JP5839854B2 - Microbial culture method - Google Patents

Description

  The present invention relates to a method for producing polyhydroxyalkanoic acid (hereinafter referred to as PHA) by microorganisms industrially efficiently using fats and oils and / or fatty acids having a high lauric acid content as a carbon source.

  PHA is a polyester-type organic molecular polymer produced by a wide range of microorganisms. PHA is a biodegradable thermoplastic polymer. PHA can also be produced from renewable resources. For these reasons, attempts have been made to industrially produce PHA as an environmentally conscious material or a biocompatible material and use it in various industries.

  To date, many microorganisms are known to accumulate PHA as an energy storage substance in the cells. A typical example of PHA is poly-3-hydroxybutyric acid (hereinafter referred to as P (3HB)), which is a homopolymer of 3-hydroxybutyric acid (hereinafter referred to as 3HB). P (3HB) was first discovered in 1925 in Bacillus megaterium. P (3HB) is a thermoplastic polymer and is biologically decomposed in the natural environment, and thus has attracted attention as an environmentally friendly plastic. However, P (3HB) has a hard and brittle nature because of its high crystallinity, so its practical application range is limited. In order to expand the application range, it was necessary to give flexibility to P (3HB).

  Therefore, a method for producing a copolymer of 3HB and 3-hydroxyvaleric acid (hereinafter referred to as 3HV) (hereinafter referred to as P (3HB-co-3HV)) is disclosed (Patent Documents 1 and 2). ). P (3HB-co-3HV) is a kind of PHA. Since P (3HB-co-3HV) is more flexible than P (3HB), it was considered that P (3HB-co-3HV) can be applied to a wide range of uses. However, actually, even if the 3HV mole fraction in P (3HB-co-3HV) is increased, the accompanying change in physical properties is scarce, and it is particularly required for processing into films, sheets, flexible packaging containers, etc. Since flexibility does not improve as much, it is used only in limited fields such as shampoo bottles and disposable razor handles.

  In order to increase the flexibility of P (3HB), research on a copolymer of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as 3HH) (hereinafter referred to as P (3HB-co-3HH)) and its production method (Patent Documents 3 and 4). P (3HB-co-3HH) is also a kind of PHA. The production method of P (3HB-co-3HH) in these reports uses Aeromonas caviae and Aeromonas hydrophila isolated from soil, and uses fatty acids such as lauric acid, oleic acid, palmitic acid, and glucose as carbon sources. It was the method that was. P (3HB-co-3HH) obtained by any method had a 3HH composition of 10 mol% or more, and the flexibility was improved. However, since the productivity of P (3HB-co-3HH) was about 50 g / L or much lower than that, a method for further increasing the productivity for practical use has been searched (Non-Patent Documents 1 to 3). . In addition, fatty acids such as lauric acid, oleic acid, and palmitic acid used in these culture productions are expensive, so that an inexpensive carbon source has been demanded from the viewpoint of production cost.

  For this reason, in order to achieve both high flexibility and productivity using vegetable oils and fats, which are cheaper carbon sources, the following efforts have been made. A transformant in which the polyhydroxyalkanoate synthase gene cloned from A. caviae was introduced into Cupriavidus necator (formerly Ralstonia eutropha or Alcaligenes eutrophus) was used, and vegetable oils such as olive oil and palm oil were used as carbon sources. As a result, the 3HH composition was 4 to 5 mol%, and the bacterial cell amount was 4 g / L and the polymer content was 80% (Non-patent Document 4). These manufacturing methods use inexpensive vegetable oils and fats as a carbon source and have a high polymer content, but the amount of cells is low, so the polymer productivity is low and the 3HH composition is 4 to 5 mol%, and it is soft enough to be used for applications such as films. It was not a thing.

  Furthermore, 3HH composition improvement examination using fats and oils, such as palm kernel oil and palm oil, is performed. However, in this study, P (3HB-co-3HH) having a 3HH composition of 8.2 to 8.6 mol% was obtained at a productivity of 30 g / L or less in 72 hours of culture. Therefore, in order to further increase the 3HH composition, expensive hexanoic acid is mixed with oil and cultured, but the 3HH composition increases as the amount of hexanoic acid increases, but P (3HB-co-3HH) The production amount of the product significantly decreased (Patent Document 5). In addition, research on production of PHA using only palm kernel olein oil, palm kernel oil, and palm oil was also conducted (Patent Document 6). In this production method, the use of palm kernel oil, palm oil, palm kernel olein oil alone or mixed fats and oils of palm kernel olein oil and soybean oil was investigated, but P (3HB-co -3HH) productivity (72 g / L) and low productivity. In addition, when using a fat having a relatively high lauric acid content such as palm kernel oil or palm oil, especially more than 41% by weight, the production amount of P (3HB-co-3HH) decreases, and the carbon source is used. It has been difficult to achieve both a high 3HH composition (for example, 6 mol% or more, particularly 7 mol% or more, more preferably 10 mol% or more, which is highly flexible) and high productivity.

  In addition, production research of a copolymer of 3HB, 3HV, and 3HH (hereinafter referred to as P (3HB-co-3HV-co-3HH)) using a carbon source having a relatively high lauric acid content (Non-Patent Document 5) However, the examination was only at the flask level, and the productivity of P (3HB-co-3HV-co-3HH) was as low as about 4 g / L in 72 hours of culture.

  Thus, in the production of PHA such as P (3HB-co-3HH) and P (3HB-co-3HV-co-3HH), vegetable oils that are mass-produced are suitable as raw materials in consideration of economy and reproducibility. However, it is difficult to say that fats and oils having a high lauric acid content, such as the above-described palm kernel oil and palm oil, have been used effectively so far. From the viewpoint of utilizing various fats and oils and / or fatty acids as raw materials for PHA, an efficient method for producing PHA using palm kernel oil, coconut oil, lauric acid and the like has been demanded. In addition, as described above, in order to increase the 3HH composition and produce a more flexible PHA, fatty acids having a relatively small number of carbon atoms such as hexanoic acid, octanoic acid, lauric acid, and the like are included as constituents. It is preferable to use fats and oils as a carbon source, and since a large amount is contained as a constituent component of natural fats and oils such as palm oil and palm oil in particular, a method for producing PHA using these carbon sources has been demanded.

Japanese Laid-Open Patent Publication No. 57-150393 JP 59-220192 A Japanese Laid-Open Patent Publication No. 5-93049 Japanese Unexamined Patent Publication No. 7-265065 JP 2001-340078 A US Pat. No. 7,235,621

Macromolecules 28, 4822-4823 (1995). Biotechnology and Bioengineering 67, 240 (2000) Appl. Microbiol. Biotechnol. 57, 50 (2001) Appl. Microbiol. Biotechnol. 49, 333-336 (1998) Polymer Degradation and Stability 93, 17-23 (2008)

  An object of the present invention is to provide a method for effectively using a carbon source having a high lauric acid content. Specifically, the present invention provides an industrially efficient method for producing PHA, particularly P (3HB-co-3HH), using fats and / or fatty acids having a lauric acid content exceeding 41% by weight. Let it be an issue.

  The present inventors diligently studied to suitably use fats and / or fatty acids having a lauric acid content in the constituent fatty acids of more than 41% by weight for the production of PHA. As a result, it was found that by controlling the oxygen transfer rate, fats and / or fatty acids having a lauric acid content in the constituent fatty acids of more than 41% by weight can be suitably used for PHA production, and the present invention was completed.

  In the present invention, fats and oils containing lauric acid exceeding 41% by weight as constituent fatty acids and / or fatty acids are used as a carbon source, the oxygen transfer rate is controlled, and the average time productivity of PHA from the start of culture is 1.5 g / It is a culture production method of PHA controlled to L / h or more. Further, the present invention is characterized by using palm kernel oil, coconut oil, and fractionated fats and oils obtained by fractionating these fats and fats, and / or fatty acids and / or fatty acids selected from the group of fatty acids as a carbon source, This is a method for producing PHA using microorganisms.

  According to the present invention, it has become possible to effectively use fats and oils and / or fatty acids having a high lauric acid content as raw materials for PHA. Further, according to the present invention, it has become possible to produce PHA having a 3HH composition of 6 mol% or more with a productivity of 100 g / L or more by using fats and / or fatty acids having a high lauric acid content as a PHA raw material.

  Hereinafter, the present invention will be described in detail.

  Regarding the production of PHA by microorganisms, particularly PHA obtained by polymerizing monomer units containing 3HH, if the 3HH composition ratio can be controlled in a wide range, fermentation production from hard copolymers to soft copolymers will be possible. Application to a wider range of fields can be expected. Until now, the manufacturing technology of PHA using fats and / or fatty acids as raw materials has been developed using various microorganisms. However, when fats and oils with a high lauric acid content such as palm kernel oil and palm oil are used, for example, palm oil, soybean oil, cottonseed oil, rapeseed oil, corn oil, and a plurality of oils and fats so that the lauric acid content is lowered. And the fall of the productivity of PHA was recognized compared with the case where the fats and oils with low lauric acid content like the composition which mixed and prepared the fatty acid were used (US Pat. No. 7,235,621).

  However, according to the present invention, a palm kernel oil, coconut oil having a high lauric acid content in the constituent fatty acid, and a mixed preparation composition of fat and / or fatty acid mixed and prepared to have a high lauric acid content as a carbon source It can be used effectively.

  The present invention is a method for producing PHA by microorganisms obtained by polymerizing monomer units containing at least 3HB and 3HH, wherein fats and / or fatty acids containing more than 41% by weight of lauric acid as a constituent fatty acid are used as a carbon source, oxygen This is a PHA culture production method in which the average time productivity of PHA from the start of culture is controlled to 1.5 g / L / h or more by controlling the moving speed. Surprisingly, according to the present invention, by controlling the oxygen transfer rate, the productivity of PHA when using a carbon source having a high content of lauric acid is low. It has been found that the improvement is equivalent to or better than the case of using a carbon source that is not included as a component. In order to control the oxygen transfer rate, a method of controlling the aeration amount, the number of stirring, the aeration oxygen concentration, the pressure in the culture tank, and the like can be appropriately selected. The present invention controls the oxygen transfer rate so that an average time productivity of PHA of 1.5 g / L / h or more is achieved, and supplies a carbon source so as to achieve the above average time productivity. To be achieved. In the present invention, the average time productivity of PHA is calculated as B / A (g / L / h) from the PHA concentration (B (g / L)) in the culture solution at the culture end point (A (h)). . The carbon source may be added in a large amount at a time, dividedly added, continuously or intermittently fed, etc. In the present invention, continuous feeding or intermittent flow may be considered. It is preferable to add. In the present invention, the oxygen transfer rate can be determined by a sodium sulfite oxidation method, a DO electrode method, a direct method, an exhaust gas analysis method, or the like. The oxygen transfer rate (OTR) is as shown in the following equation.

OTR (molO ₂ / m ³ /h)={7.317 (Pii iQi / Ti) − (Poyo Qo / To)} / V
P is the pressure (unit: atm), y is the oxygen mole fraction (%), Q is the air flow rate (L / min), V is the liquid volume (m ³ ), and T is the gas temperature (K). Further, “i” indicates a value at the entrance of the culture tank, and “o” indicates a value at the exit of the culture tank. 7.317 is a conversion constant from the percentage notation of 60 min / h, gas constant R = 0.082, and oxygen mole fraction.

A preferable oxygen transfer rate in the present invention is 70 molO ₂ / m ³ / h or more, more preferably 80 molO ₂ / m ³ / h or more, further preferably 100 molO ₂ / m ³ / h or more, and 120 molO ₂ / m ^3. / H or more is particularly preferable.

  The oxygen transfer rate in the actual liquid does not strictly indicate the same value depending on the properties of the liquid, for example, water, medium, culture liquid, etc. In doing so, there is virtually no problem with using oxygen transfer rates measured in water. The oxygen transfer rate in the present invention is a value in water of the oxygen transfer rate from the start of culture to the end of culture.

According to our study, if the OTR measured in water is 70 molO ₂ / m ³ / h or more, it is surprising to use a carbon source having a lauric acid content of 41 wt% or more throughout the culture, As with carbon sources having a lauric acid content of 41% by weight or less, such as palm kernel olein oil and coconut oil, it was possible to produce PHA satisfactorily. However, the above conditions are only examples, and culture methods such as changing the aeration and stirring conditions during culture and culture methods that change the amount of the culture solution are also conceivable. Therefore, the average time productivity of PHA is 1.5 g / All the culture conditions for achieving L / h, particularly oxygen transfer rate conditions, are included in this patent.

  As the carbon source to be used, it is preferable to use fats and oils having a lauric acid content in the constituent fatty acid of 41% by weight or more, for example, palm kernel oil or palm oil, or lauric acid derived from vegetable fats and oils. Further, from the viewpoint of effectively using palm kernel oil and palm oil having a high lauric acid content, the lauric acid content is preferably 41% by weight or more, more preferably 42% by weight or more, and further preferably 45% by weight or more. By using, the effects of the present invention can be obtained more remarkably.

  Needless to say, not only the above fats and / or fatty acids can be used alone, but also a mixture of a plurality of fats and / or fatty acids can be used.

  Further, by using the present invention, a PHA having a 3HH composition of 6 mol% or more can be efficiently produced.

  Moreover, as a microorganism used for production of the PHA of the present invention, a vegetable oil or fatty acid containing lauric acid or a fatty acid can be assimilated, and the gene encoding the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence There is no particular limitation as long as a gene encoding a polypeptide having a sequence identity of 85% or more and having a polyhydroxyalkanoic acid polymerization activity is incorporated. Manipulated microorganisms can be preferably used. Specifically, the genus Ralstonia, the genus Capriavidus, the genus Wautersia, the genus Aeromonas, the genus Escherichia, the genus Alcaligenes, the genus B, Pseudomonas, It is preferable to use microorganisms such as genus, genus Azotobacter, genus Nocardia, genus Sphingomonas, genus Comamonas. Of course, mutant strains obtained by artificial mutation treatment of the microorganisms and strains that have been mutant-treated by genetic engineering techniques can also be used.

  An example of the microorganism belonging to the genus Capyrididas is, for example, Capriavidus necator. Examples of microorganisms belonging to the genus Alkagenes include Alcaligenes latas. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas eudonosa (ex. it can. Examples of Bacillus microorganisms include Bacillus megaterium. Examples of microorganisms belonging to the genus Aeromonas include Aeromonas caviae and Aeromonas hydrophila. Microorganisms described in Microbiological Reviews, pages 450-472 (1990) can be preferably used. In addition to these microorganisms and the like, it is also possible to use biological cells that have been modified to artificially produce PHA by introducing a PHA synthase gene or the like using genetic engineering techniques. For example, Gram-negative bacteria such as Escherichia, Gram-positive bacteria such as Bacillus, yeasts such as Saccharomyces, Yarrowia, and Candida can be used. .

  For example, the PHA synthase gene can be obtained by using a method using a microorganism that originally produces 3HB and 3HH PHA such as Aeromonas caviae and Aeromonas hydrophila, or a genetic engineering technique for a microorganism that does not originally produce 3HB and 3HH PHA. It is also possible to use biological cells that have been modified to artificially produce P (3HB-co-3HH) by introducing. As the host microorganism into which the gene is introduced, for example, Cupriavidus necator can be suitably used.

  In addition, as the PHA synthase gene, PHA synthase genes derived from Aeromonas caviae or Aeromonas hydrophila or their modified forms can be used. As the modification, a base sequence encoding a PHA synthase in which an amino acid group is deleted, added, inserted, or substituted can be used. Any gene containing a base sequence encoding a polypeptide having PHA synthase activity can be used in the present invention. For example, a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 12 can be used. In addition, a gene having a base sequence encoding a polypeptide having a sequence identity of 85% or more with the amino acid sequence of wild-type PHA synthase and having a PHA synthase activity is preferably used in the present invention. I can do it. The sequence identity is preferably 90% or more, more preferably 95% or more, and still more preferably 97% or more. The number of amino acid residues to be deleted, added, inserted or substituted is preferably 80 or less, more preferably 60 or less, still more preferably 40 or less, and even more preferably 30 or less. Yes, most preferably 20 or less, 10 or less, and 5 or less.

  The types of PHA composed of 3HB and 3HH are composed of 3HB and 3HH (P (3HB-co-3HH)), 3HB and 3HV, and 3HH (P (3HB-co-3HV-co-3HH)), 3HB, 3HH, and 4-hydroxybutyric acid (hereinafter sometimes referred to as 4HB) (P (3HB-co-3HH-co-4HB)), 3HB and 3HH, 3-hydroxyoctanoic acid (hereinafter referred to as 3HO) (P (3HB-co-3HH-co-3HO))) and the like.

  In the method for producing PHA of the present invention, PHA may be accumulated in microorganisms by the culture method described above, and then PHA may be recovered from the cells using a well-known method. For example, it can be performed by the following method. After completion of the culture, the cells are separated from the culture solution with a centrifuge, and the cells are washed with distilled water, methanol, or the like and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. The bacterial cell component is removed from the solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, a method of removing the supernatant liquid by filtration or centrifugation and drying it to recover PHA is exemplified.

  The present inventors have confirmed that the present invention can be suitably implemented on a 10 L scale, and can be suitably reproduced even on a scale of 16000 L or more. The achievement of the scale-up of 5,000 times or more means that the effects of the present invention are suitably and maximally exhibited in production on a larger industrial scale.

  Hereinafter, the present invention will be described more specifically with reference to comparative examples and examples. However, the present invention is not limited to these examples. General gene manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press, (1989)). In addition, enzymes, cloning hosts, and the like used for gene manipulation can be purchased from a market supplier and used according to the instruction manual. The enzyme is not particularly limited as long as it can be used for gene manipulation.

(Comparative Example 1)
In the experiment, the KNK-005 strain (see US Pat. No. 7,384,766) was used.

The composition of Tanehaha medium 1w / v% Meat-extract, 1w / v% Bacto-Tryptone, 0.2w / v% Yeast-extract, 0.9w / v% Na 2 HPO 4 · 12H 2 O, 0.15w / V% KH ₂ PO ₄ (pH 6.8).

The composition of the preculture medium is 1.1 w / v% Na ₂ HPO ₄ · 12H ₂ O, 0.19 w / v% KH ₂ PO ₄ , 1.29 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w / _{v% MgSO 4 · 7H 2 O} , 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl _{/ v% FeCl 3 · 6H 2} O, 1w / v% CaCl 2 · 2H 2 O, 0 0.02 w / v% CoCl ₂ .6H ₂ O, 0.016 w / v% CuSO ₄ .5H ₂ O, 0.012 w / v% NiCl ₂ .6H ₂ O). As a carbon source, palm kernel oil was added all at a concentration of 10 g / L.

The composition of the PHA production medium is 0.385 w / v% Na ₂ HPO ₄ · 12H ₂ O, 0.067 w / v% KH ₂ PO ₄ , 0.291 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w / v% MgSO ₄ .7H ₂ O, 0.5 v / v% trace metal salt solution (1.6 W / v% FeCl ₃ .6H ₂ O in 0.1N hydrochloric acid, 1 w / v% CaCl ₂ .2H ₂ O, 0 0.02 w / v% CoCl ₂ · 6H ₂ O, 0.016 w / v% CuSO ₄ · 5H ₂ O, 0.012 w / v% NiCl ₂ · 6H ₂ O), 0.05 w / v% BIOSPUREX 200K (Antifoamer: manufactured by Cognis Japan).

  First, a glycerol stock (50 μl) of the KNK-005 strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to perform seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (MDL-300, manufactured by Maruhishi Bioengine) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 33 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and a culture was performed for 28 hours while controlling the pH between 6.7 and 6.8, followed by preculture. A 14% aqueous ammonium hydroxide solution was used for pH control.

Next, 1.0 V / v% of the preculture solution was inoculated into a 10 L jar fermenter (MDS-1000, manufactured by Maruhishi Bio-Engine) containing 6 L production medium. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 400 rpm, an aeration rate of 6.0 L / min, and a pH controlled between 6.7 and 6.8. A 14% aqueous ammonium hydroxide solution was used for pH control. The carbon source was added intermittently. As fats and oils, palm kernel olein oil, palm kernel oil, and palm kernel olein oil added with 20 parts of lauric acid were used. The oxygen transfer rate in the aqueous system under the culture conditions was 70 molO ₂ / m ³ / h. Culturing was performed for 64 hours, and after completion of the cultivation, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured.

  To 1 g of the obtained dried cells, 100 ml of chloroform was added and stirred overnight at room temperature to extract PHA in the cells. The bacterial cell residue was filtered off, concentrated with an evaporator until the total volume reached 30 ml, 90 ml of hexane was gradually added, and the mixture was allowed to stand for 1 hour with slow stirring. The precipitated PHA was filtered off and dried in vacuo at 50 ° C. for 3 hours. The weight of dry PHA was measured, and the polymer content in the cells was calculated.

  The PHA production amount was calculated from the dry cell weight and polymer content. The 3HH composition analysis of the produced PHA was measured by gas chromatography as follows. To 20 mg of dry PHA, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added and sealed, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a PHA decomposition product. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize it, and the mixture was allowed to stand until the generation of carbon dioxide gas stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography. The gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary column used was GL Science's NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as the carrier gas, the column inlet pressure was 100 kPa, and 1 μl of the sample was injected. As temperature conditions, the temperature was raised from an initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at a rate of 30 ° C./min. As a result of analysis under the above conditions, PHA and P (3HB-co-3HH) as shown in chemical formula (1) were obtained. Table 1 shows the PHA production amount, PHA time productivity, and 3HH composition.

(Example 1)
Incubation was carried out in the same manner as in Comparative Example 1 except that the stirring number of the 10 L jar was changed to 430 rpm. The oxygen transfer rate in the aqueous system under the culture conditions was 80 molO ₂ / m ³ / h. The results are shown in Table 1.

(Example 2)
The culture was carried out in the same manner as in Comparative Example 1 except that the stirring number of the 10 L jar was changed to 550 rpm. The oxygen transfer rate in the aqueous system under the culture conditions was 110 molO ₂ / m ³ / h. The results are shown in Table 1.

(Example 3)
The culture was carried out in the same manner as in Comparative Example 1 except that the stirring number of the 10 L jar was changed to 630 rpm. The oxygen transfer rate in the aqueous system under the culture conditions was 150 molO ₂ / m ³ / h. The results are shown in Table 1.

Example 4
Preparation of KNK-631 strain <Preparation of plasmid vector for gene insertion>
As an insertion DNA, a DNA comprising a base sequence including a promoter of phaC of A. caviae and a ribosome binding site was prepared as follows. PCR was carried out using the genomic DNA of A. caviae as a template and the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2. PCR was (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 20 seconds for 25 cycles. The DNA fragment obtained by PCR was terminally phosphorylated and digested with EcoRI. This DNA fragment was designated as PAc-5P + Eco. Next, the DNA insertion site is set immediately before the start codon of the bktB gene of the chromosomal DNA of the KNK-005AS strain described in JP 2008-029218 A [0038], and upstream of the start codon of the gene by the following procedure. A DNA consisting of the nucleotide sequence was prepared.

  Using the genomic DNA of the KNK-005 strain described in JP 2008-029218 A [0036] as a template DNA source, PCR was performed using the primers shown in SEQ ID NO: 3 and SEQ ID NO: 4 to initiate the bktB gene. DNA consisting of a base sequence upstream of the codon was obtained. PCR was (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 15 seconds, 64 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 25 cycles, and the polymerase used was KOD-plus-. The DNA fragment obtained by PCR was simultaneously digested with the restriction enzymes BamHI and EcoRI. This DNA fragment was designated as PbktB-Bam + Eco.

  PAc-5P + Eco and PbktB-Bam + Eco were ligated, and PCR was performed using the primers shown in SEQ ID NO: 3 and SEQ ID NO: 2 with the DNA produced in the ligation solution as the template DNA. PCR was (1) 98 ° C. for 2 minutes, 98 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 50 seconds for 25 cycles, and the polymerase used was KOD-plus-. The DNA fragment obtained by PCR was terminally phosphorylated and digested with BamHI. This DNA fragment was designated as bPac-5P + Bam.

  Next, a DNA having a base sequence downstream from the initiation codon of the gene was prepared. PCR was performed using the genomic DNA of the KNK-005 strain as a template DNA source and the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 to obtain a DNA comprising a base sequence downstream from the start codon of the bktB gene. . PCR was (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 15 seconds, 64 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 25 cycles, and the polymerase used was KOD-plus-. The DNA fragment obtained by PCR was terminally phosphorylated and digested with ClaI. This DNA fragment was designated ORF-5P + Cla.

  bPac-5P + Bam and ORF-5P + Cla were ligated, and PCR was performed using the primers shown in SEQ ID NO: 3 and SEQ ID NO: 6 with the DNA generated in the ligation solution as the template DNA. For PCR, (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 1 minute 30 seconds were repeated 25 cycles, and the polymerase used was KOD-plus-. The DNA fragment obtained by PCR was co-digested with BamHI and ClaI. This DNA fragment was subcloned into a site digested with the same restriction enzyme of the vector pBluescript II KS (-) (manufactured by TOYOBO). The obtained vector was designated as bAO / pBlu. The base sequence was determined using a DNA sequencer 3130xl Genetic Analyzer manufactured by APPLIED BIOSSYSTEMS, and it was confirmed that it was the same as the base sequence of the template DNA.

  Subsequently, the pSACKm described in JP 2008-029218 A [0037] was treated with the restriction enzyme NotI to cut out a DNA fragment of about 5.7 kb containing the kanamycin resistance gene and the sacB gene. This was inserted into a site cleaved with the same enzyme of bAO / pBlu to prepare a plasmid bAO / pBlu / SacB-Km for gene disruption / insertion.

<Preparation of gene insertion strain Pac-bktB / AS strain>
Next, using KNK-005AS as a parent strain, a strain was prepared using bAO / pBlu / SacB-Km in which a DNA comprising a phaC promoter and a base sequence containing a ribosome binding site was inserted immediately before the start codon of the bktB gene. E. coli strain S17-1 (ATCC 47005) was transformed with the gene insertion plasmid bAO / pBlu / SacB-Km. The obtained transformant was mixed and cultured on KNK-005AS strain and Nutrient Agar medium (manufactured by Difco) to perform conjugation transfer. Simmons agar medium containing 250 mg / L kanamycin (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium dihydrogen phosphate 1 g / L, dihydrogen phosphate 2 A strain that had grown on potassium 1 g / L, agar 15 g / L, pH 6.8) was selected to obtain a strain in which the plasmid was integrated on the chromosome of the KNK-005AS strain. This strain is cultured for 2 generations in Nutrient Broth medium (manufactured by Difco), then diluted and applied to Nutrient Agar medium containing 15% sucrose, and the grown strain is selected to perform the second recombination. The resulting stock was acquired. Furthermore, a desired gene insertion strain was isolated by PCR analysis.

  This gene insertion strain was named Pac-bktB / AS strain, its nucleotide sequence was determined using a DNA sequencer 3130xl Genetic Analyzer, and DNA comprising a phaC promoter and a ribosome binding site immediately before the start codon of the bktB gene Was confirmed to be an inserted strain.

<Cloning of ccr gene and expression unit construction>
The ccr gene encoding an enzyme that converts crotonyl-CoA to butyryl-CoA, a precursor of 3HH monomer, was cloned from the chromosomal DNA of Streptomyces cinnamonensis strain Okami (DSM 1042). PCR was performed using the DNAs of SEQ ID NO: 7 and SEQ ID NO: 8 as primers. The conditions were (1) 98 ° C. for 2 minutes, (2) 94 ° C. for 10 seconds, 55 ° C. for 20 seconds and 68 ° C. for 90 seconds for 25 cycles, and the polymerase used was KOD-plus-. The fragment amplified by PCR was purified and then cleaved with restriction enzymes BamHI and AflII. Subcloning the EE32d13 fragment (J. Bacteriol., 179, 4821 (1997)) into the EcoRI site of the pUC19 vector, cutting this plasmid with BglII and AflII and replacing the ccr gene with the BamHI and AflII fragments. The ccr expression unit was constructed by

<Cloning of phaC gene and preparation of expression unit>
A phaC expression unit containing SEQ ID NO: 9 was prepared as a SpeI fragment. PCR was performed using HG :: PRe-N149S / D171G-T / pBlu described in JP-A 2007-228894 [0031] as a template and primers shown in SEQ ID NO: 10 and SEQ ID NO: 11. The conditions were (1) 98 ° C. for 2 minutes, (2) 94 ° C. for 10 seconds, 55 ° C. for 30 seconds and 68 ° C. for 2 minutes for 25 cycles, and the polymerase used was KOD-plus-. The fragment amplified by PCR was purified and then cleaved with the restriction enzyme SpeI to prepare an expression unit.

<Construction of expression vector>
The expression vector pCUP2-631 was constructed as follows.

  As a plasmid vector for constructing an expression vector in C. necator, pCUP2 described in WO / 2007/049716 [0041] was used. First, the constructed ccr gene expression unit was cut out by EcoRI treatment, and this fragment was ligated with pCUP2 cut with MunI. Next, the phaC expression unit was prepared as a SpeI fragment and inserted into the SpeI site of pCUP2 containing the ccr gene expression unit to construct a pCUP2-631 vector.

<Preparation of transformed cells>
The introduction of the pCUP2-631 vector into cells was carried out by electrical introduction as follows. A gene pulsar manufactured by Bio-rad was used as the gene introduction device, and a gap 0.2 cm manufactured by Bio-rad was used as the cuvette. Into the cuvette, 400 μl of Pac-bktB / AS strain competent cells and 20 μl of the expression vector were injected and set in a pulse device, and an electric pulse was applied under the conditions of capacitance 25 μF, voltage 1.5 kV, and resistance 800 Ω. After pulsing, the bacterial solution in the cuvette was cultured with shaking on Nutrient Broth medium (DIFCO) at 30 ° C. for 3 hours, and then selected plate (Nutient Agar medium (DIFCO), kanamycin 100 mg / L) at 30 ° C. And cultured for 2 days to obtain the transformed transformant KNK-631 strain.

(Comparative Example 2)
Culturing was performed in the same manner as in Comparative Example 1 except that the KNK-631 strain was used for the experiment.

(Example 5)
Culturing was performed in the same manner as in Example 3 except that the KNK-631 strain was used for the experiment.

Claims (5)

A method for producing a polyhydroxyalkanoic acid obtained by polymerizing a monomer unit containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid by a microorganism, wherein the fat contains 41% by weight or more of lauric acid as a constituent fatty acid, and / Or characterized in that the microorganism is cultured with a fatty acid as a carbon source and an oxygen transfer rate of 110 molO ₂ / m ³ / h or more, and the microorganism is an amino acid sequence represented by SEQ ID NO: 12 A polyhydroxyalkanoic acid polymerase encoding a gene, or a polyhydroxyalkanoic acid polymerase encoding a polypeptide having a sequence identity of 90% or more to the amino acid sequence and having polyhydroxyalkanoic acid polymerization activity gene is integrated, a microorganism of Cupriavidus genus, polyhydroxy Al Production methods by microorganisms of phosphate.   The production method according to claim 1, wherein the 3HH composition ratio in the polyhydroxyalkanoic acid is 6 mol% or more.   The production method according to claim 1, wherein the carbon source is palm kernel oil, coconut oil, and fractionated fats and oils obtained by fractionating these fats and oils, at least one fat selected from the group of fatty acids, and / or fatty acids.   The production method according to claim 1, wherein the microorganism is Cupriavidus necator. The production method according to claim 1, wherein the polyhydroxyalkanoic acid is P (3HB-co-3HH).