KR101900152B1 - Methods for producing diocic acids using candida infanticola sp. - Google Patents

Abstract

The present invention relates to a method for producing dioic acid using Candida albicans, and it is possible to improve the conversion of dioic acids from a carbon source by optimizing medium and conditions for producing dioic acid.

Description

METHODS FOR PRODUCING DIOCIC ACID USING CANDIDA INFANTICOLA SP.

The present invention relates to a method for producing dioic acids using Candida albicans.

Dioic acids are very important chemicals in the chemical industry. They are used not only in petroleum-derived nylon used in engineering resins, automobile parts, sporting goods, carpets and toothbrushes, but also in other polymeric plasticizers, adhesives, lubricants, It is used in various industrial applications such as antifoaming agents, coating agents, processed plastics, perfumes and medicine products. Of these dioic acids, about 15,000,000,000 pounds of dodecanedioic acid per year are being synthesized from petrochemical raw materials. These petrochemical raw materials are mainly deficient natural raw materials, and their use as a global source of environmental destruction and environmental change , And these petrochemical raw materials are sensitive to price fluctuations and increase the burden on environmental pollution.

On the other hand, when wild-type strain cultivation is adopted in the conversion of dioic acids through omega-oxidation, both omega-oxidation and beta-oxidation are reacted Therefore, there is a problem that the conversion yield of dioic acid, which is the final product, is low and the concentration is lowered.

Therefore, there is a need for an optimal method for effectively producing dioxin acid, which is sustainable and reduces environmental burden.

It is an object of the present invention to provide a method for producing dioic acid by using Candida pannicola strains in order to solve the problems of the prior art as described above.

In order to solve the above-mentioned problems, the present invention provides a method for producing dioic acids using a Candida peanut tricolor strain that does not use glycerol as a carbon source, comprising the steps of: 1) a medium containing a hydrocarbon, a fatty acid or glucose, and glycerol A primary culture step in which a Candida infanticola strain is cultured; 2) inducing an omega-oxidation reaction by adding a carbon source to the culture medium of step 1); And 3) a second culture step in which the substrate and the glucose-containing medium are added to the reaction solution of step 2) and cultured.

According to one embodiment, the strain may be Candida infantola DS02 (KCTC 12820BP, deposited by Korea Research Institute of Bioscience and Biotechnology, entitled: 2015.05.29).

In addition, the above-mentioned strain is a mutant strain (Candida infanticola LC-DA01; KCTC13099BP, deposited by Korea Research Institute of Bioscience and Biotechnology, Korea), which has blocked the β-oxidation pathway of Candida infanticola DS02 (KCTC 12820BP) , Entrusted date: 2016.09.08).

According to one embodiment, the concentration of glycerol relative to the medium may range from 0.0001 to 30%.

According to one embodiment, the mutant strain can produce dioic acids using a substrate comprising selected from hydrocarbons, fatty acids, and combinations thereof.

According to one embodiment, the carbon source may include those selected from the group consisting of glucose and derivatives thereof.

According to one embodiment, the dioic acid is selected from the group consisting of ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, It is also possible to use hexanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid, acid, hexadecanedioic acid, and combinations thereof.

Other details of the embodiments of the present invention are included in the following detailed description.

According to the method for producing dioic acid using the Candida pannicola strain according to the present invention, it is possible to improve the conversion ratio of the dioic acid from the carbon source by optimizing the medium and conditions for producing the dioic acid.

Figure 1 shows the 18s rRNA nucleotide sequence of a wild-type strain.
FIG. 2 is a schematic view for producing solid medium using steam. FIG.
FIG. 3 is a graph showing DDDA conversion according to whether glycerol is added to a wild-type strain.
FIG. 4 is a graph showing the conversion of DDDA according to whether beta-oxidation is blocked and glycerol is added to a mutant strain.
FIG. 5 is a graph showing the DDDA conversion according to the glycerol concentration for the wild type strain. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, a method for producing dioic acids using the Candida papillocyte strain according to an embodiment of the present invention will be described in detail.

As used herein, the term " blocking " can be used interchangeably with " inhibition "

Also, 'hydrocarbons' can refer to organic compounds composed only of carbon and hydrogen.

In addition, 'fatty acid' may refer to saturated or unsaturated monocarboxylic acids in the form of chains.

In addition, 'omega-oxidation' may mean a reaction in which the methyl group end of a fatty acid is oxidized to dicarboxylic acid, and 'beta-oxidation' It may be a reaction that is self-oxidized and decomposes while releasing acetyl-CoA.

In general, the oxidation of fatty acids is the main reaction from β-oxidation in which the carboxy group is cleaved to two carbon units, and omega-oxidation is the main reaction for intermediate chain fatty acids having 8 to 14 carbon atoms It is understood as a path.

According to the present invention, a mutant strain in which beta-oxidation of a Candida infanticola DS02 (KCTC 12820BP) and a Candida infanticola strain, which do not use glycerol as a carbon source, is blocked (Candida infanticola LC-DA01; KCTC13099BP), the productivity of dioic acids can be improved in a medium containing glycerol. Specifically, the strain is selected from the group consisting of Candida infanticola DS02 (KCTC 12820BP), a mutant strain in which β-oxidation is blocked (Candida infanticola LC-DA01; KCTC13099BP), and combinations thereof ≪ / RTI > Candida infanticola strains can be isolated, for example, from wastewater from petrochemical plants. The mutant strains of DS02 can be derived by treating EMS (ethyl methanesulfonate), UV, and the like. In the case of the above-mentioned wild-type strain (Candida infanticola DS02; KCTC 12820BP), in the case of a mutant strain in which alkane, for example, dodecane can be used as a single carbon source but the beta-oxidation pathway is blocked It does not grow substantially using dodecane as a single carbon source. The substantially inability to grow may mean that the mutant strain does not grow or the growth is insignificant and the growth can not be judged effectively. Therefore, it is possible to select a mutant strain by comparing the growth of the strain in a solid medium containing glucose, dodecane or the like as a carbon source.

Such wild-type strains, mutant strains, or a combination thereof can be used to produce dioic acid. Examples of the substrate include a hydrocarbon or a fatty acid having 6 to 30 carbon atoms, for example, an alkane or a fatty acid having 8 to 20 carbon atoms . Specifically, the substrate may be dodecane, methyl laulate, lauric acid, a derivative thereof, or a combination thereof. The derivative of lauric acid may be C1-8 alkyl Laurate, and may include one or more selected from the group consisting of methyl laurate, ethyl laurate, propyl laurate, and the like.

According to one embodiment, there is provided a method for the production of Candida infantola, comprising: 1) a primary culture step of culturing a Candida infanticola strain in a medium containing a hydrocarbon, a fatty acid or glucose, and glycerol;

2) inducing an omega-oxidation reaction by adding a carbon source to the culture medium of step 1); And 3) a second culture step in which the substrate and the glucose-containing medium are added to the reaction solution of step 2) and cultured, and dioleic acids can be produced.

The dioic acid may include dioic acid having 8 to 14 carbon atoms, for example, ethanedioic acid, propanedioic acid, butanedioic acid, But are not limited to, pentanedioic acid, hexanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, undecanedioic acid, undecanedioic acid, dodecanedioic acid, hexadecanedioic acid, and combinations thereof. Specific examples of the dodecanedioic acid include dodecanedioic acid (dodecanedioic acid, DDDA).

According to one embodiment, when dioleic acids are produced by the method of the present invention, an alkane-derived material may be added to induce omega-oxidation. Specifically, in the culturing step of the primary culture and the secondary culture, the adding time of the alkane-derived material may be between the primary culture and the secondary culture.

In addition, glycerol may be added to the medium to improve the conversion of dioic acid. The optimum concentration of glycerol may be from 0.0001 to 30%, for example from 5 to 8%, for example from 6 to 7.5%, relative to the medium.

Candida infantola wild type strains used in the present invention can be grown using a hydrocarbon or a fatty acid as a carbon source, and examples thereof include dodecane, methyl laurate, glucose, Or a combination thereof. However, since glycerol is not used as a carbon source, the presence or absence of glycerol may have a characteristic that growth of cells is not affected. In addition, the mutant strain of the present invention can be used as a substrate without using a hydrocarbon or a fatty acid as a carbon source. For example, a mutant strain of the present invention grows using glucose as a carbon source and uses hydrocarbons or fatty acids as a substrate, dioic acids. < / RTI >

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Production Example 1: Isolation of Candida infanticola strain

Candida infantola DS02 (KCTC 12820BP) was isolated from a wastewater treatment facility at a petrochemical plant (Lotte Chemical Daesan Plant).

The isolated Candida infantola isolate (Candida infanticola DS02; KCTC 12820BP) was analyzed by 18s rRNA sequencing. Genomic DNA was extracted using yeast gDNA prep kit (PureHelix ™, NANOHELIX), and the extracted genomic DNA was amplified by PCR using the 18s ITS ¼ primer shown in Table 1, followed by TA vector cloning, followed by DNA sequencing The 18s rRNA nucleotide sequence was obtained through the reaction, and the nucleotide sequence was shown in FIG. 1 as SEQ ID NO: 1.

1, the homology of the strain was examined using a BLAST (Basic Local Alignment Search Tool) of NCBI (National Center for Biotechnology Information). As a result, as shown in Table 2, the homology of Candida albicans CBS11940 with high homology It is possible to confirm that it is the nearest species having.

Preparation Example 2: Preparation of Candida infanticola mutant strain

(Candida infanticola DS02; KCTC 12820BP) isolated from Candida infantola DS02 (KCTC 12820BP) isolated from the culture broth of the present invention was isolated by using phosphate buffered saline (PBS buffer) ) Was prepared and treated with mutagen EMS (ethyl methanesulfonate) at a concentration of 2% to prepare 1 ml. The reaction mixture was reacted at 30 ° C and 150 rpm for 120 minutes, centrifuged to remove the supernatant, and 20% sodium thiosulfate and then washed twice with sodium thiosulfate to remove EMS. Then, the strain was inoculated in 1 ml PBS buffer, 10 ul of which was applied to a YPD solid medium and cultured at 30 ° C. for 3 days to obtain a primary mutant which survived within 10%.

The strains treated with EMS mutants were treated with 10 μl of YPD solid medium for 0.01 sec, and then irradiated with UV (ultraviolet 254 nm) for 120 sec. After incubation at 30 ° C. for 3 days, about 10% Surviving secondary mutant strains were obtained. The mutagenesis process can be performed by UV irradiation after EMS treatment, EMS treatment after UV irradiation, or EMS, UV alone.

In order to select mutant strains which have been blocked by β-oxidation, we first compared the growth of strains in a solid medium containing glucose and dodecane as a single carbon source in a solid medium . The solid medium used was a solid medium (YNB, 6.7 g / L, glucose 10 g / L, YNB) in which glucose was used as a single carbon source and a solid (YNB (Yeast nitrogen base without amino acid) 6.7 g / L, Dodecane 10 g / L). In the case of solid medium containing dodecane, it is not easy to identify the colonies grown in opaque white color of the solid medium. Therefore, the growth performance of the strain was efficiently compared through the production of solid medium using dodecane vapor , And is simply shown in Fig.

A filter made of sterilized paper in a solid medium was put in a filter, and a predetermined amount of dodecane was applied to the filter. Dodecane was spread in the solid medium with the steam in the solid culture, and the strain was used. The candidate mutant strain was inoculated in PBS buffer to prepare an OD 0.01 strain suspension, and then inoculated with 10 μl of each of the two solid mediums described above using a micropipette, followed by culturing at 30 ° C. for 3 days. In the glucose solid medium, The primary selection was carried out by selecting strains which were not oxidized (β-oxidation) and could not grow in the dodecane solid medium.

Secondly, in order to select a mutant strain which is blocked by β-oxidation in a liquid medium, a mutant strain selected firstly is subjected to β-oxidation through a strain growth in a liquid medium containing dodecane as a single carbon source ) Blocking strains were screened secondarily. In this experiment, a total of six selected strains were subjected to liquid culture. The liquid culture used was inoculated in a 250 ml erlenmeyer flask with 70 ml of dodecane as a single carbon source and inoculated with each selected strain to the initial culture OD 1 and cultured at 30 ° C and 150 rpm for 6 days. The composition of the liquid medium used was a medium (YNB (Yeast nitrogen base without amino acid) 20 g / L, Dodecane 20 g / L) using dodecane as a single carbon source. The culture results are shown in the following figure as a graph of a mutant strain using dodecane as a carbon source and a mutant strain using dodecane, respectively. The strains that did not use dodecane were judged to be strains that were blocked from β-oxidation and completed the second screening to determine the presence of Candida infantica (Candida infantola), which was blocked by β-oxidation LC-DA01; KCTC13099BP).

Experimental Example 1: Analysis of carbon atomization ability of wild-type and mutant Candidia pantotychoi strains

To investigate the carbonization ability of Candida infantola LC-DA01 (KCTC13099BP), Candida infantola DS02 (KCTC 12820BP) and beta-oxidation-blocked Candida infantola LC-DA01 (KCTC13099BP), API 20c AUX ). The results of the analysis using the API 20c AUX (Biomerieux) were compared with those of the conventional Candida infantica kurtzman and Candida infantica sp., And the results are shown in Table 3 below. The wild-type and mutant Candida pantotrichicola strains used in Example 2 have the characteristics of not having glycerol as a carbon source, unlike the previously reported homozygotes, and have only glucose as a carbon source And the difference is in using.

delete

Example 1: Conversion of Dioic acid into wild type Candida intanthicola strain by glycerol addition

To investigate the conversion of dodecanedioic acid (DDDA) from an alkane (C12) substrate to Candida infantola coli wild type strain (Candida infanticola DS02; KCTC 12820BP), 20 g / L YNB, 20 g / L Dodecane , 0% and 3% of Glycerol were cultured in a flask under the condition of Candida infanticola DS02 at 30 ° C and 150 rpm, and the effect of glycerol on the DDDA conversion was compared and shown in FIG. 3 and Table 4.

 As shown in FIG. 3 and Table 4, since DS02 does not use glycerol as a carbon source, it can be confirmed that optical density values indicating cell growth are almost the same regardless of addition of glycerol.

The concentration of DDDA converted was significantly higher than that of the culture supplemented with 3% glycerol, which was about 5 times higher than that of DDDA. In addition, the DDDA detection rate was decreased by 2 days in the culture medium containing 3% glycerol . It can be seen from the present Example that the added glycerol was not used as a carbon source but was used for improving DDDA conversion.

Experimental Example 2: Determination of glycerol optimum concentration for DDDA conversion culture

To determine the optimal concentration of glycerol for the DDDA conversion culture of Candida infantola DS02 (KCTC 12820BP), the DDDA conversion of alkane (C12) substrate was compared. The composition of the medium used was 20 g / L YNB, 20 g / L dodecane, and 0% to 12% glycerol at 1.5% intervals. The optimum concentration of glycerol for DDDA conversion was investigated by culturing Candida infantola DS02 at 30 ° C and 150 rpm, and the results are shown in FIG. 4 and Table 5.

 As shown in Fig. 4 and Table 5, 0.2 g / L of DDDA was converted in the culture not containing glycerol, showing the lowest conversion. Glycerol showed the optimum concentration for DDDA conversion at 6.0 to 7.5% of the total amount of the medium and a concentration of 2.2 to 2.3 g / L at the DDDA conversion concentration, which is about 10 times higher than the condition without glycerol. The concentration of DDDA increased with increasing concentration of glycerol until the concentration of glycerol was increased to 7.5%, and gradually decreased thereafter.

Example 2: Culture of Dioic Acid Conversion of Candida Infant Ticola Mutant Strain According to Glycerol Addition

For Candida infantola LC-DA01 (KCTC13099BP), in order to obtain the initial cell mass for examining the effect of glycerol, a mixture containing 6% glycerol-free conditions and no glycerol- (100%) according to the DO value at 30 ° C, 1 v / v / m aeration amount and 30% dissolved oxygen (DO) in a yeast extraction medium containing 50 g / 900 rpm) and pH 5 for 24 to 48 hours, followed by induction of omega-oxidation at pH 7 for 12 to 20 hours using 1% dodecane. Then, lauric acid The cells were cultured for 96 to 144 hours at a concentration of 0.5 to 1.0 ml / L / h of methyl and 0.5 to 3.0 g / L / h of glucose, and DDDA conversion was carried out at pH 7 to 8.

As shown in Table 6 above, the optical density value and DDDA concentration of 70.3 and 86.9 g / L in the 6% glycerol-containing culture were lower than the cell concentration value of 69.6 and DDDA concentration of 67.8 g / L in the glycerol- The concentration was improved by about 25%, and the cell concentration was similar regardless of whether or not glycerol was contained in the characteristics of the strain used in the present patent which does not use glycerol as a carbon source.

As described above, a strain that does not use glycerol as a carbon source, that is, a Candida infanticola DS02 (KCTC 12820BP) and a Candida infantola LC-DA01 (Beta-oxidation-blocked Candida infantola LC-DA01; KCTC13099BP) can be used to improve the productivity of dioic acids in a culture medium or medium containing glycerol.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> LOTTE CHEMICAL CORPORATION <120> METHODS FOR PRODUCING DIOCIC ACIDS USING CANDIDA INFANTICOLA SP. <130> DPA-0819 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 434 <212> DNA <213> Candida <400> 1 tccgtaggtg aacctgcgga aggatcatta ttgagattca tattacacct gtgaaacaac 60 taaattgctt ggccgaaagg ccaatgtaac aaaaactatt ttacctatta tatctgaaaa 120 acgaaatcaa aagtttcaac aacggatctc ttggttctcg catcgatgaa gaacgcagca 180 aagcgcgata gttagtgtga attgcagacg tgaatcattg agtttttgaa cgcacattgc 240 accttctggt attccgggaa gtatacttgt gcgagcgtca tttcatcttc ataaagcaat 300 ttatgtgttg gggctgtagc cagccttgaa aaagatgata gagtacatgt tagacacaat 360 gtgcttttct atatttttga cctcgtatca agcaagatta cccgctgaac ttaagcatat 420 caataagcgg agga 434 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 18s ITS quarter forward primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s ITS quarter reverse primer <400> 3 tcctccgctt attgatatgc 20

Claims (6)

A method for producing dodecanedioic acid using a Candida albicola strain which does not use glycerol as a carbon source,
1) a primary culture step of culturing a Candida infanticola strain in a medium containing hydrocarbons, fatty acids or glucose together with glycerol;
2) inducing an omega-oxidation reaction by adding a carbon source to the culture medium of step 1); And
3) a secondary culture step in which the substrate and glucose containing hydrocarbon, fatty acid or a combination thereof are added to the reaction solution of step 2) and cultured,
The Candida albicans strains are characterized by the fact that the beta-oxidation pathway of Candida infanticola DS02 (KCTC 12820BP) or Candida infanticola DS02 (KCTC 12820BP) is blocked A Candida infantola LC-DA01; KCTC13099BP, or a combination thereof. &Lt; RTI ID = 0.0 &gt; delete delete The method according to claim 1,
Wherein the concentration of the glycerol is 0.0001 to 30% with respect to the culture medium. The method according to claim 1,
Wherein the substrate is selected from the group consisting of dodecane, lauric acid and C1-8 alkyl laurate. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; delete

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