CN114806910B

CN114806910B - Candida tropicalis engineering bacteria for producing L-limonene and construction method thereof

Info

Publication number: CN114806910B
Application number: CN202210187114.6A
Authority: CN
Inventors: 陈献忠; 杨海泉; 郭晋蓉; 夏媛媛; 沈微; 曹钰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2023-07-04
Anticipated expiration: 2042-02-28
Also published as: CN114806910A

Abstract

The invention discloses candida tropicalis engineering bacteria for producing L-limonene and a construction method thereof, and belongs to the technical field of microorganisms. The invention takes candida tropicalis ATCC 20336 as an original strain, and a limonene synthase from spearmint and a site-directed mutagenesis farnesyl pyrophosphate synthase are integrated on the genome of candida tropicalis CU-200 through a CRISPR-Cas9 technology. After the expression, the L-limonene yield of the recombinant candida tropicalis reaches 141.48mg/L. The preparation method has the characteristics of low cost, high efficiency, simple operation, wide prospect and the like.

Description

Candida tropicalis engineering bacteria for producing L-limonene and construction method thereof

Technical Field

The invention relates to candida tropicalis engineering bacteria for producing L-limonene and a construction method thereof, belonging to the technical field of microorganisms.

Background

L-limonene, also known as limonene, is an important monocyclic monoterpene compound that is widely distributed in nature, has been found to be present in over 300 plants, and is identified by the FDA as a safe compound. L-limonene has various pharmacological activities such as bacteriostasis, anti-inflammation, anticancer, antioxidation and the like, but the chemical property of the L-limonene is unstable, so that the application has certain limitation. There are three forms of L-limonene found in nature: dextro-L-limonene (D-limonene) and levo-L-limonene (L-limonene) are optical isomers, and a racemate (D/L-limonene). L-limonene is mainly contained in volatile oil of spearmint, peppermint, pine needle, oak and other plants or trees, has aromatic smell similar to pine oil and rosin, can be widely used as a high-quality spice in foods, beverages and cosmetics, and has high market value.

Currently, the industrial production of L-limonene is mainly achieved by direct extraction from plant materials, although L-limonene prepared directly by plant extraction is considered to be natural and more popular with consumers. However, this method has many disadvantages such as limited sources of raw materials, low contents of target substances, and great difficulty in separation and extraction. In addition, L-limonene can be synthesized chemically, but the chemical synthesis requires not only high cost (e.g., high pressure and temperature) but also environmental problems (e.g., use of a large amount of organic solvents). In recent years, as people pay more attention to health, energy and environmental problems, research on natural oxygen-containing monoterpenes with important biological functions is being carried out.

Therefore, the production of L-limonene by microbial metabolic engineering technology is a new idea and method. The microbial transformation method has the advantages of high transformation rate, mild reaction condition, low carbon, sustainable development and the like, so that the microbial transformation method has wide development prospect.

Disclosure of Invention

In order to obtain the method for producing the limonene with high yield, which has the advantages of simple operation, high conversion efficiency, low production cost and wide industrial application prospect, the invention provides the recombinant candida tropicalis (Candida tropicalis), expressing the mutant ERG20WW of the farnesyl pyrophosphoric acid synthase and the limonene synthase from spearmint (Mentha spicata).

In one embodiment, the mutant ERG20WW comprises the amino acid sequence shown in SEQ ID No. 3.

In one embodiment, the gene encoding the ERG20WW mutant is obtained by mutating phenylalanine encoding position 95 and asparagine encoding position 126 on the basis of the ERG20 nucleotide sequence shown in SEQ ID No. 1.

In one embodiment, the mutation is a mutation of both phenylalanine at position 95 and asparagine at position 126 to tryptophan.

In one embodiment, the NCBI accession number of the limonene synthase gene LS derived from Mentha spicata is GeneID:L13459.

In one embodiment, candida tropicalis (Candida tropicalis) ATCC 20336 is used as the host.

In one embodiment, the candida tropicalis ATCC 20336 further knocks out the acyl-coa oxidase genes POX5 and the D-lactate dehydrogenase gene DLD1.

The invention also provides a method for constructing the recombinant candida tropicalis, which comprises the following steps:

(1) Synthesizing a limonene synthase gene shown as a GeneID L13459, and connecting the limonene synthase gene to a plasmid to obtain a recombinant plasmid;

(2) Integrating an ERG20WW gene with a nucleotide sequence shown as SEQ ID NO.1 onto a candida tropicalis genome through CRISPR-Cas9 to obtain an expression host;

(3) And (3) transforming the linearized recombinant plasmid constructed in the step (1) into the recombinant candida tropicalis constructed in the step (2) by adopting a lithium acetate transformation method to construct the limonene-producing engineering bacteria.

In one embodiment, the nucleotide sequence of the limonene synthase is shown as SEQ ID NO. 2;

the invention also provides a method for producing limonene, which is to ferment the recombinant candida tropicalis in a fermentation medium to prepare the limonene.

In one embodiment, the fermentation medium is a YPD medium.

In one embodiment, the method comprises the steps of:

(1) Inoculating the single colony of the recombinant candida tropicalis into a seed culture medium, and culturing for 24 hours at 30 ℃ and 200rpm to obtain seed liquid;

(2) The seed liquid obtained in the step (1) is treated byAccording to OD ₆₀₀ 1.0 was inoculated into YPD medium containing 6%12% n-dodecane and fermented at a temperature of 25℃and a rotation speed of 150250r/min for 46d.

In one embodiment, the seed medium and the fermentation medium are both YPD medium.

The invention provides application of the recombinant candida tropicalis in agriculture, food industry, medicine and health field or cosmetics field.

In one embodiment, the use includes, but is not limited to, the production of L-limonene containing products.

The beneficial effects are that:

(1) The invention provides a method for producing limonene, which is simple to operate and high in conversion efficiency, and the method adopts candida tropicalis (C.tropicalis) ATCC 20336 as a host, has high resistance to limonene, can reach 141.48mg/L, and is beneficial to the application of the strain in agriculture, food industry, medicine and health fields and cosmetics.

(2) The recombinant candida tropicalis strain constructed by the invention is subjected to metabolic engineering transformation, the acyl-coa oxidase gene POX5 and the D-lactate dehydrogenase gene DLD1 are knocked out, the heterologous L-limonene synthase gene LS and the farnesyl pyrophosphoric acid synthase mutant gene ERG20WW are integrated and expressed, the lemon yield can be obviously improved, the yield of limonene is improved from 0.092mg/L to 141.48mg/L of the original strain GJR-LS-01, the yield is improved by more than 1000 times, and the idea is provided for the study of the limonene production strain while the yield is improved.

Drawings

FIG. 1 shows the metabolic pathways of producing limonene by tropical yeast after introducing mevalonate metabolic pathway genes and limonene synthesis genes.

FIG. 2 is a graph showing the results of shake flask fermentation of strain GJR-LS-01 to produce limonene using glucose as the sole carbon source and strain GJR-tLS-01.

FIG. 3 is a graph showing the results of producing limonene from strain GJR-EW-tLS-04 at different fermentation times.

FIG. 4 is a graph showing the results of shake flask fermentation of strain GJR-EW-tLS-04 to produce limonene using different concentrations of glucose as a carbon source.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The media referred to in the examples are as follows:

YPD Medium (g/L): glucose 20, yeast extract 10 and peptone 20.

MM medium (g/L): glucose 20, yeast basal nitrogen source medium (yeast nitrogen base, YNB) 6.7 and ammonium sulfate 10.

SM medium (g/L): 60mg/L uracil was added on the basis of MM medium.

LB medium (g/L): peptone 10, yeast extract 5 and sodium chloride 10.

And (3) detection of the content of limonene:

the upper n-dodecane layer was taken for gas-mass spectrometry (GC-MS) detection. The detection conditions are as follows: TG-5MS gas chromatographic column (30 m. Times.0.25 mm. Times.0.25 μm) with carrier gas He, temperature program: after maintaining the temperature at 50 ℃ for 1min, the temperature is increased to 100 ℃ according to 6 ℃/min, and then the temperature is increased to 280 ℃ according to 20 ℃/min, and the operation is carried out for 5min.

The primer sequences involved in the following examples are shown in Table 1:

TABLE 1 primer sequences

EXAMPLE 1 construction of limonene producing engineering bacteria

Construction of the knockout box: taking a POX5 gene knockout box as an example, taking a C.tropicalis ATCC 20336 genome as a template, and taking POX5-F and POX5-R as primers for amplification, and carrying out agarose gel electrophoresis verification on the PCR product, and carrying out gel recovery after the verification is correct, wherein the product is marked as POX5. Fragment POX5 was ligated with pM19T simple vector using solution I, the successfully ligated product fragment was transformed into E.coli JM109 competent cells, the transformants were diluted and plated on LB solid medium supplemented with ampicillin and positive transformants were selected (using colony PCR method). The correct single colony of colony PCR is picked up, inoculated in liquid LB culture medium added with ampicillin for 8-12h, a small amount of plasmid extraction kit is used for extracting recombinant plasmid, mlu I and Pst I are utilized for enzyme digestion verification, then sequencing analysis is carried out on the plasmid, and the correct plasmid is confirmed to be named as Ts-POX5. The recombinant plasmid Ts-POX5 is used as a template, reverse primers rPOX5-F and rPOX5-R (built-in PstI and XbaI cleavage sites) are used for inverse PCR to obtain a fragment POX5U-Ts-POX5D, and the fragment is recovered by nucleic acid electrophoresis gel and purified by a PCR cleaning kit. Plasmid Ts-gda324-URA3 is digested by using PstI and Mlu I and subjected to restriction enzyme PstI and Xba I, and after gel recovery, the linear vector POX5U-Ts-POX5D and the DNA fragment gda324-URA3 are subjected to one-step cloning and connection by using a one-step connection kit, so that recombinant plasmid Ts-POX5-gda324-URA3 is obtained. The recombinant plasmid was digested with restriction enzyme Mlu I to obtain knockout cassette POX 5-gda-324-URA 3-POX5. Constructing a DLD1 knockout box by using a similar strategy and using Ts-ERG20WW as a template and primers rDLD1-F and rDLD 1-R; POX5 knockout cassette and DLD1 knockout cassette were used to construct Candida tropicalis with modified uracil deficiency type C.tropicalis ATCC 20336, designated CU-200.

Linearization vector preparation: synthesizing a limonene synthase LS with a nucleating amino acid sequence shown as SEQ ID NO. 2;

the method comprises the steps of using a full-length L-limonene synthase LS Gene as a template, using LS-F and LS-R as primers, amplifying the L-limonene synthase LS Gene shown as a Gene ID L13459 by PCR, purifying a PCR product, and connecting the purified product with a fragment POX5-gda-URA3-PGAPDH-carRP-TENO1A by a one-step cloning kit to obtain a recombinant plasmid Ts-POX5-gda-URA3-PGAPDH-LS-TENO1A. POX5-PGAPDH-LS-TENO1A-gda324-URA3-POX5 linearization fragment was obtained by single cleavage with Mlu I, and the obtained fragment was purified.

The LiCl transformation method is adopted, uracil deficiency strain C.tropicalis CU-200 is used as an initial strain, URA3 gene is used as a screening mark, the DNA purification fragment is transferred into a host, and the culture is carried out for 3d by coating on MM solid culture medium in a 30 ℃ incubator.

Picking up the transformant on the MM solid medium, extracting genome, carrying out PCR verification, identifying the correct transformant, coating the transformant on the medium containing 5-FOA, popping up the screening marker gene URA3, carrying out preliminary PCR verification, popping up the URA3 gene, sequencing a PCR product, and selecting the correct strain for preservation. Re-use URA3 on the basis of the above, knock-out of DLD1 gene, and integration of subsequent genes.

Site-directed mutagenesis of ERG20 (mutation of phenylalanine 95 to tryptophan at asparagine 126): the Ts-ERG20 shown in SEQ ID No.1 is used as a template, primers ERG20WW-F and ERG2WW-R are designed to carry out full plasmid PCR, the PCR products are subjected to enzyme digestion identification and gel recovery, and the PCR purification kit is used for product purification. The purified product was transformed into E.coli JM109 competent cells, the transformants were diluted and plated on LB solid medium supplemented with ampicillin and positive transformants were selected (using colony PCR method). The correct single colony of colony PCR is picked up, inoculated in liquid LB culture medium added with ampicillin for 8-12h, a small amount of plasmid extraction kit is used for extracting recombinant plasmid, the plasmid is sequenced and analyzed, the correct plasmid is selected and named as Ts-ERG20WW, and the transformant is preserved in a refrigerator at-80 ℃.

EXAMPLE 2 construction of recombinant candida tropicalis and fermentation production of limonene

Transformation of recombinant plasmid Ts-ERG20WW of example 1 into Candida tropicalis CU-200 constructed in example 1, streaking cultivation of the obtained recombinant strain GJR-tLS-04 on YPD plates, picking single colony from solid plates, transferring into 10 mL YPD liquid medium, culturing at 200r/min at 30deg.C for about 1d, and obtaining OD ₆₀₀ The seed solution is transferred to 30mL YPD medium to make the initial OD ₆₀₀ 1.0 and 10% n-dodecane by volume was added for the purpose of preventing volatilization of the target product L-limonene and alleviating toxicity of L-limonene to C.tropicalis, and then placed in a shaking table at 30deg.C for 200r/min cultureEach single colony was incubated for 120h with 3 biological replicates.

The biomass change in the fermentation process is characterized by utilizing the light absorption intensity OD600 of an ultraviolet-visible light spectrophotometer under the condition of 600nm, the measurement range is kept between 0.2 and 0.8, and the biomass change is diluted and measured when the thallus concentration is too high. The residual glucose content in the fermentation broth is measured by an SBA-90 biosensor, the measurement range is 40-00mg/Dl, and the fermentation broth is diluted and then measured when the concentration of the sample is too high.

The results showed that the L-limonene yield of the overexpressed recombinant strain of ERG20WW enzyme mutant was 125.31, 7.33, 2.58mg/L in cytoplasm, peroxisome and mitochondria, resulting in L-limonene yield compared to the unmutated control strain GJR-tLS-03 (0.832 mg/L). The technical scheme of the invention has obvious effect of enhancing the synthesis of the limonene, and the over-expression of the genes of the synthesized precursor substances is beneficial to the synthesis of the limonene.

EXAMPLE 3 recombinant candida tropicalis production of limonene under different fermentation conditions

The specific embodiment is the same as in example 2, with recombinant strain GJR-tLS-04 as fermenting microorganism, except for the fermentation time and the initial glucose concentration, respectively. The method comprises the following steps:

the fermentation time is respectively controlled as follows: 24h, 48h, 72h, 96h, 120h, 144h.

The initial glucose concentration in the fermentation medium was controlled as follows: 20g/L, 40g/L, 60g/L, 80g/L, 100g/L.

As shown in FIG. 3, the recombinant strain GJR-tLS-04 is fermented for 120 hours, and the yield of L-limonene reaches 100.22mg/L; compared with the yield of 22.7mg/L in 24 hours of fermentation, the method improves 354.54 percent.

As shown in FIG. 4, the recombinant strain GJR-tLS-04 was fermented in a medium with an initial glucose concentration of 60g for 120h to yield 141.48mg/L of L-limonene; compared with the concentration of 20g, the yield is 100.22mg/L, which is improved by 41.48 percent.

Comparative examples different recombinant candida tropicalis production of limonene

The following recombinant bacteria constructed according to the strategy of example 1 were used as control strains, respectively, and fermented according to the method of example 2, and the biomass and L-limonene yield during the fermentation were examined.

Recombinant strain GJR-LS-01: the acyl-CoA oxidase gene POX5 is knocked out on the basis of the candida tropicalis uracil (URA 3) deficient strain CU-200, and the heterologous L-limonene synthase gene LS shown in SEQ ID NO.1 is integrated and expressed.

Recombinant strain GJR-tLS-01: the acyl-CoA oxidase gene POX5 is knocked out on the basis of the candida tropicalis uracil (URA 3) deficient strain CU-200, and the truncated heterologous L-limonene synthase gene tLS shown in SEQ ID NO.4 is integrated and expressed.

Recombinant strain GJR-tLS-03: based on recombinant strain GJR-tLS-01, farnesyl pyrophosphoric acid synthetase gene ERG20 shown in SEQ ID NO.1 is expressed.

Recombinant strain GJR-tLS-04: the farnesyl pyrophosphoric acid synthetase mutant gene ERG20WW shown in SEQ ID NO.3 is expressed on the basis of recombinant strain GJR-tLS-01.

The result shows that the yield of the recombinant strain GJR-LS-01 is only 0.092mg/L; the yield of the recombinant strain GJR-tLS-01 is only 0.257mg/L; the GJR-tLS-03 yield was 0.832mg/L; the GJR-tLS-04 yield was 100.222mg/L.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> engineering strain of candida tropicalis producing L-limonene and construction method thereof

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aaactcctgg caatccattt ctggtccatg tatgttgacc cacattttct tcagagttac 1260

tgattccttc accaaggaaa ctgttgattc cttgtacaag taccatgatt tggttagatg 1320

gtcctccttt gttttgagat tggctgatga tttgggtacc tctgttgaag aagtttcaag 1380

aggtgatgtt ccaaagtcct tgcaatgtta catgtctgat tacaatgctt ctgaagctga 1440

agctagaaag catgttaagt ggttgattgc tgaagtttgg aagaagatga atgctgaaag 1500

agtttccaag gattccccat ttggtaagga tttcattggt tgtgctgttg atttgggtag 1560

aatggctcaa ttgatgtacc acaatggtga tggtcatggt acccaacacc caattattca 1620

ccaacaaatg actagaacct tgtttgaacc atttgcttag gtgtactgaa attggtgg 1678

Claims

1. Recombinant candida tropicalisCandida tropicalis) Characterized in that the mutant ERG20WW of farnesyl pyrophosphoric acid synthetase is expressed and is derived from spearmint @ isMentha spicata) Is a limonene synthase of (2); the amino acid sequence of the mutant ERG20WW is shown as SEQ ID NO. 3; the NCBI accession number of the Gene encoding the limonene synthase is Gene ID: L13459; using candida tropicalisCandida tropicalis) ATCC 20336 as host; the candida tropicalis ATCC 20336 also knocks out the acyl-CoA oxidase genePOX5And D-lactate dehydrogenase geneDLD1。

2. A method of constructing the recombinant candida tropicalis of claim 1 comprising the steps of:

(1) Synthesizing a limonene synthase Gene shown as a Gene ID L13459, and connecting the limonene synthase Gene to a plasmid to obtain a recombinant plasmid;

(3) And (3) transferring the recombinant plasmid obtained in the step (1) into the expression host obtained in the step (2) by adopting a lithium acetate conversion method, and constructing to obtain the recombinant candida tropicalis.

3. A process for producing limonene, characterized in that the process comprises fermenting the recombinant candida tropicalis of claim 1 in a fermentation medium at a temperature of 25-30 ℃ and at 200-250 rpm of at least 24 h.

4. A method according to claim 3, comprising the steps of:

(1) Inoculating the single colony of the recombinant candida tropicalis into a seed culture medium, and culturing for 20-24 hours at the temperature of 25-30 ℃ and the speed of 200-250 rpm to obtain seed liquid;

(2) Inoculating the seed liquid obtained in the step (1) into a YPD culture medium containing 6% -12% of n-dodecane, and fermenting for 4-6 d at the temperature of 25-30 ℃ and the rotating speed of 150-250 r/min.

5. Use of the recombinant candida tropicalis of claim 1 or the method of claim 3 or 4 in the production of L-limonene in agriculture, food industry, in the medical and hygienic field or in the cosmetic field.