Yarrowia lipolytica engineering bacterium for producing 3-hydroxy-beta-ionone as well as construction method and application of Yarrowia lipolytica engineering bacterium

Abstract

The invention discloses yarrowia lipolytica engineering bacteria for producing 3-hydroxyl-beta-ionone as well as a construction method and application thereof, and belongs to the field of gene engineering. According to the invention, a CsCCD1 gene with a nucleotide sequence as shown in SEQ ID NO.1 is expressed in yarrowia lipolytica HA, and an ERG12 gene and an HMG1 gene are over-expressed, so that the yarrowia lipolytica engineering bacterium for producing 3-hydroxy-beta-ionone is obtained. A fermentation experiment result shows that the yarrowia lipolytica engineering bacterium provided by the invention takes glucose as a carbon source, the extracellular yield of 3-hydroxy-beta-ionone is high, and the yield in a 5L fermentation tank reaches 1.66 g / L. A new biological material and a synthesis strategy are provided for fermentation production of the 3-hydroxyl-beta-ionone, the production cost is low, the method is simple, the yield is high, a foundation is laid for industrial production of the 3-hydroxyl-beta-ionone, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to an engineered Yarrowia lipolytica strain producing 3-hydroxy-β-ionone, a construction method and an application thereof. Background Art

[0002] Yarrowia lipolytica is a dimorphic, non-pathogenic Ascomycete yeast commonly found in hydrophobic substrate-rich environments such as dairy products and oily wastes.

[0003] Carotenoid cleavage dioxygenases (CCDs) belong to the non-heme iron-dependent oxidase family. In plants, they can be divided into two major classes based on their functional characteristics: Class I includes CCD1, CCD2, CCD4, CCD7, CCD8, and CCD10, and Class II consists of 9-cis-epoxycarotenoid dioxygenases (NCEDs). Studies have shown that CCD1 and CCD4 have similar substrate selectivity and can catalyze the oxidative cleavage of double bonds at the C5-C6 (C5′-C6′), C7-C8 (C7′-C8′), and C9-C10 (C9′-C10′) positions in carotenoid molecules, generating apocarotenoid products of varying chain lengths.

[0004] Apocarotenoids are derivative metabolites produced by the oxidative cleavage of carotenoids. Their synthesis pathways and functional regulation have become cutting-edge areas of synthetic biology research. In nature, apocarotenoids are primarily produced through the specific catalysis of carotenoid cleavage dioxygenases or non-enzymatic reactions mediated by reactive oxygen species. Their carbon chain length and diversity of functional groups confer unique biological activities. Apocarotenoids themselves have shown potential as high-value-added products (such as flavors and drug lead compounds) or signaling molecules in crop stress tolerance enhancement and the construction of microbial cell factories.

[0005] 3-Hydroxy-β-ionone (molecular formula C 13 H 20 β-ionone (O2) is an apocarotenoid with a structure similar to β-ionone. Currently, research on 3-hydroxy-β-ionone is limited. Existing reports indicate that this compound has been isolated from plants including mosses, Moringa oleifera, and Spirulina platensis. Furthermore, reports have confirmed that 3-hydroxy-β-ionone possesses anti-inflammatory and anti-cancer properties, suggesting promising application prospects in anticancer drugs and health foods.

[0006] 3-Hydroxy-β-ionone can be produced through plant extraction or chemical synthesis, but both methods are associated with environmental pollution and damage to nature. Biosynthesis is an environmentally friendly production method, but research on the production of 3-Hydroxy-β-ionone using microorganisms has not yet been reported. Summary of the Invention

[0007] The purpose of the present invention is to provide an engineered Yarrowia lipolytica strain that produces 3-hydroxy-β-ionone, and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a new biomaterial and synthesis strategy for the fermentation production of 3-hydroxy-β-ionone, with low production cost, simple method and high yield, laying the foundation for the industrial production of 3-hydroxy-β-ionone.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for constructing an engineered Yarrowia lipolytica strain that produces 3-hydroxy-β-ionone, comprising the following steps:

[0010] The CsCCD1 gene was expressed in the starting strain of Yarrowia lipolytica to obtain the genetically engineered strain HCS;

[0011] Overexpressing the ERG12 gene and the HMG1 gene in the genetically engineered bacteria HCS to obtain the Yarrowia lipolytica producing 3-hydroxy-β-ionone;

[0012] Wherein, the nucleotide sequence of the CsCCD1 gene is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the ERG12 gene is shown in SEQ ID NO.2;

[0014] The nucleotide sequence of the HMG1 gene is shown in SEQ ID NO.3.

[0015] Preferably, the expressing the CsCCD1 gene in the starting strain of Yarrowia lipolytica comprises the step of transferring the plasmid pINA1312-CsCCD1 into the starting strain of Yarrowia lipolytica.

[0016] Preferably, the starting strain of Yarrowia lipolytica is a genetically engineered strain of Yarrowia lipolytica that produces zeaxanthin.

[0017] Preferably, the overexpression of ERG12 gene and HMG1 gene in the genetically engineered bacteria HCS comprises the step of introducing plasmid pINA1269-HMG1-MBP-ERG12 into the genetically engineered bacteria HCS.

[0018] The present invention also provides an engineered Yarrowia lipolytica strain producing 3-hydroxy-β-ionone obtained according to the above construction method.

[0019] The present invention also provides a use of the above-mentioned Yarrowia lipolytica engineered bacteria in the preparation of products producing 3-hydroxy-β-ionone.

[0020] The present invention also provides a starter for producing 3-hydroxy-β-ionone, wherein the starter contains the above-mentioned engineered Yarrowia lipolytica as a main active ingredient.

[0021] The present invention also provides a use of the above-mentioned Yarrowia lipolytica engineered bacteria or the above-mentioned starter in producing 3-hydroxy-β-ionone.

[0022] The present invention also provides a method for producing 3-hydroxy-β-ionone, comprising the following steps:

[0023] The Yarrowia lipolytica engineered bacteria are inoculated into a culture medium containing glucose, peptone and yeast extract for fermentation production.

[0024] Preferably, in the culture medium, the mass fraction of peptone is 4% and the mass fraction of yeast extract is 2%;

[0025] The initial concentration of glucose in the culture medium is 20 g / L, and glucose is fed during the fermentation production to ensure that the concentration of glucose in the culture medium is not less than 1 g / L and not more than 5 g / L.

[0026] The present invention discloses the following technical effects:

[0027] The present invention first constructs a heterologous synthesis pathway of 3-hydroxy-β-ionone in Yarrowia lipolytica; by overexpressing the ERG12 gene fused with the HMG1 gene and the MBP tag, the present invention allows as much carbon flux as possible to flow into the 3-hydroxy-β-ionone synthesis pathway, reduces substrate waste, and can significantly increase the yield of 3-hydroxy-β-ionone.

[0028] The genetically engineered Yarrowia lipolytica bacteria producing 3-hydroxy-β-ionone provided by the present invention has a high extracellular yield, which can achieve a 3-hydroxy-β-ionone yield of 506.3 mg / L in a shake flask and a yield of 1.66 g / L in a 5L fermentor. The genetically engineered Yarrowia lipolytica bacteria producing 3-hydroxy-β-ionone provided by the present invention can secrete 3-hydroxy-β-ionone extracellularly, and this process can significantly reduce production costs. The present invention provides new biomaterials and synthetic strategies for the fermentation production of 3-hydroxy-β-ionone, with low production costs, simple methods, and high yields, laying the foundation for the industrial production of 3-hydroxy-β-ionone and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The following are statistical graphs of 3-hydroxy-β-ionone production and product appearance of Yarrowia lipolytica HA transformed with different genes in Example 1; HA is the starting strain; β-carotene is β-carotene; Zeaxanthin is zeaxanthin; and 3-hydroxy-β-ionone is 3-hydroxy-β-ionone.

[0031] Figure 2 Statistical graph of intracellular and extracellular 3-hydroxy-β-ionone production of strain HCS transformed with different plasmids in Example 2; Intracellular means intracellular, and Extracellular means extracellular;

[0032] Figure 3 1 is a liquid chromatogram of the fermentation product of strain HCS2 before and after purification in Example 4;

[0033] Figure 4 The following are statistical graphs of 3-hydroxy-β-ionone production and product appearance of strain HCS2 in a fermenter in Example 5; wherein, 3-hydroxy-β-ionone is 3-hydroxy-β-ionone; Zeaxanthin is zeaxanthin; β-carotene is β-carotene; and Glucose is glucose. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The starting strain used in the embodiment of the present invention is Yarrowia lipolytica HA (a genetically engineered strain of Yarrowia lipolytica that produces zeaxanthin), and its construction method has been disclosed in the patent "CN115369048A, a genetically engineered strain of Yarrowia lipolytica that produces zeaxanthin, its construction method and application", and the publication date is November 22, 2022.

[0040] The plasmid pINA1269-HMG1-MBP-ERG12 used in the examples of the present invention is disclosed in patent "CN112300952A, A Genetically Engineered Yarrowia lipolytica Strain Producing α-Pinene and Its Applications," published on February 2, 2021. The applicant commits to distributing the above-mentioned biological material to the public within 20 years from the filing date of this invention.

[0041] The YPD culture medium of the present invention comprises 2% glucose, 2% peptone and 1% yeast extract, with the remainder being water; the percentages are by mass.

[0042] The CCD gene of the present invention is a carotenoid cleavage dioxygenase gene.

[0043] Example 1 Construction of strains HOF, HPH, HPA, HVV, HRD, HGJ, HBD, HPP, HCC and HCS

[0044] 1. Plasmid Construction

[0045] OfCCD1 from Osmanthus fragrans (NCBI accession number: OM256439.1), PhCCD1 from Petunia hybrida (NCBI accession number: AY576003.1), PaCCD1 from Prunus armeniaca (NCBI accession number: MH824416.1), VvCCD1 from Vitis vinifera (NCBI accession number: KF008001.1), RdCCD1 from Rosa damascena (NCBI accession number: EU327776.1), GjCCD4 from Gardenia jasminoides (NCBI accession number: KY631925.1), and GjCCD5 from Buddleja The codon-optimized sequences of BdCCD4 from davidii (NCBI accession number: KX374548.1), PpCCD4 from Prunus persica (NCBI accession number: JX309999.1), CsCCD2 from Crocus sativus (NCBI accession number: KJ541749.1), and CsCCD1 (as shown in SEQ ID NO. 1) were constructed into plasmid pINA1312 via PmlⅠ and BamHI, respectively.

[0046] SEQ ID NO.1:

[0047]

[0048] The plasmids pINA1312-OfCCD1, pINA1312-PhCCD1, pINA1312-PaCCD1, pINA1312-VvCCD1, pINA1312-RdCCD1, pINA1312-GjCCD4, pINA1312-BdCCD4, pINA1312-PpCCD4, pINA1312-CsCCD2 and pINA1312-CsCCD1 were obtained accordingly.

[0049] 2. Construction and screening of genetically engineered bacteria

[0050] The plasmids obtained in step 1 were transformed into Yarrowia lipolytica HA to obtain recombinant strains HOF, HPH, HPA, HVV, HRD, HGJ, HBD, HPP, HCC (transformed with pINA1312-CsCCD2), and HCS (transformed with pINA1312-CsCCD1). Transformation was performed using the Frozen EZYeast Transformation II™ kit (purchased from Zymo Research) according to the kit instructions.

[0051] Among them, the strains that could be detected to produce 3-hydroxy-β-ionone included HOF, HPH, HPA, HVV, HRD and HCS, with yields of 347.65 mg / L, 155.36 mg / L, 378.24 mg / L, 133.28 mg / L, 41.62 mg / L and 416.83 mg / L, respectively. Figure 1 After comparison, the HCS strain had the highest 3-hydroxy-β-ionone production and will be used for subsequent transformation.

[0052] Example 2 Construction of strain HCS2

[0053] The linearized plasmid pINA1269-HMG1-MBP-ERG12 was transformed into the Yarrowia lipolytica genetically engineered strain HCS obtained in Example 1 to overexpress the ERG12 gene (SEQ ID NO. 2) and the HMG1 gene (SEQ ID NO. 3) to obtain strains HCS2 and HCS3.

[0054] SEQ ID NO.2:

[0055]

[0056] SEQ ID NO.3:

[0057]

[0058] The extracellular and intracellular 3-hydroxy-β-ionone production of HCS2 were 506.3 mg / L and 46.1 mg / L, respectively. Figure 2 As shown, HCS2 will be used for subsequent experiments.

[0059] Example 3: Determination of 3-Hydroxy-β-ionone

[0060] 1. Yarrowia lipolytica HA, strains HOF, HPH, HPA, HVV, HRD, HGJ, HBD, HPP, HCC and HCS prepared in Example 1, and strain HCS2 prepared in Example 2 were inoculated into 2 mL YPD medium and cultured for 24 h. The initial OD 600 The inoculum size was 0.01 and the culture was inoculated into new 50 mL YPD medium for 4 days.

[0061] 2. Take 1 mL of fermentation broth into a centrifuge tube and centrifuge at 12000 rpm for 10 min to separate the bacteria and the supernatant.

[0062] 3. Remove the supernatant and filter it through a 0.22 μm aqueous filter to obtain a sample.

[0063] 4. Using LC-20 high performance liquid chromatograph and C 18 A reversed-phase column (ZORBAX SB-Aq, 4.6 × 250 mm, 5-Micron) was used for qualitative and quantitative analysis of precursor carotenoids and 3-hydroxy-β-ionone. The program parameters were as follows:

[0064] Mobile phase: acetonitrile (phase A) and water (phase B); gradient elution program: 0-1 min 50% phase A, 1-11 min 0%-100% phase A, 11-20 min 100% phase A; detection wavelength: 285 nm; flow rate: 0.5 mL / min; injection volume: 20 μL.

[0065] Example 43 Purification of hydroxy-β-ionone

[0066] 1. The strain HCS2 prepared in Example 2 was inoculated into 2 mL YPD medium and cultured for 24 h. 600 The inoculum size was 0.01 and inoculated into new 500 mL YPD medium for fermentation for 4 days.

[0067] 2. Take all the fermentation liquid and divide it into centrifuge tubes. Centrifuge at 12000 rpm for 10 minutes to separate the bacteria and the supernatant.

[0068] 3. Use a rotary evaporator to concentrate the supernatant to 50 mL, then add 50 mL of n-hexane to the concentrate, vortex and oscillate for 5 minutes, repeat the extraction three times, and combine the organic phases.

[0069] 4. Take the organic phase and use a rotary evaporator to evaporate the n-hexane in the organic phase. After spin drying, add 5 mL of acetonitrile to dissolve the residual solid.

[0070] 5. Using the Preparative Liquid Phase System and ESIL LP-C 18 The solution obtained in the previous step was separated using a reverse phase chromatography column (10×250 mm) with the program setting parameters consistent with those in Example 3.

[0071] 6. Use a rotary evaporator to evaporate and prepare the liquid sample to obtain pure 3-hydroxy-β-ionone. The results are as follows: Figure 3 shown.

[0072] Example 5 5 L fermentation tank scale-up of strain HCS2

[0073] The strain HCS2 prepared in Example 2 was fermented in a 5 L fermentor in a fed-batch medium of 2×YPD (4% peptone and 2% yeast extract, the remainder being water, the percentages being by mass). The initial glucose concentration in the fermentor was 20 g / L. When the glucose concentration in the fermentor was lower than 1 g / L, 600 g / L of glucose was added to a glucose concentration of 5 g / L. The pH was set to 5.5, the temperature was set to 30°C, and the aeration rate was set to 3 L / min.

[0074] After 80 h of fermentation, the 3-hydroxy-β-ionone yield of strain HCS2 reached 1.66 g / L, and the precursor carotenoids zeaxanthin and β-carotene were almost completely converted. Figure 4 shown.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing an engineered strain of Yarrowia lipolytica that produces 3-hydroxy-β-ionone, characterized in that: The following steps are involved: The CsCCD1 gene was expressed in the starting strain of Yarrowia lipolytica to obtain the genetically engineered strain HCS; Overexpressing the ERG12 gene and the HMG1 gene in the genetically engineered bacteria HCS to obtain the 3-hydroxy-β-ionone-producing Yarrowia lipolytica engineered bacteria; Wherein, the nucleotide sequence of the CsCCD1 gene is shown in SEQ ID NO.1; The nucleotide sequence of the ERG12 gene is shown in SEQ ID NO.2; The nucleotide sequence of the HMG1 gene is shown in SEQ ID NO.

3.

2. The construction method according to claim 1, wherein The method of expressing the CsCCD1 gene in the starting strain of Yarrowia lipolytica comprises the step of transferring the plasmid pINA1312-CsCCD1 into the starting strain of Yarrowia lipolytica.

3. The construction method according to claim 1 or 2, wherein: The Yarrowia lipolytica starting strain is a genetically engineered Yarrowia lipolytica strain that produces zeaxanthin.

4. The construction method according to claim 1, wherein The overexpression of ERG12 gene and HMG1 gene in the genetically engineered bacteria HCS comprises the step of introducing plasmid pINA1269-HMG1-MBP-ERG12 into the genetically engineered bacteria HCS. 5 . An engineered Yarrowia lipolytica strain producing 3-hydroxy-β-ionone obtained according to the construction method according to any one of claims 1 to 4 .

6. Use of the engineered Yarrowia lipolytica according to claim 5 in the preparation of a product for producing 3-hydroxy-β-ionone.

7. A fermentation agent for producing 3-hydroxy-β-ionone, characterized in that The fermentation agent contains the engineered Yarrowia lipolytica according to claim 5 as a main active ingredient.

8. Use of the engineered Yarrowia lipolytica according to claim 5 or the starter culture according to claim 7 in the production of 3-hydroxy-β-ionone.

9. A method for producing 3-hydroxy-β-ionone, characterized in that: The following steps are involved: The engineered Yarrowia lipolytica according to claim 5 is inoculated into a culture medium containing glucose, peptone and yeast extract for fermentation production.

10. The method according to claim 9, wherein The mass fraction of peptone in the culture medium is 4%, and the mass fraction of yeast extract is 2%; The initial concentration of glucose in the culture medium is 20 g / L, and glucose is fed during the fermentation production to ensure that the concentration of glucose in the culture medium is not less than 1 g / L and not more than 5 g / L.

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