Yarrowia lipolytica engineering strain for producing cocoa butter equivalent and construction method and application of Yarrowia lipolytica engineering strain

Abstract

The invention provides a Yarrowia lipolytica engineering strain for producing cocoa butter equivalent and a construction method and application thereof, and relates to the technical field of biology, the construction method comprises the following steps: inserting an anti-translation PFAD2 promoter into a Yarrowia lipolytica chassis strain to adjust the composition of target fatty acid, so that the contents of palmitic acid and stearic acid are respectively increased to 27.3% and 19.8%; the contents of palmitoleic acid and oleic acid are reduced to 4.5% and 36.8%; the twelve-site fatty acid desaturase gene FAD2 is knocked out, and linoleic acid is completely eliminated; a plant-derived diacylglycerol acyltransferase gene DGAT or phospholipid is introduced; the diacylglycerol acyltransferase gene PDAT is used for synthesizing cocoa butter equivalent (CBE). The yield of cocoa butter equivalent of the constructed yarrowia lipolytica engineering strain finally reaches 1.00 + / -0.09 g / L and accounts for 30% of the total fat content. The technical problem of low cocoa butter equivalent production efficiency of engineering modified yeast in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an engineering strain of Yarrowia lipolytica for producing cocoa butter-like yeast, a construction method and an application thereof. Background Art

[0002] Cocoa butter is the main raw material for the production of chocolate, candy and sugar coating, with an annual global demand of up to 5 million tons. Cocoa butter is mainly composed of three triglycerides: 10% 1,3-dipalmitoyl-2-oleoylglycerol (POP), 40% 1-palmitoyl-2-2-oleoyl-3-stearoylglycerol (POS) and 40% 1,3-distearoyl-2-oleoylglycerol (SOS). Due to the over-cultivation of cocoa fruit, major cocoa producing countries are facing serious environmental problems such as deforestation and climate change in the past three decades. Naturally produced cocoa butter cannot meet the growing demand of the date, so cocoa butter equivalents produced by physical mixing, fractionation and enzymatic lipid exchange have emerged to replace cocoa butter.

[0003] At present, the main method for producing cocoa butter analogs is to process low-cost oils and fats through mixing, fractionation and enzymatic lipolysis. Physical mixing is a low-cost and efficient method to adjust the physical properties of oils and fats by simply mechanically stirring palm oil intermediate fractions, mango seed oil, shea butter and sunflower oil, which have similar physical properties to cocoa butter, as substrates. However, the cocoa butter analogs produced by physical mixing only achieve physical properties similar to cocoa butter, and the variable components of these oil substrates require long-term and multiple adjustments of the mixing ratio. Cocoa butter analogs can be efficiently produced through enzymatic lipolysis reactions, but such reactions usually require multiple pretreatment steps, such as fractionation and hydrogenation of the substrate to ensure the purity of the substrate. The hydrogenation step often introduces unhealthy trans fatty acids; activation and fixation of the enzymes used in the reaction to ensure the activity of the enzyme. Furthermore, enzymatic lipolysis reactions often use organic solvents, which increases the difficulty of downstream separation and purification.

[0004] Compared with traditional cocoa butter equivalent production methods, engineered microbial factories have gradually become a popular method for synthesizing cocoa butter equivalents due to their advantages such as environmental friendliness, low cost and freedom from regional climate restrictions. However, the current engineered yeast production of cocoa butter equivalents mainly uses Saccharomyces cerevisiae as the host, which can only produce about 0.1 g / L of cocoa butter equivalents in shake flask fermentation, accounting for only 10% of the total oil. Its production efficiency is low, which affects its industrial application.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide an engineered strain of Yarrowia lipolytica for producing cocoa butter equivalent, so as to solve the technical problem of low efficiency of producing cocoa butter equivalent by engineered yeast in the prior art.

[0007] The second object of the present invention is to provide the use of the above-mentioned Yarrowia lipolytica engineered strain in the preparation of a strain with the function of biosynthesizing cocoa butter-like organisms.

[0008] The third object of the present invention is to provide a method for constructing a strain having the function of biosynthesizing cocoa butter.

[0009] A fourth object of the present invention is to provide a method for producing cocoa butter equivalent.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] In a first aspect, the present invention provides an engineered strain of Yarrowia lipolytica for producing cocoa butter-like yeast, wherein the genome of the engineered strain of Yarrowia lipolytica includes: a fat modification gene and a reverse transcription factor for inhibiting the translation of a gene OLE1 encoding a nine-position fatty acid desaturase. FAD2 Promoter;

[0012] The fat modification gene includes a codon-optimized gene DGAT encoding diacylglycerol acyltransferase or a codon-optimized gene PDAT encoding phospholipid: diacylglycerol acyltransferase, wherein the gene DGAT encoding diacylglycerol acyltransferase and the gene PDAT encoding phospholipid: diacylglycerol acyltransferase are both derived from cocoa trees;

[0013] The Yarrowia lipolytica engineered strain is a Yarrowia lipolytica strain in which the gene FAD2 encoding the 12-position fatty acid desaturase is knocked out.

[0014] Natural Yarrowia lipolytica mainly accumulates five fatty acids: 20% palmitic acid, 7% palmitoleic acid, 12% stearic acid, 50% oleic acid and 11% linoleic acid, while cocoa butter mainly contains 26% palmitic acid, 35% stearic acid and 35% oleic acid. The production of cocoa butter using Yarrowia lipolytica is mainly achieved through the following transformations:

[0015] (1) By inserting reverse translation P FAD2 The promoter was used to inhibit the nine-position fatty acid desaturase gene OLE1 to adjust the target fatty acid composition, which could increase palmitic acid and stearic acid to 27.3% and 19.8%, respectively, while reducing palmitoleic acid and oleic acid to 4.5% and 36.8%.

[0016] (2) Knock out the 12-position fatty acid desaturase gene FAD2 to completely eliminate the unnecessary linoleic acid and transform the fat synthesis pathway of Yarrowia lipolytica.

[0017] (3) Introducing exogenous fat modification genes, namely the diacylglycerol acyltransferase gene DGAT or the phospholipid:diacylglycerol acyltransferase gene PDAT.

[0018] Through the above transformation, fatty acid desaturase was successfully inhibited and knocked out in Yarrowia lipolytica, and plant-derived acyltransferase was expressed to synthesize cocoa butter analogue.

[0019] Further, the reverse translation P FAD2 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0020] Furthermore, the nucleotide sequence of the gene FAD2 encoding the 12-fatty acid desaturase is shown as SEQ ID NO.2.

[0021] Furthermore, the gene DGAT encoding diacylglycerol acyltransferase includes TcDGAT1 or TcDGAT2.

[0022] Furthermore, the nucleotide sequences of TcDGAT1 and TcDGAT2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0023] Furthermore, the gene PDAT encoding phospholipid:diacylglycerol acyltransferase includes one of TcPDAT1, TcPDAT2 or TcPDAT3.

[0024] Furthermore, the nucleotide sequences of TcPDAT1, TcPDAT2 and TcPDAT3 are shown in SEQ ID NOs. 5-7.

[0025] TcDGAT1, TcDGAT2, TcPDAT1, TcPDAT2 and TcPDAT3 from the cocoa tree Theobroma cacao were codon-optimized and expressed for Yarrowia lipolytica. These acyltransferases are key enzymes for the production of cocoa butter equivalent, and their successful expression in Yarrowia lipolytica resulted in the synthesis of cocoa butter equivalent (CBE).

[0026] It should be noted that Yarrowia lipolytica is a non-traditional yeast that is certified as GRAS (generally recognized as safe) and can be used in the food and pharmaceutical industries. This yeast can grow rapidly under simple conditions using a variety of low-cost substrates, has no Crabtree effect (does not produce ethanol), and can accumulate more than 60% of the cell dry weight of oil under nitrogen source-limited conditions. Therefore, this yeast is an ideal host for producing cocoa butter equivalents.

[0027] In a second aspect, the present invention provides the use of the above-mentioned Yarrowia lipolytica engineered strain in the preparation of a strain having the function of biosynthesizing cocoa butter-like substances.

[0028] In a third aspect, the present invention provides a method for constructing a strain having the function of biosynthesizing cocoa butter-like substances, the method comprising:

[0029] A. Insert reverse translation P FAD2 Promoter;

[0030] B. Knockout of the 12-position fatty acid desaturase gene FAD2;

[0031] C. Insert the codon-optimized gene encoding diacylglycerol acyltransferase DGAT or the codon-optimized gene encoding phospholipid:diacylglycerol acyltransferase PDAT.

[0032] Among them, insert the reverse translation P FAD2 The promoter includes the reverse-translation P obtained by PCR amplification using the Yarrowia lipolytica genome as a template. FAD2 Promoter DNA fragment, construct expression vector, and insert into OLE1 gene.

[0033] Knocking out the 12-position fatty acid desaturase gene FAD2 includes performing PCR amplification using the Yarrowia lipolytica genome as a template to obtain a homologous arm fragment of the desired FAD2 gene, constructing an expression vector, and knocking out FAD2.

[0034] Inserting the codon-optimized gene DGAT encoding diacylglycerol acyltransferase or the codon-optimized gene PDAT encoding phospholipid: diacylglycerol acyltransferase includes using the codon-optimized gene DGAT encoding diacylglycerol acyltransferase or the codon-optimized gene PDAT encoding phospholipid: diacylglycerol acyltransferase as a template, PCR amplification, constructing an expression vector containing the codon-optimized gene DGAT encoding diacylglycerol acyltransferase or the codon-optimized gene PDAT encoding phospholipid: diacylglycerol acyltransferase, constructing a plasmid vector under the control of the TEF promoter and the XPR2 terminator, and transferring and integrating it into the Yarrowia lipolytica genome. The order of steps A and B is not limited, and C must be performed after A and B are completed.

[0035] Furthermore, the transfer is mediated by lithium acetate-PEG for gene introduction, and the URA3 gene is used as a screening marker to achieve the screening of yeast colonies containing the target plasmid vector.

[0036] In a fourth aspect, the present invention provides a method for producing cocoa butter equivalent, comprising culturing the above-mentioned Yarrowia lipolytica engineered strain or the above-mentioned constructed strain to obtain cocoa butter equivalent;

[0037] Preferably, the culturing comprises inoculating the engineered strain of Yarrowia lipolytica into a culture medium for culturing;

[0038] Preferably, the carbon-nitrogen ratio of the culture medium is 100;

[0039] Preferably, the culture medium is CSM-Leu fermentation medium.

[0040] The CSM-Leu fermentation medium includes the following components in concentrations: 40-100 g / L glucose, 1.7 g / L yeast nitrogen base without amino acids and ammonium sulfate, 1.1 g / L ammonium sulfate ((NH4)2SO4) and 0.69 g / L CSM-Leu.

[0041] The invention provides a method for constructing an engineered strain of Yarrowia lipolytica for producing cocoa butter-like yeast, by inserting a reverse-translation P FAD2 The promoter adjusts the target fatty acid composition, so that the palmitic acid and stearic acid contents are increased to 27.3% and 19.8% respectively, while the palmitoleic acid and oleic acid contents are reduced to 4.5% and 36.8%; the 12-position fatty acid desaturase gene FAD2 is knocked out to completely eliminate the unnecessary linoleic acid and transform the fat synthesis pathway of Yarrowia lipolytica; the introduction of exogenous fat modification genes diacylglycerol acyltransferase gene DGAT or phospholipid: diacylglycerol acyltransferase gene PDAT enables the successful expression of acyltransferase in Yarrowia lipolytica to synthesize cocoa butter equivalent (CBE). The fatty acid desaturase is successfully inhibited and knocked out in Yarrowia lipolytica and the acyltransferase from plant sources is expressed. The experiment proves that after five days of fermentation using CSM-Leu fermentation medium with a carbon-nitrogen ratio of 100, the cocoa butter equivalent production of the constructed Yarrowia lipolytica engineered strain finally reaches 1.00±0.09g / L, accounting for 30% of the total fat content. The invention solves the technical problem of low efficiency of producing cocoa butter by engineering yeast in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 A schematic diagram of the construction process of the pUrlp-FAD2 recombinant expression plasmid provided in Example 1 of the present invention;

[0044] Figure 2 Insertion back translation P provided by embodiment 1 of the present invention FAD2 The location of the promoter and a schematic diagram of its mechanism;

[0045] Figure 3 pUrlp-reverse translation P provided in Example 1 of the present invention FAD2 Schematic diagram of the construction process of promoter recombinant expression plasmid;

[0046] Figure 4 This is a comparison chart of the fatty acid compositions of the Yarrowia lipolytica engineered strains with different transformation treatments provided in Experiment 1 of the present invention. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] CSM-Leu / CSM-Ura selective agar plates: include 20 g / L glucose, 1.7 g / L yeast nitrogen base without amino acids and ammonium sulfate, 5 g / L ammonium sulfate ((NH4)2SO4), 16 g / L agar and 0.69 g / L CSM-Leu / 0.77 g / L CSM-Ura.

[0049] YPD-5'-FOA selective agar plate: includes 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 1 g / L 5'-FOA and 16 g / L agar.

[0050] YPD liquid medium: includes 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose.

[0051] CSM-Leu-Seed seed culture medium: 20 g / L glucose, 1.7 g / L yeast nitrogen base without amino acids and ammonium sulfate, 5 g / L ammonium sulfate ((NH4)2SO4) and 0.69 g / L CSM-Leu.

[0052] CSM-Leu fermentation medium included components at the following concentrations: 50 g / L glucose, 1.7 g / L yeast nitrogen base without amino acids and ammonium sulfate, 1.1 g / L ammonium sulfate ((NH4)2SO4), and 0.69 g / L CSM-Leu.

[0053] PEG4000: polyethylene glycol 4000;

[0054] DTT: Dithiothreitol;

[0055] Salmon sperm ssDNA: salmon sperm single-stranded DNA;

[0056] Chassis cells: Yarrowia lipolytica po1fk strain, from the Laboratory of Synthetic Biology and Intelligent Control, Guangdong Technion-Israel Institute of Technology;

[0057] Expression vector construction plasmids: pUrlp and pYLXP', from the Laboratory of Synthetic Biology and Intelligent Control, Guangdong Technion-Israel Institute of Technology.

[0058] Example 1

[0059] 1. Construction of expression vector

[0060] 1. Synthesis and amplification of target genes:

[0061] The codon-optimized TcDGAT1 was obtained by gene synthesis technology;

[0062] The Yarrowia lipolytica genome was used as a template and PCR amplified to obtain the reverse-translated P FAD2 Promoter DNA fragment;

[0063] The DNA fragment of TcDGAT1 was obtained by PCR amplification using the codon-optimized TcDGAT1 sequence as a template.

[0064] PCR amplification was performed using the Yarrowia lipolytica genome as a template to obtain the homologous arm fragment of the desired FAD2 gene.

[0065] PCR amplification was performed using the Yarrowia lipolytica genome as a template to obtain the desired OLE1 gene homology arm and a T2A replacement TAA stop codon sequence fragment, and the T2A sequence is shown in SEQ ID NO.20.

[0066] 2. Construction of recombinant expression plasmid

[0067] 1) Obtain the pUrlp vector. The pUrlp vector is a circular plasmid containing a uracil Ura3 tag. The expression of the uracil Ura3 gene is driven by the TEF promoter and regulated by the XPR2 terminator. In addition, LoxP sites are provided before the TEF promoter and after the XPR2 terminator, so that the Ura3 screening marker gene can be recovered using the Cre-LoxP system. The pUrlp plasmid vector can also be inserted into the homologous arm fragments upstream and downstream of the site-specific integration site, which can realize the site-specific insertion, knockout and recombination of genes in chromosomes.

[0068] The pYLXP' vector was obtained. The vector is a circular plasmid containing a leucine Leu2 tag for screening in Yarrowia lipolytica, a strong promoter TEF and terminator XPR2 that function in Yarrowia lipolytica, and about 1000 bp sequences upstream and downstream of the NotI restriction site are homologous arms of the pBR322 docking platform, which can integrate the carried gene into the pBR322 platform of the chassis strain.

[0069] 2) The pUrlp plasmid vector was double-digested to obtain a pUrlp linearized vector with sticky ends.

[0070] In this example, the pUrlp plasmid vector was double-digested using AvrII (endonuclease 1) and SalI (endonuclease 2).

[0071] In this example, the pYLXP' plasmid vector was double-digested with KpnI (endonuclease 3)-SnaBI (endonuclease 4).

[0072] The double enzyme digestion reaction system is shown in Table 1. The reaction conditions are 37°C and the reaction time is 60 min.

[0073] Table 1: Plasmid double restriction enzyme digestion reaction system

[0074] Reagents Usage amount (μL) Plasmid vector 8 10X Buffer 2 Endonuclease 1 / 3 1 Endonuclease 2 / 4 1 Double distilled water 8

[0075] After the restriction digestion reaction was completed, the linearized pUrlp and pYLXP' vector fragments were separated by agarose gel electrophoresis, and the target fragments were recovered from the linearized pUrlp and pYLXP' vector fragments using a DNA gel recovery kit to obtain purified plasmid linearized vector fragments.

[0076] 3) The linearized plasmid vector fragment obtained in step 2) is combined with the reverse-translated P obtained in step 1) FAD2 The DNA fragment of the promoter and the homology arm of the OLE1 gene, as well as the sequence fragment of the T2A replacement of the TAA stop codon, the DNA fragment of TcDGAT1 and the homology arm fragment of the FAD2 gene were fused with the loxP-URA3-loxP sequence and the linearized pYLXP' by seamless cloning technology (Gibson Assembly) Gibson Assembly to construct pUrlp-reverse translation P FAD2 Promoter (such as Figure 2 and Figure 3 As shown), pYLXP'-TcDGAT1 and pUrlp-FAD2 recombinant plasmids (as shown Figure 1 The primers involved in plasmid construction are shown in Table 2, and the PCR reaction program is shown in Table 3.

[0077] Table 2: Primers involved in plasmid construction

[0078]

[0079]

[0080] Table 3 PCR reaction program

[0081]

[0082] Example 2

[0083] Different from Example 1, the codon-optimized TcDGAT2 was obtained by gene synthesis technology; the DNA fragment of TcDGAT2 was obtained by PCR amplification using the codon-optimized TcDGAT2 sequence as a template, and the pYLXP'-TcDGAT2 recombinant plasmid was constructed to replace the pYLXP'-TcDGAT1 recombinant plasmid.

[0084] Example 3

[0085] Different from Example 1, codon-optimized TcPDAT1 was obtained by gene synthesis technology; the DNA fragment of TcPDAT1 was obtained by PCR amplification using the codon-optimized TcPDAT1 sequence as a template, and the pYLXP'-TcPDAT1 recombinant plasmid was constructed to replace the pYLXP'-TcDGAT1 recombinant plasmid.

[0086] Example 4

[0087] Different from Example 1, codon-optimized TcPDAT2 was obtained by gene synthesis technology; the DNA fragment of TcPDAT2 was obtained by PCR amplification using the codon-optimized TcPDAT2 sequence as a template, and the pYLXP'-TcPDAT2 recombinant plasmid was constructed to replace the pYLXP'-TcDGAT1 recombinant plasmid.

[0088] Example 5

[0089] Different from Example 1, codon-optimized TcPDAT3 was obtained by gene synthesis technology; the DNA fragment of TcPDAT3 was obtained by PCR amplification using the codon-optimized TcPDAT3 sequence as a template, and the pYLXP'-TcPDAT3 recombinant plasmid was constructed to replace the pYLXP'-TcDGAT1 recombinant plasmid.

[0090] Comparative Example 1

[0091] Different from Example 1, only pUrlp-reverse translation P FAD2 Promoter recombination plasmid.

[0092] Comparative Example 2

[0093] Different from Example 1, the pYLXP'-TcDGAT1 recombinant plasmid was not constructed.

[0094] Comparative Example 3

[0095] The difference from Example 1 is that only the pYLXP'-TcDGAT1 recombinant plasmid was constructed.

[0096] Example 6

[0097] 1. Prepare competent cells of Yarrowia lipolytica chassis strain.

[0098] The Yarrowia lipolytica chassis strain is polfk.

[0099] The bottom plate strain was inoculated into a test tube containing 3 mL of YPD (Yeast extract Peptone Dextrose; 1% yeast extract, 2% glucose and 20% peptone) medium and cultured overnight in a shaking incubator at 30, 250 rpm;

[0100] After low-speed centrifugation of 1 mL of the bottom plate yeast solution, discard the supernatant and thoroughly mix the cell pellet with 95 μL of 50% PEG4000 and 5 μL of 2M lithium acetate solution;

[0101] The chassis competent cells of the Yarrowia lipolytica engineering strain for preparing high-yield cocoa butter equivalent are obtained.

[0102] 2. Preparation of an engineered strain containing the exogenous gene TcDGAT1 for biosynthesis of cocoa butter

[0103] 1. The recombinant expression vector pUrlp-reverse PFAD2 promoter and pUrlp-FAD2 obtained in Example 1 were digested by AvrII (endonuclease 5), and then linearized vectors were obtained by agarose gel electrophoresis and DNA gel recovery;

[0104] The recombinant expression vector pYLXP'-TcDGAT1 obtained in Example 1 was digested with NotI (endonuclease 6), and then the linearized vector was obtained by agarose gel electrophoresis and DNA gel recovery.

[0105] 2. Lithium acetate PEG4000 method. After culturing in YPD liquid medium for 24 hours, take 1 ml of the cell pellet by centrifugation and wash twice with sterile double distilled water. Then resuspend in transformation solution (prepared as shown in Table 4), culture at 39°C for one hour, and shake for 15 seconds every 15 minutes. Finally, spread on CSM-Leu selective agar plate for screening.

[0106] Table 4 Yeast transformation system

[0107] Reagents Dosage 50%PEG4000 80μl 2M Lithium Acetate 5μl 2M DTT 5μl Salmon sperm ssDNA 5μl Cassette (Linearization Vector) 200ng

[0108] An engineered strain containing the exogenous gene TcDGAT1 and capable of biosynthesizing cocoa butter was obtained.

[0109] Embodiments 7 to 10

[0110] Different from Example 6, the recombinant plasmids constructed in Examples 2 to 5 were transformed into the chassis competent cells in step 1, respectively, to obtain the engineered strains containing the exogenous gene TcDGAT2, the exogenous gene TcPDAT1, the exogenous gene TcPDAT2 and the exogenous gene TcPDAT3 for biosynthesis of cocoa butter.

[0111] Comparative Examples 4 to 6

[0112] Different from Example 6, the recombinant plasmids constructed in Comparative Examples 1 to 3 were transformed into the chassis competent cells in step 1, respectively, to obtain OLE1(Δ9)↓ engineering strain, OLE1(Δ9)↓ΔFAD2(Δ12) engineering strain and TcDGAT1↑ engineering strain.

[0113] Examples 11 to 15

[0114] The Yarrowia lipolytica engineered strains constructed in Examples 6 to 10 were used to ferment and produce cocoa butter equivalent. The cocoa butter equivalent was produced by the following steps:

[0115] 1) Seed culture: Inoculate the colony into CSM-Leu-Seed seed culture medium and culture at 30°C for 24 hours.

[0116] 2) Fermentation culture: The seed suspension was fermented at an initial concentration of 0.01 OD 600 The culture medium was inoculated into CSM-Leu fermentation medium and fermented at 30°C for 5 days.

[0117] Comparative Examples 7 to 9

[0118] The difference from Example 2 is that the Yarrowia lipolytica engineered strains constructed in Comparative Examples 1 to 3 were used to ferment and produce cocoa butter equivalent.

[0119] Comparative Example 10

[0120] Unlike Example 11, a wild-type Yarrowia lipolytica strain (Yarrowia lipolytica chassis strain) was used to produce cocoa butter equivalent.

[0121] Experiment 1: Quantitative analysis of fatty acids:

[0122] Take the 5-day fermentation broth of Comparative Example 7, Comparative Example 8 and Comparative Example 10 respectively, and detect according to the following steps:

[0123] 4OD of cells (e.g. 200 μl of culture medium at 20OD) will be transferred to a 1.5 ml centrifuge tube and washed twice with double distilled water.

[0124] Fatty acid transesterification and determination process: Add 500μl of 0.5N methanol-sodium hydroxide solution and 100μl of 2g / L tridecanoic acid dissolved in n-hexane as internal standard to the bacteria, and shake at 1200rpm for 2 hours. Then 40μl of 98% concentrated sulfuric acid and 400μl of n-hexane are added to the sample in sequence, and shake at 1200rpm for 15 minutes. Finally, centrifuge the sample and take the upper n-hexane to GC-FID using HP-INNOWAX (30m×0.25mm) capillary column for quantification.

[0125] Whole-cell lipid extraction and determination process: Take 1 ml of the bacteria in the fermentation broth and wash it twice with double distilled water. Then add 300 μl of 4M hydrochloric acid and incubate at 78 degrees Celsius for 2 hours. Then add 100 μl of 2g / L triglyceride heptadecanoic acid dissolved in chloroform as an internal standard, 200 μl chloroform and 300 μl methane, and shake at 1200 rpm for 20 minutes. Recover the lower layer solution, add 600 μl chloroform to the remaining sample and shake at 1200 rpm for 20 minutes to ensure complete extraction of lipids. Dry the extracted lipids with a centrifugal concentrator, then re-dissolve them with acetone and quantitatively analyze them using high-pressure high-performance liquid chromatography-mass spectrometry. The specific results are as follows. Figure 4 As shown, WT is comparative example 10, OLE1(Δ9)↓ is comparative example 7, and OLE1(Δ9)↓ΔFAD2(Δ12) is comparative example 8.

[0126] Depend on Figure 4 It can be seen that compared with Comparative Example 10, Comparative Example 7 only inserts the reverse translation P FAD2 The promoter was used to inhibit the nine-position fatty acid desaturase gene OLE1, which increased palmitic acid and stearic acid to 27.3% and 19.8%, respectively, while palmitoleic acid and oleic acid decreased to 4.5% and 36.8%; Comparative Example 8 further knocked out the twelve-position fatty acid desaturase gene FAD2, completely eliminating the unnecessary linoleic acid. FAD2 The promoter and knockout of the 12-position fatty acid desaturase gene FAD2 achieved the purpose of adjusting the target fatty acid composition.

[0127] Experiment 2 Quantitative analysis of cocoa butter produced by different recombinant yeast fermentations

[0128] The 5-day fermentation broths of Examples 11 to 15 and Comparative Examples 7 to 10 were respectively taken and tested according to the whole cell lipid extraction and determination process steps of Experiment 1. The results showed that no cocoa butter equivalent could be obtained in Comparative Examples 7 to 9, and the cocoa butter equivalent content in the fermentation broths of Examples 11 to 15 increased by more than 30% compared with Comparative Example 10 (WT).

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered strain of Yarrowia lipolytica for producing cocoa butter analogs, characterized in that: The genome of the Yarrowia lipolytica engineered strain includes: a lipid modification gene and a reverse translation P of the inhibitory gene OLE1; FAD2 Promoter; The fat modification gene includes a codon-optimized gene DGAT or a codon-optimized gene PDAT, and both the gene DGAT and the gene PDAT are derived from cocoa fruit trees; The Yarrowia lipolytica engineered strain is a Yarrowia lipolytica strain in which the FAD2 gene is knocked out.

2. The Yarrowia lipolytica engineered strain according to claim 1, characterized in that The reverse translation P FAD2 The nucleotide sequence of the promoter is shown in SEQ ID NO.

1.

3. The Yarrowia lipolytica engineered strain according to claim 1, characterized in that The nucleotide sequence of the gene FAD2 is shown in SEQ ID NO.

2.

4. The Yarrowia lipolytica engineered strain according to claim 1, characterized in that The gene DGAT includes TcDGAT1 or TcDGAT2.

5. The engineered strain of Yarrowia lipolytica according to claim 4, characterized in that The nucleotide sequences of TcDGAT1 and TcDGAT2 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The engineered strain of Yarrowia lipolytica according to claim 1, characterized in that The gene PDAT includes one of TcPDAT1, TcPDAT2 or TcPDAT3.

7. The engineered strain of Yarrowia lipolytica according to claim 6, characterized in that The nucleotide sequences of TcPDAT1, TcPDAT2 and TcPDAT3 are shown in SEQ ID NOs. 5-7.

8. Use of the engineered strain of Yarrowia lipolytica according to any one of claims 1 to 7 in preparing a strain having the function of biosynthesizing cocoa butter-like substances.

9. A method for constructing a strain with the function of biosynthesizing cocoa butter, characterized in that: The method comprises: A. Insert reverse translation P FAD2 Promoter; B. Knockout gene FAD2; C. Insert the codon-optimized gene DGAT or the codon-optimized gene PDAT.

10. A method for producing cocoa butter equivalent, characterized in that: The method comprises culturing the Yarrowia lipolytica engineered strain according to any one of claims 1 to 7 or the strain constructed by the construction method according to claim 9 to obtain cocoa butter analog; Preferably, the culturing comprises inoculating the engineered strain of Yarrowia lipolytica into a culture medium for culturing; Preferably, the carbon-nitrogen ratio of the culture medium is 80-200; Preferably, the culture medium is CSM-Leu fermentation medium.

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