JP2019041712A - Recombinants and methods for producing squalene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant capable of efficiently producing squalene and a method for producing squalene. SOLUTION: This is a recombinant of Saccharomyces cervisier and consists of a group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1 and ADH4. A recombinant in which one or more selected genes have been knocked out and tHMGR has been introduced. A method for producing squalene, in which the recombinant is cultured and squalene is extracted from the cultured recombinant. [Selection diagram] None

Description

  The present disclosure relates to recombinants and methods of producing squalene.

  Squalene is a type of lipid. Squalene is excellent in lubricity. In addition, squalene has a low pour point and freezing point and a high flash point. In addition, squalene is highly biodegradable. Therefore, squalene is a commercially important substance. Currently, squalene is produced by extraction from olive oil or liver oil of sharks that inhabit cold water. This manufacturing method increases the manufacturing cost of squalene.

  Methods for producing squalene using yeast are being investigated. For example, Patent Document 1 discloses that in yeast, overexpression or high activation of an enzyme involved in squalene synthesis and reduction of the activity of an enzyme that converts squalene to another substance are disclosed.

JP, 2017-6121, A

  In the method described in Patent Document 1, it has been difficult to efficiently produce squalene. The present disclosure provides a recombinant that can efficiently produce squalene and a method of producing squalene.

One aspect of the present disclosure is a recombinant of Saccharomyces cerevisiae, which is prepared from SERl, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4. It is a recombinant in which one or more genes selected from the group consisting of: are knocked out, and tHMGR is introduced. According to the recombinant which is one aspect of the present disclosure, squalene can be efficiently produced.
Another aspect of the present disclosure is a recombinant of the Saccharomyces cerevisiae strain BY4742, which comprises: SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, And one or more genes selected from the group consisting of ADH4 are knocked out, tHMGR is introduced, and the recombinant is higher in squalene production amount than the BY4742 strain of Saccharomyces cerevisiae. According to the recombinant that is another aspect of the present disclosure, squalene can be efficiently produced.

  Another aspect of the present disclosure is one or more selected from the group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4. This is a method for producing squalene, which comprises the steps of: culturing a recombinant of Saccharomyces cerevisiae in which a gene has been knocked out and tHMGR has been introduced, and then squalene is extracted from the cultured recombinant. According to the method of producing squalene which is another aspect of the present disclosure, squalene can be efficiently produced.

It is explanatory drawing showing the composition of SC culture medium.

Embodiments of the present disclosure will be described.
1. Recombinant The recombinant of the present disclosure is a recombinant of Saccharomyces cerevisiae. Saccharomyces cerevisiae is a type of budding yeast. Saccharomyces cervisiae is Saccharomyces cerevisiae.

  In the recombinant of the present disclosure, one or more genes selected from the group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4 Has been knocked out.

  The recombinant of the present disclosure has tHMGR introduced. tHMGR is a gene that causes the enzyme of HMG1 to function in the cytoplasm by deleting the transmembrane site of a membrane protein. Examples of tHMGR include tHMG1 (SEQ ID NO: 1) and the like.

2. Method of Producing Squalene In the method of producing squalene of the present disclosure, a recombinant is cultured, and squalene is extracted from the cultured recombinant. The recombinants are those described in the section "1. Recombinant" above.

  The culture conditions can be set appropriately. The medium used for culture can be appropriately selected from, for example, known media. Examples of the medium used for culture include SC medium.

The method of extracting squalene can be appropriately selected from known extraction methods. Examples of the extraction method include the methods used in the examples described later.
3. Example (1) Production of Recombinant A commercially available knockout clone of Saccharomyces cerevisiae was prepared. The prepared knockout clones are shown in Table 1.

これらのノックアウトクローンは、それぞれ、サッカロマイセス・セルビシエにおいて、１つの遺伝子がノックアウトされたものである。表１に、それぞれのノックアウトクローンにおいてノックアウトされた遺伝子を示す。表１に示すノックアウトクローンのうち、YGR244Cは、Dharmacon社製の商品名Yeast Mat alpha Knock Out Strainである。その他のノックアウトクローンは、transOMIC Technologies社製の商品名Yeast Knockout Collection Haploid Mat-alphaである。表１に示す遺伝子は、周知のデータベースであるKEGG（Kyoto Encyclopedia of Genes and Genomes)やSGD（Saccharomyces Genome Database）に登録されたものである。

Each of these knockout clones is one in which one gene has been knocked out in Saccharomyces cerevisiae. Table 1 shows the genes knocked out in each knockout clone. Among the knockout clones shown in Table 1, YGR244C is a trade name of Yeast Mat alpha Knock Out Strain manufactured by Dharmacon. Another knockout clone is trade name Yeast Knockout Collection Haploid Mat-alpha manufactured by transOMIC Technologies. The genes shown in Table 1 are those registered in the well-known databases KEGG (Kyoto Encyclopedia of Genes and Genomes) and SGD (Saccharomyces Genome Database).

  In each knockout clone, tHMGR was introduced. The method is as follows. As primers to which a restriction enzyme site was added, tHMG1 SalI_fw (SEQ ID NO: 2) and tHMG1 NotI_rv (SEQ ID NO: 3) were prepared. Using tHMG1 SalI_fw and tHMG1 NotI_rv, tHMG1 (SEQ ID NO: 1) was replicated by PCR using the genome of budding yeast as a template. tHMG1 corresponds to tHMGR.

  Plasmid pAT425 for gene expression was digested with HMG1 SalI_fw and tHMG1 NotI_rv. Next, the cloned tHMG1 was inserted into a gene expression plasmid pAT425 using a DNA Ligation Kit (Takara) to construct a plasmid for tHMG1 expression. In addition, pAT425 is disclosed in a publicly known document (Ishii et al., FEMS Yeast Res 14, 399 (2014)).

  Next, using the lithium acetate method, transformation of a plasmid for tHMG1 expression into a knockout clone was performed. The lithium acetate method is disclosed in the publicly known document (Gietz and Woods, Methods in Mol Biool, 313, 107 (2002)).

  Specifically, first, frozen yeast stock was applied to YPD agar medium and cultured at 30 ° C. for 2 days. Next, single colonies on agar medium were inoculated into 5 mL of YPD liquid medium in a test tube, and shake cultured at 30 ° C. for 1 day.

  Next, 500 μL of the above YPD liquid medium was centrifuged to obtain pelleted cells. Next, the pelleted cells were washed twice with sterile water. Next, 240 μL of 50 w / v% polyethylene glycol, 36 μL of 1.0 M lithium acetate solution, 50 μL of salmon sperm DNA, and 34 μL of plasmid are added to the pelleted cells, suspended, and incubated at 42 ° C. for 1 hour did. The molecular weight of the above polyethylene glycol is 3350. The concentration of salmon sperm DNA above is 2 mg / mL. The amount of the above plasmid is 1 μg.

After incubation, the cells were centrifuged and the transformation solution was discarded to obtain pelleted cells. The pelleted cells were added with 100 μL of sterile water, suspended, and applied to SC (-Leu) selective agar medium. The agar medium coated with the cells was static cultured at 30 ° C. for 3 to 5 days to obtain colonies. The introduction of tHMG1 into a knockout clone was confirmed by PCR using the obtained colonies as a template. The recombinant was produced by the above steps.
(2) Production of squalene
Each recombinant was used to produce squalene. The method is shown below. First, the recombinant was cultured under the following conditions. Hereinafter, this culture is referred to as the first culture.

Type of medium: SC medium Amount of medium: 5 mL
Shaking conditions of medium: 150 rpm
Medium temperature: 30 ° C.
Culture time: 48 hours The SC medium used has the composition shown in FIG. Next, the medium after the first culture was transferred to another medium and cultured under the following conditions. Hereinafter, this culture is referred to as a second culture. At the beginning of the second culture, the concentration of the recombinant in the medium was OD600 = 0.05.

Type of medium: SC medium Amount of medium: 5 mL
Shaking conditions of medium: 150 rpm
Medium temperature: 30 ° C.
Culture time: 72 hours After the second culture, the recombinant was recovered. Next, lipid components were extracted from the recombinants. The extraction method is as follows.

  2 mL of culture was transferred to a 2 mL Eppendorf tube, centrifuged at 15000 g for 15 seconds, and the supernatant was discarded. 1 mL of deionized water was added to the above Eppendorf tube, suspended, centrifuged under the same conditions, and the supernatant was discarded. As a result, pelleted cells were obtained. 20% (w / v) potassium hydroxide solution (20% (w / v) KOH in 50) dissolved in 50% (v / v) ethanol containing 10 μg / ml cholesterol as an internal standard in pelleted cells 0.4 mL of (v / v) ethanol containing 10 μg / ml cholesterol (internal standard) was added and suspended, and heated in boiling water for 10 minutes.

  After cooling the sample with running water, add 0.4 mL of dodecane, and shake it under the conditions of 25 ° C and 180 rpm for 1 hour using a constant temperature shaking incubator (Deep Well Maximizer Bioshaker, manufactured by Taitec Co., Ltd.) Was extracted into dodecane. The extracted lipid components include squalene. Through the above steps, squalene could be produced.

  Next, after centrifuging the sample at 3000 g for 5 minutes, 0.1 mL of the dodecane layer (upper layer) was taken out and analyzed using CG-MS. Then, the amount of squalene contained in the lipid component was quantified. The control was also cultured in the same manner as above, the lipid component was extracted, and the amount of squalene was quantified. The control is one obtained by introducing tHMGR into BY4742 strain. The BY4742 strain is Saccharomyces cervisiae in which no gene has been knocked out. Controls correspond to wild strains.

  Table 1 above shows the average value of the amount of squalene produced using each of the recombinants and the standard deviation of the amount of squalene. The amount of squalene is a relative value where the amount 1 of squalene produced by the control is 1. As shown in Table 1, the recombinant was able to produce squalene more efficiently than the control.

4. Other Embodiments Although the embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments, and can be variously modified and implemented.

  (1) The recombinants used in the examples are two or more of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, PAE1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4. The gene may be knocked out. Also in this case, the same effect can be obtained.

(2) In the example, tHMGR introduced into a knockout clone may be other than tHMG1. Even in this case, the same effect can be obtained.
(3) The plurality of functions of one component in the above embodiment may be realized by a plurality of components, or one function of one component may be realized by a plurality of components . Also, a plurality of functions possessed by a plurality of components may be realized by one component, or one function realized by a plurality of components may be realized by one component. In addition, part of the configuration of the above embodiment may be omitted. In addition, at least a part of the configuration of the above-described embodiment may be added to or replaced with the configuration of the other above-described embodiment. In addition, all the aspects contained in the technical thought specified from the wording described in the claim are an embodiment of this indication.

  (4) In addition to the above-mentioned recombinants, the present disclosure can also be realized in various forms such as a system having the recombinants as a component, a method of producing the recombinants, and the like.

Claims (5)

A recombinant of Saccharomyces cerevisiae,
One or more genes selected from the group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4 are knocked out and tHMGR is introduced Recombinants. A recombinant according to claim 1, wherein
A recombinant that produces more squalene than the wild strain of Saccharomyces cerevisiae. A recombinant of Saccharomyces cerevisiae BY4742 strain,
One or more genes selected from the group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4 are knocked out and tHMGR is introduced And
Recombinants that produce more squalene than the BY4742 strain of Saccharomyces cerevisiae.   One or more genes selected from the group consisting of SER1, LSC1, AAT2, LEU9, DPP1, LSC2, PYC1, MAE1, SRY1, PNP1, PDE2, ALT2, GPP2, NQM1, FRD1, and ADH4 are knocked out and tHMGR is introduced A method for producing squalene, which comprises culturing the above-mentioned recombinant of Saccharomyces cerevisiae and extracting squalene from the cultured recombinant. A method for producing squalene according to claim 4, wherein
The manufacturing method of squalene which culture | cultivates the said recombinant using SC culture medium.

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