Application of caprylic acid tolerance related gene in improving caprylic acid tolerance of saccharomyces cerevisiae strain

Abstract

The invention discloses application of a caprylic acid tolerance related gene in improving caprylic acid tolerance of a saccharomyces cerevisiae strain, and belongs to the technical field of biology. Five key genes, namely an ALG2 gene, a THI4 gene, a PHO5 gene, an SOD1 gene and a CCP1 gene, capable of improving the caprylic acid tolerance of the saccharomyces cerevisiae strain are excavated. According to the invention, the gene is expressed in saccharomyces cerevisiae BY4742, and a plurality of saccharomyces cerevisiae engineering strains capable of tolerating octanoic acid are constructed, so that the constructed strains can tolerate 0.45 mM octanoic acid. The octanoic acid high-tolerance saccharomyces cerevisiae engineering strain constructed by the invention can normally grow in a high-concentration octanoic acid culture medium and produce high-added-value chemicals, can be used as a chassis cell of a high-added-value chemical production strain, and has a huge application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to application of caprylic acid tolerance related genes in improving caprylic acid tolerance of a Saccharomyces cerevisiae strain and belongs to the technical field of biotechnology. BACKGROUND

[0002] Medium-chain fatty acids (hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10) and lauric acid (C12)) have important application values in the fields of daily chemical industry, medicine and food. With more and more attention paid to the application research of medium-chain fatty acids, the market demand for the medium-chain fatty acids is increasing year by year. At present, the main way to obtain the medium-chain fatty acids is to extract them from coconut oil and palm kernel oil, and both coconut trees and palm trees are tropical trees and are difficult to be widely cultivated in China, so the supply of the medium-chain fatty acids in China has been mainly dependent on import from abroad. In addition, the plant extraction method has low yield and is easily affected by seasonal factors and natural disasters, which seriously limit the research and wide application of the medium-chain fatty acids. The use of microbial cell factories to produce the medium-chain fatty acids in a green, efficient and low-cost manner has become a very potential sustainable alternative method. A plurality of studies have constructed and optimized the Saccharomyces cerevisiae strains for producing the medium-chain fatty acids, and it is found that the low tolerance of the Saccharomyces cerevisiae to caprylic acid and capric acid is a key problem limiting the further improvement of the yield. In order to further improve the yield of the medium-chain fatty acids, it is necessary to break through the bottleneck of the low tolerance of the Saccharomyces cerevisiae to the medium-chain fatty acids, and the tolerance mechanism of the yeast strain to the caprylic acid and the capric acid needs to be analyzed, and a plurality of key genes that can be used as a rational design strategy are excavated, so as to lay a foundation for the breakthrough improvement of the yield of the medium-chain fatty acids. SUMMARY

[0003] In order to overcome the defects and deficiencies of the prior art, the purpose of the present application is to provide application of five caprylic acid tolerance related genes in improving caprylic acid tolerance of Saccharomyces cerevisiae.

[0004] The application provides a genetically engineered bacterium with improved caprylic acid tolerance, which expresses one or more genes of the following: a mannose transferase gene ALG2, a thiazole synthase gene THI4, an acid phosphatase gene PHO5, a superoxide dismutase gene SOD1 and a cytochrome C peroxidase gene CCP1.

[0005] In one embodiment, the starting strain is Saccharomyces cerevisiae BY4742.

[0006] In one embodiment, the genes are expressed by a pRS413 plasmid as a carrier.

[0007] In one embodiment, the gene ALG2 has the nucleotide sequence shown in Genbank accession number: NM_001180930.1.

[0008] In an embodiment, the gene THI4 has the nucleotide sequence shown in Genbank Accession No. NM_001181273.1.

[0009] In an embodiment, the gene PHO5 has the nucleotide sequence shown in Genbank Accession No. NM_001178441.3.

[0010] In an embodiment, the gene SOD1 has the nucleotide sequence shown in Genbank Accession No. NM_001181762.1.

[0011] In an embodiment, the gene CCP1 has the nucleotide sequence shown in Genbank Accession No. XM_022820242.1.

[0012] The present application also provides a method for constructing the recombinant Saccharomyces cerevisiae, comprising the following steps:

[0013] (a) using pRS413 plasmid as a carrier to construct a recombinant plasmid containing the coding gene of the protein;

[0014] (b) transforming the recombinant plasmid constructed in (a) into Saccharomyces cerevisiae cells.

[0015] The present application also provides a method for improving the tolerance of Saccharomyces cerevisiae to octanoic acid, which is to overexpress one or more genes of ALG2, THI4, PHO5, SOD1 and CCP1 in Saccharomyces cerevisiae.

[0016] In an embodiment, the Saccharomyces cerevisiae includes but is not limited to Saccharomyces cerevisiae BY4742.

[0017] In an embodiment, the expression is carried out using pRS413 plasmid as a carrier.

[0018] The present application also provides the use of the recombinant Saccharomyces cerevisiae as a chassis cell for constructing genetically engineered bacteria.

[0019] The present application also provides the use of the recombinant Saccharomyces cerevisiae in the field of fermentation for preparing octanoic acid or its derivatives.

[0020] Advantages:

[0021] The application takes the Kluyveromyces marxianus screened from Guizhou sour meat which can tolerate caprylic acid as the research object, and five key genes ALG2 gene (mannosyltransferase), THI4 gene (thiazole synthase), PHO5 gene (acid phosphatase), SOD1 gene (superoxide dismutase) and CCP1 gene (cytochrome C peroxidase) which can improve the caprylic acid tolerance of Saccharomyces cerevisiae strains are mined. The overexpression is carried out in Saccharomyces cerevisiae BY4742 cells by using molecular biology technology, and a plurality of Saccharomyces cerevisiae engineering strains which can tolerate caprylic acid are constructed, wherein the strain expressing pRS413-ALG2 can tolerate 0.45mM caprylic acid.

[0022] The Saccharomyces cerevisiae engineering strain with high caprylic acid tolerance constructed by the application can grow normally in a high-concentration caprylic acid culture medium and produce high-value chemicals, and can be used as a chassis cell of a high-value chemical production strain, and has great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the growth state of the engineering strain in the SD liquid culture medium with the concentration of 0.45mM caprylic acid.

[0024] Figure 2 is the growth state of the engineering strain in the SD solid culture medium with the concentration of 0.45mM caprylic acid.

[0025] Figure 3 is the growth state of the engineering strain which does not tolerate caprylic acid in the SD solid culture medium with the concentration of 0.45mM caprylic acid. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with the embodiments and drawings, but the embodiments of the application are not limited thereto. In order to make the purpose, technical scheme and effect of the application more clear, the application will be described in detail below in combination with the drawings and specific embodiments, the following embodiments are illustrative and not limiting, and the person skilled in the art can improve and change them according to the spirit of the application, and the improvements and changes should be regarded as within the scope of the application, the scope and essence of the application are limited by the claims.

[0027] SD-His-0 mM C8 auxotrophic solid medium: Yeast base nitrogen source 6.7 g / L, glucose 20 g / L, adenine sulfate 0.04 g / L, L-arginine hydrochloride 0.02 g / L, L-aspartate 0.1 g / L, L-glutamic acid g / L, L-leucine 0.06 g / L, L-lysine 0.03 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-serine 0.375 g / L, L-threonine 0.2 g / L, L-tryptophan 0.04 g / L, L-tyrosine 0.03 g / L, L-valine 0.15 g / L, uracil 0.02 g / L, agar 15 g / L.

[0028] SD-His-0.45 mM C8 auxotrophic solid medium: Yeast base nitrogen source 6.7 g / L, glucose 20 g / L, adenine sulfate 0.04 g / L, L-arginine hydrochloride 0.02 g / L, L-aspartate 0.1 g / L, L-glutamic acid g / L, L-leucine 0.06 g / L, L-lysine 0.03 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-serine 0.375 g / L, L-threonine 0.2 g / L, L-tryptophan 0.04 g / L, L-tyrosine 0.03 g / L, L-valine 0.15 g / L, uracil 0.02 g / L, agar 15 g / L, octanoic acid 0.45 mM.

[0029] SD-His-0.45 mM C8 auxotrophic liquid medium: Yeast base nitrogen source 6.7 g / L, glucose 20 g / L, adenine sulfate 0.04 g / L, L-arginine hydrochloride 0.02 g / L, L-aspartate 0.1 g / L, L-glutamic acid g / L, L-leucine 0.06 g / L, L-lysine 0.03 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-serine 0.375 g / L, L-threonine 0.2 g / L, L-tryptophan 0.04 g / L, L-tyrosine 0.03 g / L, L-valine 0.15 g / L, uracil 0.02 g / L, octanoic acid 0.45 mM.

[0030] Construction of recombinant plasmid of Example 1

[0031] (1) The gene fragments of ALG2, THI4, PHO5 and SOD1 were derived from Saccharomyces cerevisiae BY4742, and the gene fragment of CCP1 was from Kluyveromyces marxianus.

[0032] The ALG2, THI4, PHO5 and SOD1 genes were amplified using the BY4742 genome as a template, and the CCP1 gene was amplified using the K. marxianus genome as a template. The nucleotide sequence of the ALG2 gene is shown in GenBank No. NM_001180930.1, the nucleotide sequence of the THI4 gene is shown in GenBank No. NM_001181273.1, the nucleotide sequence of the PHO5 gene is shown in GenBank No. NM_001178441.3, the nucleotide sequence of the SOD1 gene is shown in GenBank No. NM_001181762.1, and the nucleotide sequence of the CCP1 gene is shown in GenBank No. XM_022820242.1.

[0033] The primers used for amplifying the above genes are as follows:

[0034] ALG2-F: 5'-CAAGCAAAAATGATTGAAAAGG-3';

[0035] ALG2-R: 5'-TTATATTTCTTCATAAGGGTAGGAG-3';

[0036] PHO5-F: 5'-CCAATGTTTAAATCTGTTG-3';

[0037] PHO5-R: 5'-CTATTGTCTCAATAGACTGGCG-3';

[0038] THI4-F: 5'-CATGTCTGCTACCTC-3';

[0039] THI4-R: 5'-CCTAAGCAGCAAAGTGTT-3';

[0040] SOD1-F: 5'-ATGGTTCAAGCAGTCGCAG-3';

[0041] SOD1-R: 5'-TTAGTTGGTTAGACCAATGAC-3';

[0042] CCP1-F: 5'-ATGTCGAGTTTTAGAGCAGC-3';

[0043] CCP1-R: 5'-TTACAGATCTTGTTCGTCAAG-3'.

[0044] (2) Using the genome of Saccharomyces cerevisiae BY4742 as a template, a ALG2 gene fragment containing a homologous arm, a THI4 gene fragment containing a homologous arm, a PHO5 gene fragment containing a homologous arm, and a SOD1 gene fragment containing a homologous arm were amplified; and using the genome of Kluyveromyces marxianus as a template, a CCP1 gene fragment containing a homologous arm as shown in SEQ ID NO. 1 was amplified.

[0045] The specific primers used are as follows:

[0046] pRS413-ALG2-F:

[0047] 5'-CACCAAGAACTTAGTTTCGAgaattcCAAGCAAAAATGATTGAAAAGG-3';

[0048] pRS413-ALG2-R:

[0049] 5'-CTAATTACATGAaggcctATTATATTTCTTCATAAGGGTAGGAG-3';

[0050] pRS413-PHO5-F:

[0051] 5'-CACCAAGAACTTAGTTTCGAgaattcCCAATGTTTAAATCTGTTG-3';

[0052] pRS413-PHO5-R:

[0053] 5'-GACATAACTAATTACATGAaggcctCTATTGTCTCAATAGACTGGCG-3';

[0054] pRS413-THI4-F:

[0055] 5'-CTTAGTTTCGAgaattcCATGTCTGCTACCTC-3';

[0056] pRS413-THI4-R:

[0057] 5'-CTAATTACATGAaggcctCCTAAGCAGCAAAGTGTT-3';

[0058] pRS413-SOD1-F:

[0059] 5'-CACCAAGAACTTAGTTTCGAgaattcATGGTTCAAGCAGTCGCAG-3';

[0060] pRS413-SOD1-R:

[0061] 5'-GCGTGACATAACTAATTACATGAaggcctTTAGTTGGTTAGACCAATGAC-3';

[0062] pRS413-CCP1-F:

[0063] 5'-CACCAAGAACTTAGTTTCGAgaattcATGTCGAGTTTTAGAGCAGC-3';

[0064] pRS413-CCP1-R:

[0065] 5'-GCGTGACATAACTAATTACATGAaggcctTTACAGATCTTGTTCGTCAAG-3';

[0066] Wherein, gaattc is EcoRI restriction site, gaattc is StuI restriction site.

[0067] (3) Construction of recombinant plasmids pRS413-ALG2, pRS413-THI4, pRS413-PHO5, pRS413-SOD1 and pRS413-CCP1: pRS413 vector was digested with EcoRI and StuI at 37°C for 2h, the recovered products were ligated with the ALG2, THI4, SOD1, PHO5 or CCP1 gene fragments containing homologous arms constructed in step (2) at 50°C for 5min, and then immediately cooled on ice. The ligation products were transformed into DH5α super-competent cells thawed on ice, and plated on 50ng / mL carbenicillin sodium resistant plates. After single colonies grew, the correct transformants were selected for plasmid extraction and sequencing. The obtained recombinant plasmids were named as pRS413-ALG2, pRS413-THI4, pRS413-SOD1, pRS413-PHO5 and pRS413-CCP1, respectively.

[0068] Example 2 Construction of recombinant strains

[0069] The recombinant plasmids pRS413-ALG2, pRS413-THI4, pRS413-PHO5, pRS413-SOD1 and pRS413-CCP1 constructed in Example 1 were respectively transformed into Saccharomyces cerevisiae BY4742 by LiAc transformation method, and were coated on 0mM-SD-His plates, and the engineering bacteria BY4742-ALG2, BY4742-THI4, BY4742-PHO5, BY4742-SOD1 and BY4742-CCP1-K.M were screened by using SD-His-0mM C8 auxotrophic solid medium. After single colonies were grown, they were used for subsequent tolerance verification experiments.

[0070] Example 3 Caprylic acid tolerance verification of recombinant strains

[0071] The single colony liquid of the engineering bacteria BY4742-ALG2, BY4742-THI4, BY4742-PHO5, BY4742-SOD1 and BY4742-CCP1-K.M constructed in Example 2 was respectively cultured in SD-His-0mM C8 auxotrophic liquid medium, 30°C, 200rmp shaking culture for 16h. After activation, the bacterial liquid was centrifuged (5000rmp, 5min) to remove the supernatant, washed twice with normal saline, centrifuged (5000rmp, 5min), and the bacterial body was collected. BY4742 was used as a control, and the growth state in SD-His-0.45mMC8 auxotrophic liquid medium was measured at 30°C.

[0072] The results are shown in Figure 1 Under the condition of containing 0.45mM C8, the OD 600nm of BY4742, BY4742-ALG2, BY4742-THI4, BY4742-PHO5, BY4742-SOD1 and BY4742-CCP1-K.M cultured for 72h were respectively 0.1, 2.5, 1.6, 2.3, 2.1 and 2.5.

[0073] The bacterial liquid of BY4742-pRS413, BY4742-ALG2, BY4742-THI4, BY4742-PHO5, BY4742-SOD1 and BY4742-CCP1-K.M obtained by culture was respectively adjusted to OD 600nm =1, and was gradiently diluted by different times (10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5) After that, 3 uL of each was spotted on SD-His-0 mM C8 auxotrophic solid medium and SD-His-0.45 mM C8 auxotrophic solid medium, and cultured at 30°C for 72 h, and the growth of each strain was compared. BY4742-pRS413 was used as a control. In the SD-His-0 mM C8 auxotrophic solid medium C8, the growth of each engineered strain was not much different, and in the SD-His-0.45 mM C8 auxotrophic solid medium, the growth of the five engineered strains was better than that of the control strain BY4742-pRS413. Figure 2 ).

[0074] Comparative Example:

[0075] The specific implementation is the same as that in Examples 1-2, except that the CCP1 gene (nucleotide sequence as shown in Genbank accession number: NM_001179856.1) is amplified using the genome of Saccharomyces cerevisiae BY4742 as a template, and the ALG2 gene (nucleotide sequence as shown in Genbank accession number: XM_022819860.1) is amplified using the genome of Kluyveromyces marxianus as a template, and recombinant Saccharomyces cerevisiae expressing the above genes is constructed, respectively, and cultured according to the method of Example 3. The results are shown in Table 2. Figure 3 The strains do not exhibit significant caprylic acid tolerance.

[0076] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A recombinant Saccharomyces cerevisiae having improved tolerance to octanoic acid, characterized in that: Based on the starting strain, one or more of the following proteins are overexpressed: mannosyltransferase ALG2, thiazole synthase THI4, acid phosphatase PHO5, superoxide dismutase SOD1 and cytochrome C peroxidase CCP1.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that The starting strain is Saccharomyces cerevisiae BY4742.

3. The recombinant Saccharomyces cerevisiae according to claim 1 or 2, characterized in that The pRS413 plasmid is used as a vector to express the protein or the gene encoding the protein.

4. The recombinant Saccharomyces cerevisiae according to claim 1 or 3, characterized in that The ALG2 gene has the nucleotide sequence shown in Genbank Accession No. NM_001180930.1; the THI4 gene has the nucleotide sequence shown in Genbank Accession No. NM_001181273.1; the PHO5 gene has the nucleotide sequence shown in Genbank Accession No. NM_001178441.3; the SOD1 gene has the nucleotide sequence shown in Genbank Accession No. NM_001181762.1; and the CCP1 gene has the nucleotide sequence shown in Genbank Accession No. XM_022820242.

1.

5. A method for constructing the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 4, characterized in that: The steps include: (a) using the pRS413 plasmid as a vector, constructing a recombinant plasmid containing the gene encoding the protein; (b) The recombinant plasmid constructed in (a) was transformed into Saccharomyces cerevisiae cells.

6. A method for improving the tolerance of Saccharomyces cerevisiae to octanoic acid, characterized in that: One or more genes among ALG2, THI4, PHO5, SOD1, and CCP1 are overexpressed in Saccharomyces cerevisiae.

7. The method according to claim 6, characterized in that The Saccharomyces cerevisiae includes but is not limited to Saccharomyces cerevisiae BY4742.

8. The method according to claim 6 or 7, characterized in that The expression is carried out using the pRS413 plasmid as a vector.

9. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3 as a chassis cell for constructing genetically engineered bacteria.

10. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3 in the production of octanoic acid or its derivatives in the fermentation field.

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