Methods for measuring activity of enzyme participating hydroxy fatty acid biosynthesis, methods for screening the enzyme, and methods for detecting hydroxy fatty acid

Abstract

To provide technology for measuring activity of enzyme participating generation of hydroxy fatty acid.SOLUTION: Provided is a method for measuring the activity of an enzyme participating the production of a hydroxy fatty acid comprising the steps of: (1) culturing a microorganism expressively comprising a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein that is induced by the hydroxy fatty acid and a nucleotide sequence encoding a hydroxy fatty acid-discharging pump protein; and (2) determining the activity using the expressed amount of the reporter protein as an indicator.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to a method for measuring the activity of an enzyme involved in the production of hydroxy fatty acids, a method for screening the enzyme, a method for detecting hydroxy fatty acids, and a microorganism used in these methods. More specifically, the present disclosure relates to a method for measuring the activity of an enzyme involved in the production of hydroxy fatty acids using a biosensor, etc. [Background technology]

[0002] A gene-inducible biosensor is a biosensor that places a reporter such as a fluorescent protein downstream of the transcriptional control mechanism induced by the substance to be detected. The gene-inducible biosensor can be used to evaluate the activity of intracellular enzymes using the concentration of metabolites as an index, and can be applied to screening mutant enzymes with improved activity.

[0003] For example, Patent Document 1 discloses a method for screening enzyme activity, which comprises the following steps (see claim 13). (1) A process of introducing into a cell or adding to a cell-free protein synthesis system a nucleic acid library of fusion mutants of enzyme E and actuator A obtained by introducing mutations, insertions, and / or deletions into a gene construct carrying a gene sequence encoding all or a part of enzyme E and a gene sequence encoding actuator A, and a reporter expression vector carrying a gene sequence encoding promoter P controlled by actuator A and a gene sequence encoding reporter R functionally linked to promoter P. (2) A step of adding metabolite M to the cells or cell-free protein synthesis system of (1). (3) A step of selecting an enzyme E mutant using the expression level of a reporter R under the control of the actuator A as an index.

[0004] Metabolite biosensors must first have a sensor motif specific to the metabolite to be detected, and must also have practical performance in terms of response concentration, S / N ratio, output intensity, etc. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-136867 Summary of the Invention [Problem to be solved by the invention]

[0006] A primary object of the present disclosure is to provide a technique for measuring the activity of an enzyme involved in the production of hydroxy fatty acids using a biosensor. [Means for solving the problem]

[0007] To solve the above problems, this disclosure provides the following [1]-

[41] . [1] A method for measuring the activity of an enzyme involved in the production of hydroxy fatty acids, comprising: (1) culturing a microorganism having, in an expressible manner, a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein; (2) determining the activity using the expression level of the reporter protein in a microorganism as an index; The method includes: [2] The method of [1], wherein the culturing of the microorganism in step (1) is carried out in a microreactor containing a single-cell microorganism. [3] The method according to [2], wherein the microreactor is formed by entrapping the single-cell microorganism in a hydrophobic solvent or gel. [4] The method of [3], wherein the hydrophobic solvent is a fluorocarbon oil. [5] Any of the methods [1] to [4], wherein the concentration of the hydroxy fatty acid accumulated in the culture medium in the step (1) is greater than 1 mM. [6] Any of the methods [1] to [5], wherein the hydroxy fatty acid is 3-hydroxyisobutyric acid and / or 3-hydroxypropionic acid. [7] Any of the methods [1] to [6], wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67. [8] The microorganism has a nucleotide sequence encoding a hydroxy fatty acid responsive protein and a promoter sequence that is positively controlled by the hydroxy fatty acid responsive protein; Any of the methods according to [1] to [7], wherein the nucleotide sequence encoding the reporter protein is operably linked to the promoter sequence. [9] The method according to [8], wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

[10] The method according to [1]-[9], wherein the microorganism is Escherichia coli or a Pseudomonas bacterium.

[11] The method according to

[10] , wherein the microorganism is a Pseudomonas bacterium, and the hydroxy fatty acid responsive protein and the reporter protein are expressed using a construct consisting of any one of the nucleotide sequences of SEQ ID NOs: 12, 13, 14, or 15.

[0008]

[12] A method for screening an enzyme involved in the production of hydroxy fatty acids, comprising: (1) culturing a microorganism having, in an expressible manner, a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein; (2) measuring the expression level of the reporter protein in a microorganism; (3) isolating a microorganism having the expression level equal to or higher than a predetermined value; (4) identifying the nucleotide sequence encoding said enzyme contained in the isolated microorganism; The method includes:

[13] The method of

[12] , wherein, when the expression level is equal to or higher than a predetermined value, an enzyme that is actively involved in the production of hydroxy fatty acids is identified, and, when the expression level is equal to or lower than a predetermined value, an enzyme that is inhibitingly involved in the production of hydroxy fatty acids is identified.

[14] The method according to

[12] or

[13] , wherein the enzyme is an endogenous protein or an exogenous protein.

[15] Any of the methods

[12] -

[14] , wherein the culturing of microorganisms in step (1) is carried out in a microreactor containing single-cell microorganisms.

[16] The method of

[15] , wherein the microreactor is formed by entrapping the single-cell microorganism in a hydrophobic solvent or gel.

[17] The method of

[16] , wherein the hydrophobic solvent is a fluorocarbon oil.

[18] Any of the methods

[15] -

[17] , wherein the measurement of the expression level of the reporter protein in the step (2) is performed using a flow cytometer, and the microorganism is removed from the microreactor and subjected to the flow cytometer.

[19] Any of the methods

[12] -

[18] , wherein the concentration of the hydroxy fatty acid accumulated in the culture medium in the step (1) is greater than 1 mM.

[20] Any of the methods

[12] -

[19] , wherein the hydroxy fatty acid is 3-hydroxyisobutyric acid and / or 3-hydroxypropionic acid.

[21] Any of the methods

[12] -

[20] , wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

[22] The microorganism has a nucleotide sequence encoding a hydroxy fatty acid responsive protein and a promoter sequence that is positively controlled by the hydroxy fatty acid responsive protein; Any of the methods according to

[12] to

[21] , wherein the nucleotide sequence encoding the reporter protein is operably linked to the promoter sequence.

[23] The method according to

[22] , wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

[24] The method according to

[12] -

[23] , wherein the microorganism is Escherichia coli or a Pseudomonas bacterium.

[25] The method of

[24] , wherein the microorganism is a Pseudomonas bacterium, and the hydroxy fatty acid responsive protein and the reporter protein are expressed using a construct consisting of any one of the nucleotide sequences of SEQ ID NO: 12, 13, 14, or 15.

[0009]

[26] A method for detecting a hydroxy fatty acid, comprising: (1) contacting a microorganism having, in an aqueous medium, a hydroxy fatty acid and a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid and a nucleotide sequence encoding a hydroxy fatty acid release pump protein in an expressible manner; (2) detecting a hydroxy fatty acid in the microorganism based on expression of the reporter protein; The method includes:

[27] The method according to

[26] , wherein the concentration of the hydroxy fatty acid accumulated in the aqueous medium in the step (1) is greater than 1 mM.

[28] The method of

[26] or

[27] , wherein the hydroxy fatty acid is 3-hydroxyisobutyric acid and / or 3-hydroxypropionic acid.

[29] Any of the methods

[26] -

[28] , wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

[30] The microorganism has a nucleotide sequence encoding a hydroxy fatty acid responsive protein and a promoter sequence that is positively controlled by the hydroxy fatty acid responsive protein; Any of the methods according to

[26] -

[29] , wherein the nucleotide sequence encoding the reporter protein is operably linked to the promoter sequence.

[31] The method of

[30] , wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

[32] The method according to

[26] -

[31] , wherein the microorganism is Escherichia coli or a Pseudomonas bacterium.

[33] The method according to

[32] , wherein the microorganism is a Pseudomonas bacterium, and the hydroxy fatty acid responsive protein and the reporter protein are expressed using a construct consisting of a nucleotide sequence of any one of SEQ ID NOs: 12, 13, 14, and 15.

[0010]

[34] (i) a nucleotide sequence encoding a hydroxy fatty acid responsive protein; and (ii) a promoter sequence that is positively regulated by the hydroxy fatty acid responsive protein; (iii) a gene encoding a reporter protein operably linked to the promoter sequence; A nucleotide sequence; (iv) a nucleotide sequence encoding a hydroxy fatty acid efflux pump protein; and A microorganism having

[35] The microorganism of

[34] , wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any one of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

[36] The microorganism according to

[34] or

[35] , wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

[37] Any of the microorganisms

[34] -

[36] that are Escherichia coli or Pseudomonas bacteria.

[38] The microorganism of

[37] , wherein the microorganism is a Pseudomonas bacterium, and the hydroxy fatty acid responsive protein and the reporter protein are expressed by a construct consisting of the nucleotide sequences of SEQ ID NOs: 12, 13, 14, and 15.

[0011]

[39] (i) a nucleotide sequence encoding a hydroxy fatty acid responsive protein; and (ii) a promoter sequence that is positively regulated by the hydroxy fatty acid responsive protein; (iii) a gene encoding a reporter protein operably linked to the promoter sequence; A nucleotide sequence; (iv) a nucleotide sequence encoding a hydroxy fatty acid efflux pump protein; and A cell having

[40] The cell of

[39] , wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any one of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

[41] The cell of

[39] or

[40] , wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

[0012] In this disclosure, "microorganism" refers, according to the general definition, to all types of bacteria, archaea, prokaryotes such as cyanobacteria, eukaryotes such as yeasts, filamentous fungi, slime molds, protists, and viruses, as well as to undifferentiated cells and tissue cultures of animals and plants. A "derived strain" is generally a strain that is derived from a given microbial strain, either naturally or by chemical or physical treatment. "Culturing" means growing a microorganism in an artificial environment. The term "aqueous medium" refers to water, or aqueous solutions and gels (agar) that contain water as the main component, and also includes those in which undissolved liquids or solids are dispersed. "Contact" means exposing the microorganism to a substance for a period of time, even if the microorganisms grow. Effect of the Invention

[0013] The present disclosure provides techniques for measuring the activity of enzymes involved in the production of hydroxy fatty acids using biosensors. [Brief description of the drawings]

[0014] [Figure 1] The structures of the biosensor plasmid pSEN403 (A) and the control plasmid pSEN403 (B) are shown. [Diagram 2] The graph shows the response when acetic acid, lactic acid, 3-hydroxypropionic acid (3HPA), 3-hydroxyisobutyric acid (3HIBA), isobutyric acid (IBA), methacrylic acid (MAA), or 2-ketoisobutyric acid (KIV) was added to E. coli biosensor cells as a ligand. The vertical axis of the graph shows the ratio of the response value under ligand addition conditions to the response value under non-ligand addition conditions. [Diagram 3] The growth curve (A) of Pseudomonas biosensor cells (cells carrying the biosensor plasmid pSEN403) and the response to 2 mM 3HIBA (B) over time are shown. For comparison, the results for cells carrying the control plasmid pSEN403 are also shown. [Figure 4] (A) shows the response of Pseudomonas biosensor cells to different concentrations of 3HIBA. (B) shows the response when acetic acid, 3HPA, 3HIBA, IBA, MAA, or KIV was added to the Pseudomonas biosensor cells as a ligand. The vertical axis of the graph shows the ratio of the response value under the ligand addition condition to the response value under the non-addition condition. [Diagram 5] 1 shows growth curves (A) of Pseudomonas strains carrying biosensor plasmids pSEN431, pSEN432, pSEN437, and pSEN438, and changes in response values ​​over time under conditions in which 3HIBA was not added (B) and was added (C). [Figure 6] The response concentration range for Pseudomonas strain 3HIBA carrying the biosensor plasmid pSEN431 is shown. (A) shows the response value, and (B) shows the ratio of the response value under ligand addition conditions to the response value under non-addition conditions. [Figure 7] 3 shows the construction of the HIBA efflux pump expression plasmid. [Figure 8] 1 shows the response values ​​of 3HIBA efflux pump-introduced Pseudomonas biosensor cells to different concentrations of 3HIBA. [Figure 9] 1 shows the response values ​​of 3HIBA efflux pump-introduced Pseudomonas biosensor cells to different concentrations of 3HIBA. [Figure 10] The metabolic pathway that produces 3HIBA and the genes involved in the reaction (mmsB, AlsS, IlvC, IlvD, BCKDH, ACD, Ech) are shown. [Figure 11] The figures show the results of evaluating the 3HIBA production amount (A), 3HIBA responsiveness (B), and proliferation (C) of HIBA-releasing pump-introduced Pseudomonas biosensor cells into which a 3HIBA-producing gene was introduced. [Figure 12] 3 This is a histogram of fluorescence intensity obtained by droplet culture of HIBA release pump-introduced Pseudomonas biosensor cells into which a HIBA production gene has been introduced, and analyzing the cells after the droplets are destroyed using a flow cytometer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and the scope of the present disclosure should not be interpreted as being narrow.

[0016] 1. Biosensors The microorganism disclosed herein is a genetically induced hydroxy fatty acid biosensor, and is capable of expressing a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein. More specifically, the microorganisms disclosed herein include those that (i) express a hydroxy fatty acid responsive protein. (ii) a nucleotide sequence that positively regulates said hydroxy fatty acid responsive protein; (iii) a promoter sequence operably linked to the promoter sequence, (iv) a nucleotide sequence encoding a hydroxy fatty acid efflux pump. and a nucleotide sequence encoding the protein.

[0017] When a hydroxy fatty acid is present in a microorganism, the hydroxy fatty acid responsive protein binds to the hydroxy fatty acid, activates the promoter sequence, and expresses the reporter protein. Thus, in the microorganism according to the present disclosure, the presence of a hydroxy fatty acid in the microorganism is detected by the expression of the reporter protein.

[0018] Hydroxy fatty acid release pump proteins transport hydroxy fatty acids in microorganisms to the outside of the cells. By transporting hydroxy fatty acids in the cells to the outside of the cells using hydroxy fatty acid release pump proteins, the detectable range of extracellular hydroxy fatty acid concentrations (working range) can be expanded to the upper limit side. The upper limit of the working range when the hydroxy fatty acid efflux pump protein is expressed is, for example, 4-fold, preferably 20-fold, more preferably 50-fold, compared to when the hydroxy fatty acid efflux pump protein is not expressed. The working range is, for example, 0.1-1 mM when the hydroxy fatty acid efflux pump protein is not expressed, and, for example, from greater than 1 mM to 4 mM or from greater than 1 mM to 10 mM, preferably 5-20 mM or 5-50 mM, more preferably 5-100 mM or 5-140 mM, when the hydroxy fatty acid efflux pump protein is expressed.

[0019] In the present disclosure, the hydroxy fatty acid is preferably a short-chain fatty acid. The short-chain fatty acid may be, for example, 3-hydroxyisocaproic acid, 3-hydroxyvaleric acid, 3-hydroxyisovaleric acid, 3-hydroxyisobutyric acid (3HIBA), 3-hydroxypropionic acid, 3-hydroxybutyric acid, 2-hydroxyisocaproic acid, 2-hydroxyvaleric acid, 2-hydroxyisovaleric acid, 2-hydroxyisobutyric acid, 2-hydroxybutyric acid, lactic acid, etc., and is preferably 3-hydroxyisobutyric acid (3HIBA) and / or 3-hydroxypropionic acid (3HPA). The microorganism according to the present disclosure may be a biosensor that shows a high response, particularly to 3-hydroxyisobutyric acid.

[0020] The hydroxy fatty acid responsive protein may be a LysR type transcription factor protein, preferably mmsR. The promoter for expressing the hydroxy fatty acid responsive protein may be a general-purpose constitutive expression promoter (such as the J23116 promoter).

[0021] As the promoter sequence positively regulated by a hydroxy fatty acid responsive protein, a promoter sequence regulated by a LysR type transcription factor protein can be used, and preferably, the mmsA promoter sequence can be used. The mmsA promoter is activated by mmsR.

[0022] The reporter protein is not particularly limited and may be a conventionally known reporter protein, for example, an antibiotic resistance gene or a fluorescent protein. As an antibiotic resistance gene product, a tetracycline resistance gene tetA or the like may be preferably used. As a fluorescent protein, Green Fluorescence Protein (GFP), Red Fluorescence Protein (RFP), Yellow Fluorescence Protein YFP, tdTomato, mCherry, or the like may be preferably used.

[0023] As the hydroxy fatty acid release pump protein, any known protein may be used, but preferably, a protein encoded by any of the nucleotide sequences set forth in SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67 is used.

[0024] The microorganism according to the present disclosure can be prepared by introducing the nucleotide sequence and promoter sequence encoding the above-mentioned hydroxy fatty acid responsive protein, reporter protein, and hydroxy fatty acid release pump protein into a suitable host microorganism. The gene introduction may be performed by a conventionally known molecular biology technique.

[0025] The host microorganism is not particularly limited, but may be, for example, Escherichia coli, Pseudomonas bacteria, and the like.

[0026] The host microorganism is preferably a bacterium of the genus Pseudomonas, and Pseudomonas putida can be particularly preferably used. When a Pseudomonas bacterium is used as the host microorganism, the hydroxy fatty acid responsive protein and the reporter protein are preferably introduced by a construct consisting of any one of the nucleotide sequences of SEQ ID NOs: 12, 13, 14, and 15. The construct consisting of the nucleotide sequence of SEQ ID NOs: 12, 13, 14, and 15 can be introduced into a Pseudomonas bacterium to improve growth properties and enhance responsiveness to hydroxy fatty acids.

[0027] 2. Hydroxy fatty acid detection method The microorganism according to the present disclosure can be used as a hydroxy fatty acid biosensor for detecting hydroxy fatty acids. The method for detecting hydroxy fatty acids includes the following steps. (1) A procedure in which a microorganism having, in an expressible state, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid and a nucleotide sequence encoding a hydroxy fatty acid release pump protein is contacted with a hydroxy fatty acid in an aqueous medium. (2) detecting a hydroxy fatty acid in the microorganism based on the expression of the reporter protein.

[0028] Step (1) involves contacting a microorganism according to the present disclosure with a hydroxy fatty acid in an aqueous medium. When hydroxy fatty acids are present in the aqueous medium, the hydroxy fatty acid response protein binds to the hydroxy fatty acid taken up into the cells, activating the promoter sequence and causing the reporter protein to be expressed. The expression of this reporter protein is detected based on a signal (such as fluorescence) from the reporter protein in step (2), allowing the detection of hydroxy fatty acids.

[0029] The detectable hydroxy fatty acid concentration range (working range) is the concentration of hydroxy fatty acid in the aqueous medium, for example 1-4 mM or 1-10 mM, preferably 5-20 mM or 5-50 mM, more preferably 5-100 mM or 5-140 mM. It could be.

[0030] 3. Method for measuring activity of enzyme involved in production of hydroxy fatty acids and screening method The method for detecting hydroxy fatty acids according to the present disclosure can be applied to methods for measuring the activity and screening of enzymes involved in the production of hydroxy fatty acids. The method for measuring the activity of an enzyme involved in the production of hydroxy fatty acids includes the following steps. (1) A procedure for culturing a microorganism having a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein. (2) A procedure for determining the activity using the expression level of the reporter protein in a microorganism as an index. Furthermore, the screening method for an enzyme involved in the production of hydroxy fatty acids includes the following steps. (1) A procedure for culturing a microorganism having a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein. (2) A procedure for measuring the expression level of the reporter protein in a microorganism. (3) A procedure for isolating microorganisms having the expression level above or below a predetermined value. (4) Identifying the nucleotide sequence encoding said enzyme contained in the isolated microorganism.

[0031] The nucleotide sequence encoding the enzyme involved in the production of hydroxy fatty acids may be endogenous to the microorganism (wild-type enzyme) or exogenous to the microorganism. By introducing an enzyme library to be measured for activity into the microorganism according to the present disclosure, the enzyme library can be used for screening of an enzyme having a desired activity.

[0032] Step (1) is a process for culturing a microorganism, during which hydroxy fatty acids are produced under the action of endogenous or exogenous enzymes contained in the microorganism.

[0033] Microorganisms may be cultured by a conventional method, but are preferably cultured in a microreactor containing a single-cell microorganism. In enzyme screening, one or more enzymes that can exhibit different activities and constitute an enzyme library are genetically introduced into each cell of the microorganism. Hydroxy fatty acids are produced in each microbial cell under the action of the enzyme introduced into the cell, and in the culture in a single-cell microreactor, hydroxy fatty acids are accumulated in each cell in an amount that independently reflects the action of the enzyme in the cell. If cells are cultured in bulk, the hydroxy fatty acids produced by one cell may be taken up by other cells, and the amount of hydroxy fatty acids in an individual cell may not directly reflect the action of the enzyme within that cell. In addition, due to the proliferation of microorganisms, two or more cells may be contained in the microreactor. In this case, the two or more cells are clonal cells and can be regarded as one cell from the viewpoint of the action of intracellular enzymes.

[0034] Single cell culture using a microreactor may be performed by a conventional method, such as a hydrophobic solvent of fluorocarbon oil such as HFE7500 ("Using gel microdroplets to develop a simple high-throughput screening platform for oleaginous microorganisms", Tanaka et al., Journal of Biotechnology, 2022, Vol. 358, p. 46-54HYPERLINK), or a method of conjugating cells with a hydrophilic gel such as PEG or dextran ("High-throughput selection of cells based on accumulated growth and division using PicoShell particles", Zee et al. 2022, 119 (4) e2109430119). For example, a method of forming a microreactor using fluorocarbon oil is known in which a flow path for a solution containing cells and a flow path for fluorocarbon oil are formed in a microchip, and the cell solution flow is sandwiched between a sheath flow of fluorocarbon oil at the confluence of these flow paths. By entrapping the cells with a hydrophobic solvent or gel, it is possible to prevent the hydroxy fatty acids produced by the cells from permeating out of the microreactor, and to accumulate an amount of hydroxy fatty acids within the microreactor that reflects the action of enzymes within each cell.

[0035] In step (2), the expression level of the reporter protein is measured, and the activity of the enzyme is determined using the expression level as an index. For example, if the expression level of the reporter protein is increased in cells into which a nucleotide sequence encoding the enzyme has been introduced compared to cells into which the nucleotide sequence has not been introduced, the enzyme can be determined to have promoting activity in the production of hydroxy fatty acids. On the other hand, if the expression level of the reporter protein is decreased in cells into which the nucleotide sequence encoding the enzyme has been introduced compared to cells into which the nucleotide sequence has not been introduced, the enzyme can be determined to have inhibitory activity in the production of hydroxy fatty acids. The promoting or inhibitory activity of the enzyme can be quantitatively evaluated based on the expression level of the reporter protein.

[0036] Furthermore, for example, if the expression level of the reporter protein is increased in a cell into which enzyme 1 constituting an enzyme library has been introduced compared to a cell into which another enzyme 2 has been introduced, enzyme 1 can be determined to have a promoting activity on the production of hydroxy fatty acids compared to enzyme 2, and conversely, enzyme 2 can be determined to have an inhibitory activity on the production of hydroxy fatty acids compared to enzyme 1. Furthermore, for example, if the expression level of the reporter protein is increased to a certain standard value or higher in a cell into which enzyme 3 has been introduced, enzyme 3 can be determined to have a promoting activity on the production of hydroxy fatty acids, and if the expression level of the reporter protein is reduced to below that standard value, enzyme 3 can be determined to have an inhibitory activity on the production of hydroxy fatty acids.

[0037] The expression level of the reporter protein can be measured by a flow cytometer. The microorganisms cultured in the microreactor are removed from the microreactor for analysis by a flow cytometer. The cells can be removed from the microreactor by, for example, breaking the oil droplets or gel capsules by static electricity.

[0038] In step (3), microorganisms having a reporter protein level above or below a predetermined level as described above are isolated. The isolation of microorganisms can be carried out by cell sorting following measurement using a flow cytometer.

[0039] In step (4), by identifying the nucleotide sequence encoding the enzyme contained in the isolated microorganism, the enzyme involved in promoting or inhibiting the production of hydroxy fatty acids can be identified. In this case, the biosensor according to the present disclosure can expand the range of detectable intracellular hydroxy fatty acid concentrations (working range), making it possible to identify enzymes that have high stimulatory or inhibitory activity due to the production of hydroxy fatty acids. It is also possible to isolate microorganisms in which the expression level of the reporter protein is equal to or higher than a predetermined value as high hydroxy fatty acid producers in step (3), or to isolate microorganisms in which the expression level of the reporter protein is equal to or lower than a predetermined value as low hydroxy fatty acid producers, without carrying out step (4). Since microorganisms capable of producing the target hydroxy fatty acid can be separated according to the expression level of the reporter protein, it is also possible to sort a population of microorganisms having different production capabilities into multiple subpopulations based on their production capabilities. EXAMPLES

[0040] [Example 1: Preparation of 3-hydroxyisobutyric acid-responsive biosensor cells] 1. Biosensor Plasmid Construction To create a 3-hydroxyisobutyric acid (3HIBA)-responsive biosensor, we constructed a biosensor cell using mmsR, a 3-hydroxypropionic acid (3HPA)-responsive protein that is an organic acid similar to 3HIBA, and the promoter of the mmsA gene that is activated by mmsR.

[0041] A Green Fluorescence Protein (GFP) gene fragment (SEQ ID NO: 2) linked to a 160 bp upstream region of the mmsA gene containing the mmsA promoter sequence (SEQ ID NO: 1), a constitutive expression promoter J23116 promoter sequence (SEQ ID NO: 3), and an mmsR gene fragment (SEQ ID NO: 4) were artificially synthesized. The artificially synthesized gene fragment was linked to a backbone plasmid having a kanamycin resistance gene and a replication origin pBR1 using NEBuilder (New England Bio Labs). The resulting DNA fragment was used to transform E. coli JM109 strain, which was then statically cultured in LB agar medium containing 50 μg / ml kanamycin to obtain a transformant. Plasmid DNA was extracted and purified from the resulting transformant to obtain the biosensor plasmid pSEN403 (SEQ ID NO: 5, see FIG. 1).

[0042] In addition, pSEN403 was treated with the restriction enzyme AgeI to remove the region from the GFP gene to the mmsR gene, and then self-ligated to construct a control plasmid pSEN421 (SEQ ID NO: 6) (see FIG. 1). The control plasmid pSEN421 does not have biosensor activity, so it was used in the test as a negative control.

[0043] 2. Confirmation of response in E. coli The biosensor plasmid pSEN403 was used to transform competent cells of Escherichia coli JM109 (Takara Bio Inc.) to prepare biosensor cells of E. coli.

[0044] The biosensor cells were precultured overnight at 30°C using liquid LB medium. Then, main culture was started in 0.2 ml of inorganic salt medium containing 1xM9 minimal salts, 0.5 g / L yeast extract, 5 g / L glucose, 0.022 g / L calcium chloride, and 0.12 g / L magnesium sulfate, to which 50 mg / L kanamycin had been added. A 96-well plate was used as the culture vessel, and the cells were inoculated so that the initial OD630 was 0.04. Main culture was performed at 30°C with shaking.

[0045] After 3 hours of incubation, one of the following ligands was added at a concentration of 2 mM: acetic acid, lactic acid, 3-hydroxypropionic acid (3HPA), 3-hydroxyisobutyric acid (3HIBA), isobutyric acid (IBA), methacrylic acid (MAA) or 2-ketoisobutyric acid (KIV). After overnight incubation, the turbidity (OD630) and fluorescence activity (excitation wavelength 485 nm, detection wavelength 585 nm) were measured using a plate reader, and the fluorescence activity value per turbidity (fluorescence value / turbidity) was calculated as the "response value." The value was calculated from the average value of three independent samples.

[0046] The results are shown in Figure 2. The figure shows the response value under the ligand addition condition as a factor of the response value under the non-addition condition. The biosensor cells showed a response value 1.1 times higher under the 3HPA addition condition than under the non-addition condition, and a response value 1.8 times higher under the 3HIBA addition condition than under the non-addition condition. On the other hand, no increase in the response value was observed for butyric acid, lactate, MAA, KIV, and IBA. This result demonstrated that the biosensor cells function as a biosensor for fatty acids having hydroxyl groups.

[0047] 3. Confirmation of response in Pseudomonas Responsiveness was confirmed using Pseudomonas bacteria as a host for evaluating the biosensor function. The Pseudomonas bacteria used was a 3HIBA-producing bacterium, a mmsB gene disruptant encoding 3-hydroxyisobutyric acid dehydrogenase (Pseudomonas putida KT2440ΔmmsB, "Multistep Synthesis of (S)-3-Hydroxyisobutyric Acid from Glucose using Pseudomonas taiwanensis VLB120 B83 T7 Catalytic Biofilms", Lang et. al., Synthesis & Catalysis, 2015, Vol.357, Issue 8, p. 1919-1927). P. putida KT2440ΔmmsB strain was cultured in LB medium at 30°C with shaking until it reached the logarithmic growth phase. The resulting bacteria were collected and suspended in a 10% sucrose solution to prepare competent cells. The competent cells were transformed with the biosensor plasmid pSEN403 by electroporation to prepare biosensor cells of the P. putida KT2440ΔmmsB strain. The biosensor cells were cultured overnight at 30°C in liquid LB medium, and then inoculated at an initial turbidity of 0.04 into 0.3 ml of a culture solution containing 50 mg / L kanamycin in evaluation medium (1xM9 minimal salts, 0.5 g / L yeast extract, 5 g / L glucose, 0.022 g / L calcium chloride, and 0.12 g / L magnesium sulfate) to initiate main culture. Main culture was performed at 30°C with shaking.

[0048] After 3 hours of incubation, one of the following ligands was added: acetic acid, 3-hydroxypropionic acid (3HPA), 3-hydroxyisobutyric acid (3HIBA), isobutyric acid (IBA), methacrylic acid (MAA), or 2-ketoisobutyric acid (KIV). After overnight incubation, the turbidity (OD600) and fluorescence activity (excitation wavelength 485 nm, detection wavelength 585 nm) were measured using a plate reader, and the fluorescence activity value per turbidity (fluorescence value / turbidity) was calculated as the "response value." The value was calculated from the average value of three independent samples.

[0049] It was confirmed that the bacteria harboring the biosensor plasmid pSEN403 tended to grow slower than the bacteria harboring the control plasmid pSEN421 (see FIG. 3A). By incubating the bacteria with 2 mM 3HIBA, the response value increased over time in the bacteria carrying the biosensor plasmid pSEN403 (see Figure 3B). The bacteria carrying the biosensor plasmid pSEN403 showed a concentration-dependent response in the 3HIBA concentration range of 0.1-2 mM (see Figure 4A).

[0050] However, in the bacteria harboring the biosensor plasmid pSEN403, a gradual increase in the response value was observed over time even in the absence of 3HIBA (see Figure 3B). In addition, the ligand selectivity was relatively broad, and the bacteria were responsive not only to 3HIBA and 3HPA, but also to IBA, KIV, and MAA (Figure 4B).

[0051] 4. Improved response in Pseudomonas Although the pSEN403-harboring cells were responsive to 3HIBA, they had low proliferation rate and showed a gradual increase in response value over time even in the absence of ligand addition. Therefore, we improved the biosensor cells using Pseudomonas bacteria as a host. Specifically, the biosensor plasmid was optimized by introducing random mutations into the promoter region of the biosensor plasmid pSEN403 (a 300 bp region including the J23116 promoter sequence and the mmsA promoter sequence from the start codon of the mmsR gene to the start codon of the GFP gene: SEQ ID NO: 7). The artificially synthesized randomly mutated sequence was used as a template, and a forward primer (SEQ ID NO: 8) and a reverse primer (SEQ ID NO: 9) for the non-translated sequence were used to amplify the DNA fragments by PCR. The pSEN403 was used as a template, and a forward primer (SEQ ID NO: 10) and a reverse primer (SEQ ID NO: 11) for amplifying the vector sequence were used to amplify the DNA fragments by PCR. Each DNA fragment was ligated using NEBuilder (New England Bio Lab). The resulting DNA fragments were used to transform E. coli JM109 strain, and then the transformants were obtained by static culture in LB agar medium containing 50 μg / ml kanamycin. Plasmid DNA was extracted and purified from the resulting transformants to obtain a biosensor plasmid library.

[0052] The P. putida KT2440ΔmmsB strain was transformed by electroporation using the randomly mutated biosensor plasmid library. 0.5 μg of plasmid DNA was mixed with 100 μL of competent cell suspension, and transformation was performed under conditions of a voltage of 2500 V and a resistance value of 200 ohms, resulting in the production of 1.8 × 10 3 A library of transformants was obtained. The transformant library suspension was pre-cultured overnight in 50 ml of the aforementioned mineral salts medium. The pre-culture solution was diluted 100-fold and main cultured in 1 ml of mineral salts medium at 30°C and 200 rpm. After 2 hours of main culture, 3HIBA (final concentration 2 mM) was added as necessary and cultured for an additional 7 hours.

[0053] Cell sorting was carried out using a flow cytometer SH800 (Sony). As the first screening, cells cultured without 3HIBA were sorted to obtain fractions with low fluorescent activity. The obtained fraction was pre-cultured in the same manner, and as a second screening, cells cultured in the presence of 2 mM 3HIBA were sorted to obtain a fraction with high fluorescent activity. Colonies were isolated from the fractions obtained in the second screening and cultured overnight at 30°C in liquid LB medium. Then, the cells were inoculated into 1 ml of evaluation medium at an initial turbidity of 0.04 to start main culture, and growth and fluorescence activity were confirmed. The isolated strains were named pSEN431-carrying strain, pSEN432-carrying strain, pSEN437-carrying strain, and pSEN438-carrying strain. The nucleotide sequences of the biosensor plasmids pSEN431, pSEN432, pSEN437, and pSEN438 carried by each isolated strain are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0054] The results are shown in Figure 5. It was confirmed again that the pSEN403-containing strain exhibited lower growth rate compared to the control vector pSEN421-containing strain (see Figure 5A). On the other hand, the growth rate of the pSEN431-, pSEN432-, pSEN437- and pSEN438-containing strains was improved to the same level as that of the pSEN421-containing strain (see Figure 5A). It was confirmed that the growth inhibition that occurred in the pSEN403-containing strain was relieved by introducing a mutation into the promoter region of the biosensor plasmid pSEN403.

[0055] In the pSEN403-carrying strain, a gradual increase in the response value over time was again confirmed even in the absence of 3HIBA (see Figure 5B). On the other hand, in the pSEN431-, pSEN432-, pSEN437-, and pSEN438-carrying strains, no increase in the response value was observed in the absence of 3HIBA (see Figure 5B), but in the presence of 3HIBA, the response value increased until 9 hours after the addition and was then maintained at a constant level (see Figure 5C). The responsiveness of the pSEN403-carrying strain was improved by introducing a mutation into the promoter region of the biosensor plasmid pSEN403.

[0056] Furthermore, the concentration dependence of the 3HIBA response was evaluated for the pSEN431-carrying strain. The response concentration range (working range) was wider than that of the pSEN403-carrying strain, being 0.1-10 mM (see FIG. 6).

[0057] [Example 2: Preparation of 3-hydroxyisobutyric acid release pump-introduced biosensor cells] In the 3-hydroxyisobutyric acid-responsive biosensor cells prepared in Example 1, when Pseudomonas bacteria were used as hosts, the concentration range of 3HIBA that could be quantitatively detected was 0.1-2 mM. In this example, improvements were made to expand the quantitative detection range to higher concentrations. Specifically, we attempted to appropriately regulate the expression level of a reporter protein under high-concentration 3HIBA conditions by expressing a 3HIBA efflux pump in cells.

[0058] As a candidate for the 3HIBA efflux pump, we examined YohJK ("Systems evaluation reveals novel transporter YohJK renders 3-hydroxypropionate tolerance in Escherichia coli", Scientific Reports, 2020, 10, 19064), which is known to be a 3-hydroxypropionate (3HPA) efflux pump. In addition, we selected 18 types of YohJK homologue proteins from the database as candidates for the 3HIBA efflux pump. DNA fragments encoding the candidate proteins (Table 1) were artificially synthesized.

[0059] [Table 1]

[0060] The artificially synthesized DNA fragment was ligated to a backbone plasmid having a streptomycin resistance gene and a replication origin RK2 using NEBuilder (New England Bio Labs). The resulting DNA fragment was used to transform E. coli JM109 strain, which was then statically cultured on LB agar medium containing 50 μg / ml of spectinomycin to obtain a transformant. Plasmid DNA was extracted and purified from the resulting transformant to obtain a 3HIBA release pump expression plasmid (see Figure 7).

[0061] The pSEN431-carrying cells of P. putida KT2440ΔmmsB obtained in Example 1 were transformed by electroporation using the 3HIBA efflux pump expression plasmid. A transformant in which the biosensor plasmid and the 3HIBA efflux pump expression plasmid coexisted was prepared by selection on an agar medium containing 50 μg / ml kanamycin and 150 μg / ml streptomycin. The obtained transformant was used as a 3HIBA efflux pump-introduced biosensor cell in the subsequent experiments.

[0062] FIG. 8 shows the results of evaluating the concentration-dependent response to 3HIBA using 3HIBA release pump-introduced biosensor cells. In cells not expressing 3HIBA efflux pump (pHIB100), the quantitative detectable range was about 0.1-1.0 mM (see FIG. 8). On the other hand, in cells transfected with 10 types of 3HIBA efflux pump expression plasmids (pHIB103, pHIB105, pHIB109, pHIB111, pHIB112, pHIB113, pHIB115, pHIB116, pHIB117, and pHIB118), it was confirmed that the 3HIBA concentration could be detected as a concentration-dependent fluorescence value up to 20 mM (see FIG. 8). The nucleotide sequences of the 3HIBA efflux pump non-expressing plasmid pHIB100 and the 3HIBA efflux pump expression plasmids pHIB103, pHIB105, pHIB109, pHIB111, pHIB112, pHIB113, pHIB115, pHIB116, pHIB117, and pHIB118 are shown in SEQ ID NOs: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, and 83, respectively.

[0063] The concentration-dependent response to 3HIBA was evaluated in detail for the representative pumps pHIB105, pHIB113, and pHIB115, and the results are shown in Figure 9. It was shown that the working range of the 3HIBA sensor cells could be expanded to the high concentration side up to 140 mM by expression of the 3HIBA efflux pump protein gene.

[0064] [Example 3: Functional evaluation of biosensor in 3-hydroxyisobutyric acid producing strain] The biosensor was activated in the strain expressing the 3HIBA-producing gene, and the correlation between the amount of 3HIBA produced by the bacteria and the fluorescence value was evaluated.

[0065] In order to produce 3HIBA in P. putida, it is necessary to disrupt the 3HIBA degrading enzyme gene mmsB and introduce the AlsS, IlvC, and IlvD genes (see Figure 10). Plasmids for expressing AlsS, IlvC, and IlvD were introduced into the mmsB gene disruptant P. putida KT2440ΔmmsB. The AlsS, IlvC, and IlvD genes were linked under the control of a single promoter. Four types of promoter sequences were selected from the Anderson promoter series, which are constitutively expressed promoters with different strengths (J23111, J23117, J23101, and J2107). Plasmids were constructed by incorporating one of the four types of plasmids upstream of the cassettes of the AlsS gene, the IlvC gene, and the IlvD gene, and the plasmids were named pEUR012, pEUR013, pEUR014, and pEUR015, respectively. The DNA sequences of pEUR012, pEUR013, pEUR014, and pEUR015 are shown in SEQ ID NOs: 84, 85, 86, and 87, respectively. The expression plasmids pEUR012, pEUR013, pEUR014, and pEUR015 for AlsS, IlvC, and IlvD were introduced into the P. putida EUR010 strain harboring the biosensor plasmid pSEN431 and the efflux pump expression plasmid pHIB105, to obtain the pEUR012, pEUR013, pEUR014, and pEUR015 strains, respectively. The resulting transformants were cultured in test tubes, and growth, fluorescence intensity, and 3HIBA accumulation concentration were measured.

[0066] The results are shown in Figure 11. Accumulation of 3HIBA over time was observed in all transformants (see Figure 11A). The response value (fluorescence activity per turbidity) also increased over time in association with the accumulation of 3HIBA (see Figure 11B).

[0067] [Example 4: Isolation of 3HIBA high-producing strains using a flow cytometer] The strains pEUR012, pEUR013, pEUR014 and pEUR015, which were found to have different 3HIBA productivity, were mixed and cultured, and attempts were made to isolate high 3HIBA producers by sorting using the level of 3HIBA productivity as an indicator.

[0068] A mixed culture of pEUR012, pEUR013, pEUR014 and pEUR015 strains was droplet-conjugated using fluorocarbon oil and cultured overnight. One droplet conjugates one cell to form a nano-scale bioreactor. 3HIBA produced by the cells is accumulated in the nano-scale bioreactor.

[0069] The droplets were destroyed by treating the droplet culture solution with a static electricity generator. The aqueous layer of the solution from which the droplets were destroyed was diluted with PBS buffer and the fluorescence value was measured using a flow cytometer SH800.

[0070] The histograms obtained by flow cytometer analysis are shown in Figure 12. Cells were isolated from the low and high activity fractions, and their sequences were confirmed. It was demonstrated that by combining the biosensor and droplet culture, it is possible to separate cell populations based on their 3HIBA production ability. [Sequence List Free Text]

[0071] SEQ ID NO: 1: Nucleotide sequence containing the mmsA promoter SEQ ID NO:2: Nucleotide sequence of the GFP gene fragment SEQ ID NO: 3: Nucleotide sequence of the J23116 promoter sequence SEQ ID NO: 4: Nucleotide sequence of the mmsR gene fragment SEQ ID NO: 5: Nucleotide sequence of biosensor plasmid pSEN403 SEQ ID NO: 6: Nucleotide sequence of the control plasmid pSEN421 SEQ ID NO: 7: The promoter region of the biosensor plasmid pSEN403 (nucleotide sequence of the region from the start codon of the mmsR gene to the start codon of the GFP gene, including the J23116 promoter sequence and the mmsA promoter sequence) SEQ ID NO: 8: Forward primer for amplifying untranslated sequences SEQ ID NO: 9: Reverse primer for amplifying non-translated sequences SEQ ID NO: 10: Forward primer for amplifying vector sequence SEQ ID NO: 11: Reverse primer for amplifying vector sequence SEQ ID NO: 12: Nucleotide sequence of biosensor plasmid pSEN431 SEQ ID NO: 13: Nucleotide sequence of biosensor plasmid pSEN432 SEQ ID NO: 14: Nucleotide sequence of biosensor plasmid pSEN437 SEQ ID NO: 15: Nucleotide sequence of biosensor plasmid pSEN438 SEQ ID NO: 16: Nucleotide sequence of the gene AhYohJK for the HIBA efflux pump candidate protein SEQ ID NO: 17: Amino acid sequence of the YohJ homolog encoded by the gene AhYohJK SEQ ID NO: 18: Amino acid sequence of the YohK homolog encoded by the gene AhYohJK SEQ ID NO: 19: Nucleotide sequence of the gene BsYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 20: Amino acid sequence of the YohJ homolog encoded by the gene BsYohJK SEQ ID NO: 21: Amino acid sequence of the YohK homolog encoded by the gene BsYohJK SEQ ID NO: 22:3 Nucleotide sequence of the gene CkYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 23: Amino acid sequence of the YohJ homolog encoded by the gene CkYohJK SEQ ID NO: 24: Amino acid sequence of the YohK homolog encoded by the gene CkYohJK SEQ ID NO: 25: Nucleotide sequence of the gene DpYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 26: Amino acid sequence of the YohJ homolog encoded by the gene DpYohJK SEQ ID NO: 27: Amino acid sequence of the YohK homolog encoded by the gene DpYohJK SEQ ID NO: 28: Nucleotide sequence of the gene EcYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 29: Amino acid sequence of the YohJ homolog encoded by the gene EcYohJK SEQ ID NO: 30: Amino acid sequence of the YohK homolog encoded by the gene EcYohJK SEQ ID NO:31: Nucleotide sequence of the gene ElYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 32: Amino acid sequence of the YohJ homolog encoded by the gene ElYohJK SEQ ID NO: 33: Amino acid sequence of the YohK homolog encoded by the gene ElYohJK SEQ ID NO:34: Nucleotide sequence of the gene EnYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 35: Amino acid sequence of the YohJ homolog encoded by the gene EnYohJK SEQ ID NO: 36: Amino acid sequence of the YohK homolog encoded by the gene EnYohJK SEQ ID NO:37: Nucleotide sequence of the gene KpYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 38: Amino acid sequence of the YohJ homolog encoded by the gene KpYohJK SEQ ID NO: 39: Amino acid sequence of the YohK homolog encoded by the gene KpYohJK SEQ ID NO: 40: Nucleotide sequence of the gene KvYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 41: Amino acid sequence of the YohJ homolog encoded by the gene KvYohJK SEQ ID NO: 42: Amino acid sequence of the YohK homolog encoded by the gene KvYohJK SEQ ID NO: 43: Nucleotide sequence of the gene PaYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 44: Amino acid sequence of the YohJ homolog encoded by the gene PaYohJK SEQ ID NO: 45: Amino acid sequence of the YohK homolog encoded by the gene PaYohJK SEQ ID NO: 46: Nucleotide sequence of the gene PeYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 47: Amino acid sequence of the YohJ homolog encoded by the gene PeYohJK SEQ ID NO: 48: Amino acid sequence of the YohK homolog encoded by the gene PeYohJK SEQ ID NO: 49: Nucleotide sequence of the gene PhYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 50: Amino acid sequence of the YohJ homolog encoded by the gene PhYohJK SEQ ID NO: 51: Amino acid sequence of the YohK homolog encoded by the gene PhYohJK SEQ ID NO: 52: Nucleotide sequence of the gene PpYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 53: Amino acid sequence of the YohJ homolog encoded by the gene PpYohJK SEQ ID NO: 54: Amino acid sequence of the YohK homolog encoded by the gene PpYohJK SEQ ID NO: 55: Nucleotide sequence of the gene PrYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 56: Amino acid sequence of the YohJ homolog encoded by the gene PrYohJK SEQ ID NO: 57: Amino acid sequence of the YohK homolog encoded by the gene PrYohJK SEQ ID NO:58:3 Nucleotide sequence of the gene PsYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 59: Amino acid sequence of the YohJ homolog encoded by the gene PsYohJK SEQ ID NO: 60: Amino acid sequence of the YohK homolog encoded by the gene PsYohJK SEQ ID NO:61:3 Nucleotide sequence of the gene RaYohJK for the HIBA efflux pump candidate protein SEQ ID NO: 62: Amino acid sequence of the YohJ homolog encoded by the gene RaYohJK SEQ ID NO: 63: Amino acid sequence of the YohK homolog encoded by the gene RaYohJK SEQ ID NO:64:3 Nucleotide sequence of the gene SeYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 65: Amino acid sequence of the YohJ homolog encoded by the gene SeYohJK SEQ ID NO: 66: Amino acid sequence of the YohK homolog encoded by the gene SeYohJK SEQ ID NO: 67: Nucleotide sequence of the gene SpYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 68: Amino acid sequence of the YohJ homolog encoded by the gene SpYohJK SEQ ID NO: 69: Amino acid sequence of the YohK homolog encoded by the gene SpYohJK SEQ ID NO: 70: Nucleotide sequence of gene SqYohJK, a candidate HIBA efflux pump protein SEQ ID NO: 71: Amino acid sequence of the YohJ homolog encoded by gene SqYohJK SEQ ID NO: 72: Amino acid sequence of the YohK homolog encoded by gene SqYohJK SEQ ID NO:73:3 Nucleotide sequence of HIBA efflux pump non-expressing plasmid pHIB100 SEQ ID NO:74:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB103 SEQ ID NO:75:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB105 SEQ ID NO:76:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB109 SEQ ID NO:77:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB111 SEQ ID NO:78:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB112 SEQ ID NO:79:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB113 SEQ ID NO:80:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB115 SEQ ID NO:81:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB116 SEQ ID NO:82:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB117 SEQ ID NO:83:3 Nucleotide sequence of HIBA efflux pump expression plasmid pHIB118 SEQ ID NO: 84: Nucleotide sequence of plasmid pEUR012 SEQ ID NO: 85: Nucleotide sequence of plasmid pEUR013 SEQ ID NO: 86: Nucleotide sequence of plasmid pEUR014 SEQ ID NO: 87: Nucleotide sequence of plasmid pEUR015

Claims

1. 1. A method for measuring the activity of an enzyme involved in the production of hydroxy fatty acids, comprising: (1) culturing a microorganism having, in an expressible manner, a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein; (2) determining the activity using the expression level of the reporter protein in a microorganism as an index; The method includes:

2. The method according to claim 1 , wherein the culturing of the microorganism in step (1) is carried out in a microreactor containing a single-cell microorganism.

3. The method according to claim 2, wherein the concentration of the hydroxy fatty acid accumulated in the culture medium in step (1) is greater than 1 mM.

4. The method of claim 3, wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

5. The microorganism has a nucleotide sequence encoding a hydroxy fatty acid responsive protein and a promoter sequence that is positively controlled by the hydroxy fatty acid responsive protein; The method of claim 4 , wherein the nucleotide sequence encoding the reporter protein is operably linked to the promoter sequence.

6. A method for screening for an enzyme involved in the production of hydroxy fatty acids, comprising the steps of: (1) culturing a microorganism having, in an expressible manner, a nucleotide sequence encoding the enzyme, a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid, and a nucleotide sequence encoding a hydroxy fatty acid release pump protein; (2) measuring the expression level of the reporter protein in a microorganism; (3) isolating a microorganism having the expression level equal to or higher than a predetermined value; (4) identifying the nucleotide sequence encoding said enzyme contained in the isolated microorganism; The method includes:

7. The method of claim 6 , wherein the enzyme is an endogenous protein or an exogenous protein.

8. 1. A method for detecting a hydroxy fatty acid, comprising: (1) contacting a microorganism having, in an aqueous medium, a hydroxy fatty acid and a nucleotide sequence encoding a reporter protein whose expression is induced by a hydroxy fatty acid and a nucleotide sequence encoding a hydroxy fatty acid release pump protein in an expressible manner; (2) detecting a hydroxy fatty acid in the microorganism based on expression of the reporter protein; The method includes:

9. 9. The method of claim 8, wherein the detectable concentration of hydroxy fatty acid is greater than 1 mM.

10. (i) a nucleotide sequence encoding a hydroxy fatty acid responsive protein; and (ii) a promoter sequence that is positively regulated by the hydroxy fatty acid responsive protein; (iii) a gene encoding a reporter protein operably linked to the promoter sequence; A nucleotide sequence; (iv) a nucleotide sequence encoding a hydroxy fatty acid release pump protein; A microorganism having

11. The microorganism of claim 10, wherein the nucleotide sequence encoding the hydroxy fatty acid release pump protein comprises any of the nucleotide sequences of SEQ ID NOs: 22, 28, 40, 46, 49, 52, 58, 61, 64, and 67.

12. The microorganism described in claim 11, wherein the hydroxy fatty acid responsive protein is mmsR and the promoter sequence is the mmsA promoter sequence.

13. The microorganism described in claim 12, wherein the microorganism is a Pseudomonas bacterium and the hydroxy fatty acid responsive protein and the reporter protein are expressed by a construct consisting of a nucleotide sequence of any one of SEQ ID NOs: 12, 13, 14, and 15.

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