation method is as described above.

[0033] Bacteria belonging to the genus Shewanella are inherently unable to take up glucose into their cells. Therefore, by further possessing glucose kinase and glucose permease, the bacteria are able to take up glucose into their cells, where it is phosphorylated and converted to glucose-6-phosphate. The converted glucose-6-phosphate can be used, for example, in the ED pathway, allowing for increased production of NADPH within the cells. This allows for more efficient production of 6-HHA.

[0034] As used herein, "metabolism" refers to a series of chemical reactions essential for bacterial survival, such as glycolysis, the Entner-Doudoroff pathway (ED pathway), the citric acid cycle, lipid synthesis, protein synthesis, and nucleic acid synthesis.

[0035] As used herein, "metabolic inhibitory conditions" refers to conditions that inhibit any or all of the chemical reactions in metabolism, resulting in a reduced bacterial growth rate compared to when Shewanella oneidensis is cultured in a complete medium (e.g., LB medium, etc.) under aerobic conditions.

[0036] As used herein, typical culture conditions for Shewanella bacteria are aerobic conditions (oxygen concentration: 2-7 ppm) in a complete medium (rich conditions), at a pH of 6.0-9.0, a salt concentration of 0.1-1.0%, and a culture temperature of 30°C-37°C. In this specification, the term "complete medium" refers to a rich medium containing a nitrogen source, a carbon source, and minerals, and also containing, as specific components, proteins, peptides, amino acids, nucleic acids, nucleotides, and lipids. The proteins, peptides, amino acids, nucleic acids, nucleotides, and lipids are derived from, for example, tryptone or peptone, and extracts (e.g., yeast extract, meat extract, etc.).

[0037] The metabolic inhibition conditions may include, for example, one or more conditions selected from the group consisting of conditions for reducing the oxygen supply, conditions for reducing the nutrient supply, conditions for reducing the pH, conditions for reducing or increasing the salt supply, and conditions for reducing or increasing the culture temperature, compared to general culture conditions for bacteria belonging to the genus Shewanella, and preferably include one or more conditions selected from the group consisting of conditions for reducing the nutrient supply, conditions for reducing the oxygen supply, and conditions for reducing the pH.

[0038] The condition for reducing the amount of nutrient source supply may be, for example, a condition for using a nutrient-limited medium. As used herein, "nutrient-limited medium" refers to a medium that is not a complete medium, and refers to a medium that does not contain one or more selected from the group consisting of proteins, peptides, amino acids, nucleic acids, nucleotides, and lipids. The nutrient-limited medium may be, for example, a minimal medium, and is preferably a medium that does not contain a nitrogen source, and more preferably a minimal medium that does not contain a nitrogen source. As used herein, "minimal medium" refers to a medium that contains the minimum nutrients necessary for the growth of the bacterium, and is a medium that contains only inorganic substances except for sugars (e.g., glucose) as a carbon source (also referred to as an inorganic medium).

[0039] The condition of reduced oxygen supply may be, for example, an anaerobic condition. As used herein, anaerobic conditions refer to a condition in which the oxygen concentration in the culture space is sufficiently low, and may be a condition in which oxygen is completely absent or a condition in which a trace amount of oxygen is present. The anaerobic condition may be, for example, an oxygen concentration in the culture space of 1000 ppm or less, 100 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, 0.2 ppm or less, or 0 ppm.

[0040] The condition for decreasing the pH may be a condition in which the pH is 3 or more and 5.5 or less, 3 or more and 5 or less, or 3 or more and 4.5 or less.

[0041] The culture conditions for the bacteria in the culture step may be any conditions that inhibit metabolism and can be appropriately set so that the bacteria survive or grow. For example, the culture temperature may be 20°C to 40°C. The pH of the medium may be 3.0 to 9.0. The culture time may be 1 to 7 days, or 3 to 5 days.

[0042] The medium used in the culture step is not particularly limited as long as it allows the microorganism to grow. Examples include minimal media (inorganic media) such as M9 medium and MM medium, and complete media such as LB medium and 2xYT medium, with minimal media (inorganic media) being preferred.

[0043] The adipic acid content in the medium may be, for example, 0.01 g / L or more, 0.05 g / L or more, 0.1 g / L or more, 0.5 g / L or more, 1.0 g / L or more, or 1.5 g / L or more, or 25 g / L or less, 20 g / L or less, 15 g / L or less, or 10 g / L or less.

[0044] In the culturing step, the bacteria may or may not be cultured by applying a voltage. Examples of methods for culturing the microorganisms by applying a voltage include a method using an electrochemical cell having at least a pair of electrodes and a culture tank capable of accommodating a medium containing adipic acid in which the electrodes are immersed. The pair of electrodes of the electrochemical cell are connected to an external power source (voltage application device), and the bacteria can be cultured in the culture tank while applying a voltage to the electrodes.

[0045] The material for forming the electrode is not particularly limited as long as it functions as an electrode, and examples thereof include carbon (e.g., graphite felt, etc.), stainless steel, platinum, titanium, etc. The shape of the electrode is also not particularly limited, but it is preferable that the cathode be formed in a plate shape.

[0046] When bacteria are cultured by applying a voltage in the culture step, the voltage to be applied is not particularly limited, and may be set, for example, so that the potential of the working electrode relative to the reference electrode is −0.8 V or more, −0.7 V or more, −0.6 V or more, or −0.5 V or more, or may be set to −0.4 V or less, −0.5 V or less, or −0.6 V or less. Furthermore, the voltage to be applied in the culture step may be set, for example, so that the potential difference between the working electrode and the counter electrode is −1.6 V or more, −1.4 V or more, −1.2 V or more, or −1.0 V or more, or may be set to −0.8 V or less, −1.0 V or less, or −1.2 V or less.

[0047] The production method according to this embodiment may include a pre-culture step. The pre-culture step is a step of pre-culturing the bacteria before the culture step. The culture conditions for the bacteria in the pre-culture step may be any conditions that allow the bacteria to grow, and suitable culture conditions can be set as appropriate. For example, the culture temperature may be 20°C to 40°C, or 30°C to 37°C. The pH of the medium may be 6 to 8, or 7.0 to 7.5. The culture time may be 1 to 7 days, or 2 to 3 days. The same medium as that used in the culture step may be used.

[0048] The production method according to this embodiment may include a purification step. The purification step is a step of purifying the 6-HHA produced in the culture step from the culture. The culture may be the medium after the culture step, or may include the microorganism and the medium after the culture step.

[0049] In the purification step, an appropriate combination of purification methods commonly used in this technical field can be used, such as size exclusion chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, salting out, ammonium sulfate precipitation, precipitation, crystallization, etc. When the culture contains the microorganism and medium after the culture step, for example, the supernatant obtained by disrupting the microorganism and removing the cell debris by centrifugation or the like can be applied to the purification method.

[0050] [Method for improving 6-HHA production efficiency] The method for improving the production efficiency of 6-hydroxyhexanoic acid according to this embodiment (hereinafter also referred to as the "method according to this embodiment") comprises culturing a bacterium in a medium containing adipic acid under metabolically inhibiting conditions. Here, the bacterium belongs to the genus Shewanella and has a carboxylic acid reductase and an aldehyde reductase. Specific embodiments of the method can be any of the above-mentioned embodiments without particular limitation.

[0051] [Bacteria belonging to the genus Shewanella] The bacterium belonging to the genus Shewanella according to this embodiment (hereinafter also referred to as "bacterium according to this embodiment") has a carboxylic acid reductase and an aldehyde reductase. The bacterium according to this embodiment may further have a glucose kinase and a sugar permease. The bacterium belonging to the genus Shewanella, the carboxylic acid reductase, the aldehyde reductase, the glucose kinase, and the sugar permease are as described above. [Example]

[0052] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0053] [Construction of expression vector] <1. Construction of pBAD18-MYS> Based on non-patent literature (Kramer et al., 2020; Khusnutdinovaeet a., 2017), we designed codon-optimized nucleotide sequences (SEQ ID NOS: 1–3, respectively) encoding the amino acid sequences of a modified Mycobacterium avium carboxylate reductase (MavCAR N335R), a Bacillus subtilis phosphopantetheinyl transferase (Sfp), and an Escherichia coli (E. coli) aldehyde reductase (YahK). These sequences were chemically synthesized and cloned into the multiple cloning site of the expression vector (pBAD18-kan; Guzman et al., 1995) to create the expression vector (pBAD18-MYS). The nucleotide sequence encoding MavCAR N335R was ligated downstream of the araBAD promoter (Para) contained in pBAD18-kan, and the nucleotide sequences encoding YahK and Sfp were ligated downstream of it. Of these nucleotide sequences, the nucleotide sequences encoding MavCAR N335R and YahK were cloned into the region between pBAD18-kan cleaved with the restriction enzyme NheI using the Gibson assembly system. The lac promoter (Plac) from the pHSG299 plasmid (Takara Bio Inc.) was added upstream of the nucleotide sequence encoding YahK using the Gibson assembly system to promote expression of the downstream genes YahK and Sfp. The nucleotide sequence encoding Sfp was ligated into the region between the cleavage of the expression vector with the restriction enzymes XbaI and EcoRI. Figure 1 shows the vector map of the constructed expression vector (pBAD18-MYS).

[0054] 2. Construction of pBBR-glk-galP The nucleotide sequences encoding the amino acid sequences of glucose kinase (glk) and galactose permease (galP) from Escherichia coli (E. coli) (SEQ ID NOS: 4–5, respectively) were amplified by PCR using E. coli genomic DNA as a template. The resulting PCR products were cloned into the multiple cloning site of the expression vector (pBBR1MCS-5; Kovach et al., 1995, Gene 166, 175–176) to construct the expression vector (pBBR-glk-galP). The nucleotide sequence encoding glk was ligated downstream of the lac promoter contained in pBBR1MCS-5, and the nucleotide sequence encoding galP was ligated downstream of that. The nucleotide sequences encoding glk and galP were digested with the restriction enzymes KpnI and XhoI, and XhoI and PstI, respectively, and then ligated into the region between the restriction enzymes KpnI and PstI in pBBR1MCS-5.

[0055] [Creation of transformants] As described above, the expression vectors (pBAD18-MYS and pBBR-glk-galP) were constructed as hosts for the Shewanella oneidensis MR-1 strain (KlacI strain), which contained the lacI gene integrated into the chiA gene. In the KlacI strain, expression of lacI allows gene expression from the lac promoter to be induced. The expression vectors (pBAD18-MYS and pBBR-glk-galP) were introduced into the KlacI strain by conjugation with Escherichia coli to obtain a transformant (MR-1 modified strain [KlacI(pBAD18-MYS)(pBBR-glk-galP)]).

[0056] [Pre-cultivation of MR-1 modified strain and preparation of strain suspension] The MR-1 modified strain [KlacI(pBAD18-MYS)(pBBR-glk-galP)] (hereinafter simply referred to as "MR-1 modified strain") was inoculated into LB medium containing 50 μg / mL kanamycin (Km) and 15 μg / mL gentamicin (Gm) and cultured with shaking at 30°C. 600When the pH reached 0.4, L-arabinose and IPTG were added to a final concentration of 10 mM and 0.5 mM, respectively, and the culture was continued for another 3 hours with shaking. The cells were then harvested by centrifugation (7,030 × g, 5 minutes, 4°C), and the cell pellet was suspended in minimal medium (MM) and centrifuged again. This procedure was repeated three times to wash the cells. The supernatant was removed, and the cell pellet was suspended in a small amount of MM.

[0057] [Test Example 1: Conversion of adipic acid under aerobic conditions] 50 μg / mL Km, 15 μg / mL Gm, 10 mM L-arabinose, and 0.5 mM IPTG were added to 20 mL of GMM+adipic acid medium (medium with the composition shown in Table 1), and the cell suspension of the MR-1 modified strain prepared by the above method was incubated at an initial OD 600 The cells were inoculated so that the pH was 7.5 and cultured with shaking at 30°C for 24 hours. The culture medium was then centrifuged (16,200 × g, 5 minutes, 4°C), and the supernatant was collected by filtration. The concentrations of 1,6-HDO, 6-HHA, adipic acid, and glucose in the resulting culture supernatant were measured using the following method. The culture supernatant was stored at -20°C until the concentrations of the above components were measured. The culture supernatant was also measured for the concentrations of 1,6-HDO, 6-HHA, adipic acid, and glucose in the culture supernatant using the same method as above, except that a medium in which the nitrogen source was removed from the GMM+adipic acid medium (a medium with the composition listed in Table 1 except for the "nitrogen source") was used. These conditions, in which the nitrogen source was removed from the medium, were non-proliferative conditions for the cells.

[0058] The concentrations of 1,6-HDO, 6-HHA, adipic acid, and glucose in the culture medium were quantified using an HPLC (Agilent 1260 Infinity II series, Agilent Technologies) equipped with an Aminex HPX-87H column (7.8 × 300 mm). The column temperature was set to 28°C, and the mobile phase was 5 mM sulfuric acid, operated at a flow rate of 0.6 mL / min. Signals were detected using a UV detector (G7114A) set at a wavelength of 210 nm and an RI detector. The concentrations of 1,6-HDO, 6-HHA, adipic acid, and glucose in the culture supernatant were calculated based on calibration curves prepared using standard samples. The results are shown in Figures 1 and 2.

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] The MR-1 modified strain was cultured aerobically with shaking in GMM + 10 mM adipic acid medium (with or without a nitrogen source) for 24 hours. Consumption of adipic acid and glucose was confirmed, along with the production of 6-HHA and 1,6-HDO (Figures 1-2). Comparing growth and non-growth conditions, glucose consumption and 6-HHA and 1,6-HDO production were slightly enhanced under non-growth conditions compared with growth conditions. 6-HHA production was 2.3 mM under growth conditions and 2.95 mM under non-growth conditions. Meanwhile, the ratio of 6-HHA to 1,6-HDO produced was 4.8:1 under growth conditions, compared with 3.7:1 under non-growth conditions, demonstrating a higher 6-HHA production ratio under growth conditions.

[0063] Furthermore, the pH of the medium was measured before and after cultivation (Fig. 3). The pH was around 7 before cultivation, but after cultivation it decreased to around 5, indicating that the medium had become acidic. This is thought to be due to glucose and other substances in the medium being metabolized by the MR-1 modified strain and converted into organic acids such as acetic acid.

[0064] [Test Example 2: Adipic acid conversion under anaerobic conditions] A GMM+adipic acid medium (Table 4) containing 50 mM fumaric acid as an electron acceptor was added to a vial, and 50 μg / mL Km, 15 μg / mL Gm, 10 mM L-arabinose, and 0.5 mM IPTG were added. The bacterial cell suspension of the MR-1 modified strain prepared by the method described in Test Example 1 was then added to the vial at an initial OD 600 The cells were inoculated so that the pH was 3.0. The vial was sealed with a butyl rubber stopper and an aluminum cap, and then N2 gas was bubbled through it for 15 minutes to create an anaerobic environment. The cells were then cultured at 30°C. The anaerobic conditions were maintained inside the vial. Every 24 hours, 1 mL of the culture medium was centrifuged (16,200 × g, 5 minutes, 4°C), and the supernatant was collected by filtration. The concentrations of 1,6-HDO, 6-HHA, adipic acid, fumaric acid, succinic acid, acetic acid, and glucose in the resulting culture supernatant were measured using the same method as described in Test Example 1. The culture supernatant was stored at -20°C until the concentrations of the above components were measured. The results are shown in Figures 4 and 5.

[0065] [Table 4]

[0066] When the MR-1 mutant was cultured under anaerobic conditions in GMM + 10 mM adipic acid medium supplemented with fumarate (electron acceptor), 6-HHA and 1,6-HDO were produced with the consumption of adipic acid and glucose, similar to aerobic conditions (Figure 4(A)). However, the glucose consumption rate was slower than under aerobic conditions (Figure 5(A)), and 6-HHA production reached a maximum (2.7 mM) after 2 days of culture (Figure 4(B)). At this time point, the ratio of 6-HHA to 1,6-HDO produced was 11:1, indicating that anaerobic culture yielded a higher production ratio than aerobic culture. Furthermore, the concentrations of organic acids, such as acetate, in the medium were indeed confirmed to increase (Figure 5(B)). Note that fumarate can be converted to succinate by fumarate reductase (FccA), but little to acetate. Therefore, it is likely that glucose in the medium was primarily converted to acetate.

[0067] [Reference example: Conversion of adipic acid under eutrophic and aerobic conditions] 50 μg / mL Km, 15 μg / mL Gm, 10 mM L-arabinose, and 0.5 mM IPTG were added to 30 mL of GLB + adipic acid medium (LB medium supplemented with 50 mM glucose as an energy source (carbon source), 10 mM adipic acid as a substrate, and 30 mM MOPS buffer (pH 7.0)), and the MR-1 modified strain was inoculated and pre-cultured overnight with shaking at 30°C. The cells were then harvested by centrifugation (7,030 × g, 5 minutes, 4°C), and the cell pellet was suspended in LB medium and centrifuged again. This procedure was repeated three times to wash the cells. The supernatant was removed, and the cell pellet was suspended in a small amount of GLB + adipic acid medium. The cell suspension was added to 20 mL of GLB+adipic acid medium supplemented with 50 μg / mL Km, 15 μg / mL Gm, 10 mM L-arabinose, and 0.5 mM IPTG to obtain an initial OD 600 The OD of the culture medium was then increased to 1.0, and 1 mL of the culture medium was sampled every 6 hours while the medium was being shaken at 30°C. 600was measured using a spectrophotometer (UH5300, HITACHI), the culture medium was centrifuged (16,200 × g, 5 minutes, 4°C), and the culture supernatant was collected by filtration. The concentrations of 1,6-HDO, 6-HHA, adipic acid, lactic acid, acetic acid, and glucose were then measured in each of the obtained culture supernatants using the same method as in Example 1. The culture supernatants were stored at -20°C until the concentrations were measured. The results are shown in Figure 6.

[0068] When the MR-1 modified strain was cultured under aerobic conditions in GLB + adipic acid medium (rich medium), 7.2 mM of 1,6-HDO was produced 24 hours after the start of culture (Figure 6(A)). On the other hand, 6-HHA was detected transiently (approximately 1.4 mM) 12 hours after the start of culture, but then decreased, reaching 0.3 mM or less after 24 hours (Figure 6(A)). Furthermore, the pH of the culture supernatant decreased slightly with bacterial growth, but remained around 6 even 24 hours after the start of culture (Figures 7(A)-(B)).

[0069] From the results of Test Examples 1 and 2 and the Reference Example, it is believed that the metabolic activity of Shewanella oneidensis was suppressed by changing the medium composition to one that limited nutrients and / or by changing the culture to anaerobic conditions, and therefore the conversion to 1,6-HDO was suppressed. This is thought to have made it easier for the pH to decrease due to an increase in the organic acid concentration in the medium, further suppressing the metabolic activity of Shewanella oneidensis and increasing the amount of 6-HHA produced.

Claims

1. The method comprises culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically inhibited conditions, The method for producing 6-hydroxyhexanoic acid, wherein the bacterium has a carboxylic acid reductase and an aldehyde reductase.

2. 2. The method of claim 1, wherein the metabolically inhibited conditions include one or more conditions selected from the group consisting of anaerobic conditions, a condition in which the medium is a nutrient-limited medium, and a condition in which the pH of the medium is 3 or higher and 5.5 or lower.

3. The method of claim 1 or 2, wherein the medium does not contain a nitrogen source.

4. The method of claim 1 or 2, wherein the bacterium further comprises a glucose kinase and a sugar permease.

5. The method comprises culturing a bacterium belonging to the genus Shewanella in a medium containing adipic acid under metabolically inhibited conditions, The method for improving the production efficiency of 6-hydroxyhexanoic acid, wherein the bacterium has a carboxylic acid reductase and an aldehyde reductase.

6. The method of claim 5, wherein the metabolically inhibited conditions include one or more conditions selected from the group consisting of anaerobic conditions, conditions in which the medium is a nutrient-limited medium, and conditions in which the pH of the medium is 3 or higher and 5.5 or lower.

7. 7. The method of claim 5 or 6, wherein the medium does not contain a nitrogen source.

8. The method of claim 5 or 6, wherein the bacterium further comprises glucose kinase and a sugar permease.

9. A bacterium belonging to the genus Shewanella, A bacterium having carboxylic acid reductase and aldehyde reductase.

10. The bacterium of claim 9 , further comprising a glucose kinase and a sugar permease.

11. The bacterium according to claim 9 or 10, wherein the bacterium belonging to the genus Shewanella is Shewanella oneidensis.

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