Yarrowia lipolytica strain with high squalene yield, construction method of Yarrowia lipolytica strain and application method of Yarrowia lipolytica strain in squalane synthesis

Abstract

The invention belongs to the technical field of bioengineering, and relates to a yarrowia lipolytica strain with high squalene yield, a construction method of the yarrowia lipolytica strain and an application method of the yarrowia lipolytica strain in squalane synthesis. Specifically, a DGA1 gene is knocked out on the basis of constructed recombinant yarrowia lipolytica by utilizing a gene editing means, and then a squalene synthetase gene is integrated on a genome, so that a series of recombinant yarrowia lipolytica strains with high squalene yield are constructed; the high-yield strain is subjected to fermentation culture optimization, tricarboxylic acid circulation metabolism is strengthened, alkane is added into a culture solution for extraction to promote discharge of products, and the final squalene yield reaches 42.1 g / L; the squalene extracted and separated from the fermentation broth is subjected to hydrogenation reduction by taking Reshaki nickel as a catalyst to obtain squalene. The constructed recombinant yarrowia lipolytica strain has the advantages of high yield, simple product extraction, separation and conversion, low production cost, and good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Yarrowia lipolytica strain with high squalene production, a construction method thereof, and an application method thereof in squalane synthesis. Background Art

[0002] Squalene is an unsaturated triterpene compound composed of six isoprene units. It is a key precursor for the synthesis of various unsaturated triterpenes and steroids. Since squalene has multiple unsaturated conjugated double bonds, it has good antioxidant properties, which can be used to treat eczema, anti-aging and anti-wrinkle cosmetics. In addition, squalene also has good anti-cardiovascular and anti-cancer activities and can be used to treat cardiovascular diseases. In nature, many animals, plants and microorganisms can synthesize squalene autonomously. For decades, shark liver has been the main source of squalene. However, with the increase of people's environmental awareness, the large-scale extraction of squalene has become very limited. Extracting squalene from vegetable oil is an alternative method to obtain squalene from shark liver, but due to the disadvantages of too low content in vegetable oil and complicated extraction process, the industrial application of this method is limited.

[0003] Squalane is a saturated hydrocarbon oil obtained by hydrogenating squalene. It is colorless, odorless, non-toxic, chemically inert, bright and transparent; it is moisturizing but not greasy; it has high moisturizing and humectant properties; it also has good antioxidant and thermal stability; it has good compatibility with mineral oil and the raw materials of most care products, so it is widely used in cosmetics.

[0004] Due to the application potential of squalene and squalane, the global market demand for squalene is also increasing. In order to meet people's growing demand, it has become urgent to produce squalene in a renewable and sustainable way. In recent years, with the continuous development of synthetic biology, the use of microorganisms to produce some complex natural products in a green and efficient manner has become more and more popular. As an unconventional yeast, Yarrowia lipolytica can synthesize squalene autonomously, which has the following advantages: 1) It can synthesize a large amount of lipids, provide a storage environment for hydrophobic compounds, and reduce toxicity to cells; 2) It has an efficient TCA cycle and PP pathway, which provides high levels of acetyl-CoA and cofactor NADPH for the synthesis of terpenoids; 3) Yarrowia lipolytica has no Crabtree effect and can be fermented efficiently and at high density. At present, several research groups have used Yarrowia lipolytica to biosynthesize squalene. Among them, Liu Huhu's research group, on the basis of strengthening the MVA pathway, increased the content of intracellular lipids and optimized the culture conditions, and achieved a squalene production of 1.63g / L (Bioresour.Technol., 2024, 394, 130322). Xu Jianhe's research group, on the basis of strengthening the MVA pathway and increasing the level of acetyl-CoA, weakened the synthesis of sterols, balanced cell growth, and reduced the conversion of squalene to other compounds, so that the shake flask production of squalene reached 2.4g / L, and then through batch feeding culture, the production of squalene in a 5-L fermenter reached 35g / L (J.Agric.FoodChem., 2024, 72, 9984).

[0005] Squalene synthase SQS is a key enzyme for the synthesis of squalene, and doubling the enzyme is expected to further increase the production of squalene. Diacylglycerol acyltransferase DGA1 is a key enzyme for the synthesis of lipids in Yarrowia lipolytica. Liu Huhu et al. (Bioresour. Technol., 2024, 394, 130322) overexpressed the enzyme, which promoted lipid synthesis and enhanced the intracellular accumulation of squalene. However, the squalene yield obtained by this scheme is about 1600 mg / L, and the squalene yield still needs to be improved. Summary of the invention

[0006] In order to overcome the current situation that the squalene production in the prior art is still relatively low, the present invention provides a Yarrowia lipolytica strain with high squalene production, a construction method thereof, and an application method thereof in squalane synthesis.

[0007] In the process of obtaining the scheme of this application, the inventors conducted research and analysis and believed that knocking out the acylglycerol acyltransferase DGA1 is expected to reduce the flow of acetyl-CoA toward lipid synthesis, thereby increasing the metabolic flow of acetyl-CoA for the synthesis of squalene. In addition, the present application found that the addition of alkanes during the fermentation process to extract and ferment squalene can help the excretion of intracellular squalene and simplify downstream separation.

[0008] The present invention further carries out genome modification on the basis of the existing squalene production strain, provides a recombinant Yarrowia lipolytica strain with higher squalene production, and on this basis, optimizes the fermentation of the recombinant strain to efficiently produce squalene, and then hydrogenates and reduces the squalene to prepare squalane.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention provides a technical solution 1:

[0011] Provided is a Yarrowia lipolytica strain with high squalene production, which is a recombinant Yarrowia lipolytica strain in which a diacylglycerol acyltransferase DGA1 gene is knocked out on the basis of a starting strain YLSQ0, wherein the nucleic acid sequence of the diacylglycerol acyltransferase DGA1 is shown as SEQ ID No.1.

[0012] The present invention obtains a recombinant Yarrowia lipolytica engineered strain by knocking out the DGA1 gene. Knocking out the diacylglycerol acyltransferase DGA1 gene in the Yarrowia lipolytica engineered strain can reduce the flow of acetyl-CoA toward lipid synthesis, thereby increasing the metabolic flow of acetyl-CoA for squalene synthesis.

[0013] In some embodiments of the present invention, the starting strain YLSQ0 is selected from recombinant Yarrowia lipolytica ATCCMYA-2613, MATAura3-302, Leu2-270, xpr2-322, axp2-deltaNU49XPR2::SUC2, Δku70, AXP::ScRAD52+ScRAD59, A08::ERG10+ERG13, POX3::tHMG1+ERG12, POX4::ERG8+ERG19, POX6::IDI+ERG2, CIT2::ACL1, MLS1::ACS L641P ,D17::ERG10-tHM G1,ERG1::P ACL -ERG1 G29S ,ΔERG5,ZETA::P hp4d -Ura3-T XPR2 -P Leu -Leu2-T XPR2(named as Yarrowia lipolytica YLSQ0), which has been published in the document J. Agric. Food Chem., 2024, 72, 9984.

[0014] The second technical solution provided by the present invention is:

[0015] A method for constructing a Yarrowia lipolytica strain with high squalene production is provided, comprising the following steps:

[0016] 1) In the recombinant Yarrowia lipolytica ATCC MYA-2613, MATAura3-302, Leu2-270, xpr2-322, axp2-deltaNU49 XPR2::SUC2, Δku70, AXP::ScRAD52+ScRAD59, A08::ERG10+ERG13, POX3::tHMG1+ERG12, POX4::ERG8+ERG19, POX6::IDI+ERG2, CIT2::ACL1, MLS1::ACS L641P ,D17::ERG10-tHMG1,ERG1::P ACL -ERG1 G29S ,ΔERG5,ZETA::P hp4d -Ura3-T XPR2 -P Leu -Leu2-T XPR2 Based on the extracted genome of Yarrowia lipolytica YLSQ0, primers DGA1-LH-F / DGA1-LH-R and DGA1-RH-F / DGA1-RH-R were used to amplify the left and right homology arms for knocking out DGA1, respectively. Then, the left and right homology arms of DGA1 were connected with the pHR plasmid backbone amplified by primers HR-DGA1-XF / HR-DGA1-XR in a seamless cloning manner. After sequencing verification, the recombinant plasmid pHR_ΔDGA1 was obtained.

[0017] 2) Construction of plasmid pCRISPRyl_ΔDGA1: Using plasmid pCRISPRyl as a template, reverse PCR amplification was performed with primers DGA1-N20-F / DGA1-N20-R. After amplification, the amplified fragment was digested with Dpn I and transformed into Escherichia coli. After successful screening and sequencing verification, the plasmid pCRISPRyl_ΔDGA1 for gene knockout was obtained.

[0018] 3) Construction of the Yarrowia lipolytica engineered strain YLSQ10: The constructed pCRISPRyl_ΔDGA1 and pHR_ΔDGA1 plasmids were co-transfected into the recombinant Yarrowia lipolytica YLSQ0 cells by chemical transformation, and the recombinant Yarrowia lipolytica strain YLSQ10-1 with the DGA1 gene knocked out was obtained after sequencing verification. Then the strain YLSQ10-1 was streaked on a YPD plate containing 5-fluoroorotic acid to eliminate the plasmids pCRISPRyl_ΔDGA1 and pHR_ΔDGA1, and the recombinant engineered strain YLSQ10 was obtained.

[0019] The sequence of the primer DGA1-LH-F is shown as SEQ ID No.4, the sequence of the primer DGA1-LH-R is shown as SEQ ID No.5, the sequence of the primer DGA1-RH-F is shown as SEQ ID No.6, the sequence of the primer DGA1-RH-R is shown as SEQ ID No.7, the sequence of the primer HR-DGA1-XF is shown as SEQ ID No.8, the sequence of the primer HR-DGA1-XR is shown as SEQ ID No.9, the sequence of the primer DGA1-N20-F is shown as SEQ ID No.10, and the sequence of the primer DGA1-N20-R is shown as SEQ ID No.11.

[0020] The present invention provides a third technical solution:

[0021] A recombinant engineered Yarrowia lipolytica strain with a squalene synthase gene integrated into its genome is provided. The squalene synthase gene is integrated into the genome of the high-squalene-producing Yarrowia lipolytica strain described in technical solution 1 to achieve overexpression of squalene synthase and increase squalene production.

[0022] In one embodiment of the present invention, the squalene synthase is selected from squalene synthase SQS or a squalene synthase SQS mutant, wherein the amino acid sequence of squalene synthase SQS is as shown in SEQ ID No. 3, and the squalene synthase SQS mutant is a protein having the following amino acid sequence:

[0023] (1) replacing the threonine at position 37 of the amino acid sequence shown in SEQ ID No. 3 with serine;

[0024] (2) replacing the 55th alanine of the amino acid sequence shown in SEQ ID No. 3 with leucine;

[0025] (3) replacing the isoleucine at position 81 of the amino acid sequence shown in SEQ ID No. 3 with threonine;

[0026] (4) The lysine at position 137 of the amino acid sequence shown in SEQ ID No. 3 is replaced by threonine.

[0027] The nucleic acid sequence of the squalene synthase SQS gene is shown in SEQ ID No.2, wherein bases 522-1859 encode the squalene synthase SQS composed of the amino acid sequence shown in SEQ ID No.3.

[0028] The amino acid sequence of squalene synthase SQS is as follows:

[0029] MGKLIELLLHPSELSAAIHYKLWRQPLHPRDLSKESTELRRCYELLDVCSRSFAAVIRELHPEVRDAVMLFYLILRALDTIEDDMTLSRDIKIPILRDFTKCMKTPGWKFT DSDPNERDRVVLQEFPVVMTEFNKLKPKYQEVIYDITDRMGNGMADYVIDDDFNNNGVDTIAAYDLYCHHVAGIVGEGLTRITILAGFGTDVLHENPRLQESMGLFLQKVN IIRDYREDIDVNRAFWPREIWHKYAEEMRDFKDPKYSKKALHCTSDLVANALGHATDCLDYLDNVTDPSTFTFCAIPQVMAIATLDLVYRNPDVFQKNVKLRKGTTVSLIL EASNVSGVCDIFTRYARKVYKKSDPNDPNYFRVSVLCGKIEQHAALIKRQRGPPAKTIAQLEGERKEMALSLIVCLAVIFSMSGLMAYIAYVSGFRWSPREIFDSKMFPLRD

[0030] Squalene synthase SQS mutants can be obtained by protein engineering modification of squalene synthase SQS.

[0031] The nucleic acid sequences of natural squalene synthase SQS and engineered squalene synthase SQS mutants were integrated into the A3 site of the genome of the high-squalene-producing Yarrowia lipolytica strain (recombinant Yarrowia lipolytica YLSQ10) described in Technical Solution 1 to obtain recombinant Yarrowia lipolytica strains YLSQ21-YLSQ25.

[0032] The present invention provides a fourth technical solution:

[0033] A method for constructing a recombinant engineering strain of Yarrowia lipolytica with a squalene synthase gene integrated into its genome as described in technical solution 3 is provided, comprising the following steps:

[0034] 1) Construction of plasmid pHR_A3: First, the genome of Yarrowia lipolytica YLSQ0 was extracted as a template, and the left and right homology arms of the genomic A3 insertion site were amplified using primers A3-LH-F / A3-LH-R and A3-RH-F / A3-RH-R, respectively, and the left and right homology arms of the P insertion site were amplified using primers TEFin-F / TEFin-R and primers CYC1-F / CYC1-R, respectively. TEFin Promoter and T CYC1 terminator, and connected them with the pHR plasmid backbone amplified by primers HR-A3-XF / HR-A3-XR in a seamless cloning manner, transformed into Escherichia coli, and after screening and sequencing verification, the plasmid pHR_A3 with genome integration at the A3 site was obtained;

[0035] 2) Construction of plasmid pHR_A3_SQS0: Using the genome of Yarrowia lipolytica YLSQ10 as a template, the upstream and downstream primers SQS-F / SQS-R of squalene synthase were used to amplify the open reading frame of natural squalene synthase SQS, and the plasmid pHR_A3 was linearized with primers HR-SQS-XF / HR-SQS-XR. After seamless cloning, the plasmid was connected and transferred into Escherichia coli. After successful sequencing verification, the recombinant plasmid pHR_A3_SQS0 was obtained;

[0036] 3) Construction of recombinant plasmid pCRISPRyl_A3: Using plasmid pCRISPRyl as a template, pCRISPRyl was amplified by reverse PCR using primers gA3-F / gA3-R, and then digested with Dpn I. The plasmid was then transformed into Escherichia coli. After successful sequencing verification, the Cas9 plasmid pCRISPRyl_A3 that can be used for integration at the A3 site was obtained.

[0037] 4) Construction of the Yarrowia lipolytica engineered strain YLSQ21: The plasmids pCRISPRyl_A3 and pHR_A3_SQS0 were co-transformed into the recombinant Yarrowia lipolytica YLSQ10 by chemical transformation, and after gene integration, the recombinant Yarrowia lipolytica strain YLSQ21-1 was obtained;

[0038] Then, strain YLSQ21-1 was streaked on a YPD plate containing 5-fluoroorotic acid to eliminate plasmids pCRISPRyl_A3 and pHR_A3_SQS0, and the recombinant engineered strain YLSQ1021 was obtained.

[0039] Using the same method, four variant protein genes obtained by engineering SQS were integrated into the recombinant Yarrowia lipolytica YLSQ10 to obtain recombinant Yarrowia lipolytica strains YLSQ22-YLSQ25.

[0040] Wherein, the sequence of the primer A3-LH-F is shown in SEQ ID No.12, the sequence of the primer A3-LH-R is shown in SEQ ID No.13, the sequence of the primer A3-RH-F is shown in SEQ ID No.14, the sequence of the primer A3-RH-R is shown in SEQ ID No.15, the sequence of the primer TEFin-F is shown in SEQ ID No.16, the sequence of the primer TEFin-R is shown in SEQ ID No.17, the sequence of the primer CYC1-F is shown in SEQ ID No.18, the sequence of the primer CYC1-R is shown in SEQ ID No.19, the sequence of the primer HR-A3-XF is shown in SEQ ID No.20, the sequence of the primer HR-A3-XR is shown in SEQ ID No.21, the sequence of the primer SQS-F is shown in SEQ ID No.22, the sequence of SQS-R is shown in SEQ ID No.23, the sequence of HR-SQS-XF is shown in SEQ ID The sequence of primer gA3-F is shown in SEQ ID No.26, and the sequence of primer gA3-R is shown in SEQ ID No.27.

[0041] The present invention provides a fifth technical solution:

[0042] Provided is a method for preparing squalene, comprising the following steps: culturing the high-squalene-producing Yarrowia lipolytica strain described in technical solution 1 of the present invention or the recombinant engineering strain of Yarrowia lipolytica described in technical solution 3, and separating squalene from the culture solution.

[0043] The culture medium used to culture the engineered strain can be selected from conventional culture medium in the art, as long as the recombinant strain can grow and produce squalene. The specific operation of culturing the transformant can be carried out according to conventional operations in the art.

[0044] For example, in shake flask culture, pick a single colony on the YPD medium plate and inoculate it into fresh YPD liquid culture medium; culture it in a constant temperature shaker at 30°C and 220rpm for 16-24h; according to the inoculation amount of 20%, inoculate it into a 250mL Erlenmeyer flask containing 50mL fresh YPD liquid culture medium, and continue to culture it in a constant temperature shaker at 30°C and 220rpm for 3-5d.

[0045] In the fermentation tank culture, first pick a single colony on the YPD medium plate and inoculate it into fresh YPD liquid medium; culture in a constant temperature shaker at 30°C and 220rpm for 16-24h; inoculate the seed liquid into a 5-L fermentation tank filled with fermentation medium, calibrate the dissolved oxygen to 100%, adjust the ventilation to 2vvm, set the dissolved oxygen concentration to 25%, maintain the fermentation temperature at 25°C, set the stirring paddle speed at 300-800rpm, and associate the dissolved oxygen level with the stirring speed. During the fermentation process, ammonia and sulfuric acid solutions are used to maintain the pH of the culture medium at around 6.5. After the glucose in the initial culture medium is exhausted, glucose is added by flow.

[0046] The formula of the YPD culture medium is: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0047] Preferably, lactate, pyruvate, and citrate are added to the culture medium in an amount of 1-5% (w / v) to enhance tricarboxylic acid cycle metabolism;

[0048] Preferably, alkanes with a carbon chain length of 8-14 are added during the culture process in an amount of 5-30% (v / v) and the addition time is 1-4 days after inoculation to promote the timely excretion of squalene accumulated in the cells.

[0049] After the cultivation is completed, the reaction solution is extracted with a solvent, or the organic phase containing squalene is separated and then distilled under reduced pressure to obtain purified squalene.

[0050] The present invention provides a sixth technical solution:

[0051] A method for preparing squalane is provided. The method uses Raney nickel as a catalyst to hydrogenate and reduce the squalene obtained in the fifth technical solution to obtain a novel saturated alkane squalane.

[0052] In some embodiments of the present invention, squalene is dissolved in an organic solvent under nitrogen protection, stirring is started, 1-10% (w / w) Raney nickel is carefully added, and then nitrogen is replaced with hydrogen, and hydrogenation reduction reaction is carried out at 30-50° C. for 6-12 hours, cooled, replaced with nitrogen, the catalyst is filtered out, and squalane is obtained by reduced pressure distillation. The organic solvent is preferably petroleum ether.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] The present invention knocks out the DGA1 gene in Yarrowia lipolytica, and then integrates the gene of squalene synthase or its mutant into the genome to obtain recombinant Yarrowia lipolytica with higher squalene yield.

[0055] Furthermore, the present invention also effectively increases the fermentation yield of squalene and simplifies product separation by adding precursors during the fermentation process, strengthening the tricarboxylic acid cycle metabolism, and adding medium-chain alkanes for extraction fermentation; on this basis, squalane is further obtained by hydrogenation, and the overall process is simple and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : Map of plasmid pHR;

[0057] Figure 2 : Map of plasmid pCRISPRyl. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help to further understand the concept of the present invention, but do not limit the scope of use of the present invention.

[0059] The gene editing method used in the present invention is carried out with reference to the literature (ACS Synth.Biol., 2017, 6, 402).

[0060] The map of the plasmid pHR is attached Figure 1 The map of plasmid pCRISPRyl is attached. Figure 2 Plasmid pHR and plasmid pCRISPRyl can be purchased or prepared using conventional techniques in the art.

[0061] Example 1 Construction of recombinant Yarrowia lipolytica strain YLSQ10

[0062] Based on Yarrowia lipolytica YLSQ0, diacylglycerol acyltransferase DGA1 was knocked out to construct strain YLSQ10.

[0063] The specific steps are as follows:

[0064] 1) Construction of plasmid pHR_ΔDGA1

[0065] The extracted genome of Yarrowia lipolytica YLSQ0 was used as a template, and the upstream homology arm of diacylglycerol acyltransferase DGA1 was amplified with primers DGA1-LH-F / DGA1-LH-R, and the downstream homology arm of diacylglycerol acyltransferase DGA1 was amplified with primers DGA1-RH-F / DGA1-RH-R. Subsequently, the upstream and downstream homology arms of DGA1 were connected with the pHR plasmid backbone amplified with primers HR-DGA1-XF / HR-DGA1-XR in a seamless cloning manner, and transformed into Escherichia coli. After screening and sequencing verification, the recombinant plasmid pHR_ΔDGA1 was obtained.

[0066] Wherein, the nucleic acid sequence of the diacylglycerol acyltransferase DGA1 is shown in the sequence table SEQ ID No.1.

[0067] The sequence of the primer DGA1-LH-F is shown as SEQ ID No.4, the sequence of the primer DGA1-LH-R is shown as SEQ ID No.5, the sequence of the primer DGA1-RH-F is shown as SEQ ID No.6, the sequence of the primer DGA1-RH-R is shown as SEQ ID No.7, the sequence of the primer HR-DGA1-XF is shown as SEQ ID No.8, and the sequence of the primer HR-DGA1-XR is shown as SEQ ID No.9.

[0068] 2) Construction of plasmid pCRISPRyl_ΔDGA1

[0069] The map of plasmid pCRISPRyl is attached Figure 2 , based on the plasmid pCRISPRyl, the plasmid pCRISPRyl_ΔDGA1 was constructed.

[0070] The Benchling CRISPR tool (https: / / benchling.com / crispr / ) was used to design the sgRNA sequence for ΔDGA1 gene knockout, and the corresponding primers DGA1-N20-F / DGA1-N20-R were designed.

[0071] Using plasmid pCRISPRyl as a template, reverse PCR amplification was performed with primers DGA1-N20-F / DGA1-N20-R. After amplification, the gene was digested with Dpn I and transformed into Escherichia coli. After successful screening and sequencing verification, the plasmid pCRISPRyl_ΔDGA1 for gene knockout was obtained.

[0072] The sequence of the primer DGA1-N20-F is shown in SEQ ID No.10, and the sequence of the primer DGA1-N20-R is shown in SEQ ID No.11:

[0073] 3) Construction of Yarrowia lipolytica engineered strain YLSQ10

[0074] The constructed pCRISPRyl_ΔDGA1 and pHR_ΔDGA1 plasmids were transformed into the Yarrowia lipolytica recombinant engineering strain YLSQ0 by chemical transformation. After screening and sequencing verification, the Yarrowia lipolytica recombinant engineering strain YLSQ10-1 with the DGA1 gene knocked out in the genome was obtained.

[0075] The recombinant engineering strain YLSQ10-1 was streaked on a YPD plate containing 5-fluoroorotic acid (1 g / L) and cultured at 30°C for 2 days to eliminate plasmids pCRISPRyl_ΔDGA1 and pHR_ΔDGA1. Single colonies on the plate were randomly selected and inoculated into liquid culture media of YPD, YNB+Leu, and YNB+Ura, respectively, and cultured in a shaker at 30°C and 220 rpm for 2-3 days. If the single colony can grow in YPD culture medium but cannot grow in YNB+Leu and YNB+Ura culture media, it means that the selection marker has been successfully removed and the recombinant engineering strain YLSQ10 is obtained.

[0076] The YPD plate culture medium comprises: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, fixed to 1 L with ddH2O, added with 2% agar powder, sterilized at 115°C for 20 min, cooled to about 50°C, and added with 50 μg / mL chloramphenicol.

[0077] YPD liquid culture medium: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, dilute to 1 L with ddH2O, and sterilize at 115°C for 20 min.

[0078] YNB+Leu liquid medium: glucose 20g / L, Yeast Nutrition Base (Shanghai Biotechnology) 6.7g / L, leucine 100mg / L, sterilized at 115℃ for 20min

[0079] YNB+Ura liquid culture medium: glucose 20 g / L, Yeast Nutrition Base (Shanghai Biotechnology) 6.7 g / L, uracil 100 mg / L, sterilized at 115°C for 20 min.

[0080] The primer sequences are shown in Table 1.

[0081] Table 1 Primer sequence list for constructing Yarrowia lipolytica engineered strain YLSQ10

[0082]

[0083]

[0084] Example 2 Fermentation of recombinant Yarrowia lipolytica strain YLSQ10 to produce squalene

[0085] Use an inoculation loop to pick up single colonies on the YPD medium plate, inoculate them into a 250mL Erlenmeyer flask filled with 50mL fresh YPD liquid medium, and shake and culture them in a constant temperature shaker at 30°C and 220rpm for 20h. According to the inoculation amount of 20%, transfer it to a 250mL Erlenmeyer flask filled with 50mL fresh YPD liquid medium, and shake and culture it in a constant temperature shaker at 30°C and 220rpm for 4 days. The yield of squalene in the fermentation broth was measured to be 2.52g / L.

[0086] Example 3 Construction of recombinant Yarrowia lipolytica strain YLSQ21

[0087] Based on the recombinant engineering strain YLSQ10, the squalene synthase SQS of Yarrowia lipolytica or the mutant SQS01-4 of squalene synthase were integrated into the A3 site of the genome.

[0088] The specific steps are as follows:

[0089] 1) Construction of plasmid pHR_A3

[0090] Using the extracted genome of Yarrowia lipolytica YLSQ0 as a template, primers A3-LH-F / A3-LH-R and A3-RH-F / A3-RH-R were used to amplify the upstream and downstream homology arms, respectively, and primers TEFin-F / TEFin-R and primers CYC1-F / CYC1-R were used to amplify P TEFin Promoter and T CYC1 terminator, and connected them with the pHR plasmid backbone amplified by primers HR-A3-XF / HR-A3-XR in a seamless cloning manner, transformed into Escherichia coli, and after screening and sequencing verification, the plasmid pHR_A3 with genomic integration at the A3 site was obtained.

[0091] The sequence of primer A3-LH-F is shown in SEQ ID No.12, the sequence of primer A3-LH-R is shown in SEQ ID No.13, the sequence of primer A3-RH-F is shown in SEQ ID No.14, the sequence of primer A3-RH-R is shown in SEQ ID No.15, the sequence of primer TEFin-F is shown in SEQ ID No.16, the sequence of primer TEFin-R is shown in SEQ ID No.17, the sequence of primer CYC1-F is shown in SEQ ID No.18, the sequence of primer CYC1-R is shown in SEQ ID No.19, the sequence of primer HR-A3-XF is shown in SEQ ID No.20, the sequence of primer HR-A3-XR is shown in SEQ ID No.21,

[0092] 2) Construction of plasmid pHR_A3_SQS0

[0093] Using the genome of Yarrowia lipolytica YLSQ10 as a template, the upstream and downstream primers SQS-F / SQS-R of squalene synthase were used to amplify the open reading frame of SQS, and the plasmid pHR_A3 was linearized with primers HR-SQS-XF / HR-SQS-XR. After seamless cloning, the plasmid was connected and transformed into Escherichia coli. After successful screening and sequencing verification, the recombinant plasmid pHR_A3_SQS0 was obtained.

[0094] The sequence of the primer SQS-F is shown as SEQ ID No.22, the sequence of the primer SQS-R is shown as SEQ ID No.23, the sequence of the primer HR-SQS-XF is shown as SEQ ID No.24, and the sequence of the primer HR-SQS-XR is shown as SEQ ID No.25.

[0095] 3) Construction of recombinant plasmid pCRISPRyl_A3

[0096] Using plasmid pCRISPRyl as a template, the plasmid was amplified by reverse PCR using primers gA3-F / gA3-R, digested with Dpn I after amplification, and then transformed into Escherichia coli. After screening and sequencing verification, the Cas9 plasmid pCRISPRyl_A3 that can be used for integration at the A3 site was obtained.

[0097] The sequence of the primer gA3-F is shown as SEQ ID No.26, and the sequence of the primer gA3-R is shown as SEQ ID No.27.

[0098] 4) Construction of Yarrowia lipolytica engineered strain YLSQ21

[0099] The constructed plasmids pCRISPRyl_A3 and pHR_A3_SQS0 were co-transformed into the recombinant Yarrowia lipolytica YLSQ10 by chemical transformation. After screening and sequencing verification, the recombinant engineering strain YLSQ21-1 of Yarrowia lipolytica with the squalene synthase SQS gene integrated into its genome was obtained.

[0100] The recombinant engineering strain YLSQ21-1 was streaked on a YPD plate containing 5-fluoroorotic acid (1 g / L) and cultured at 30°C for 2 days to eliminate plasmids pCRISPRyl_A3 and pHR_A3_SQS0. Single colonies on the plate were randomly selected and inoculated into liquid culture media of YPD, YNB+Leu, and YNB+Ura, respectively, and cultured in a shaker at 30°C and 220 rpm for 2-3 days. Strains that could grow in YPD medium but could not grow in YNB+Leu and YNB+Ura medium were selected to obtain the recombinant engineering strain YLSQ21.

[0101] The primer sequences are shown in Table 2.

[0102] Table 2 Primer sequence list for constructing Yarrowia lipolytica engineering strain YLSQ10

[0103]

[0104] Example 4 Construction of recombinant Yarrowia lipolytica strains YLSQ22 to YLSQ25

[0105] The squalene synthase gene was mutated to obtain squalene synthase variants SQS1-SQS4, primers were designed with the corresponding mutation site sequences, and full-sequence PCR was performed to construct recombinant plasmids pHR_A3_SQS1-pHR_A3_SQS4 containing the corresponding squalene synthase mutant nucleic acid sequences.

[0106] Referring to the same method as in Example 3, the constructed plasmids pCRISPRyl_A3 and pHR_A3_SQS1-4 were co-transformed into the recombinant Yarrowia lipolytica YLSQ10 by chemical transformation, and the Yarrowia lipolytica recombinant engineering strain with squalene synthase SQS gene integrated into the genome was obtained after screening and sequencing verification. Then, the strain was streaked on a YPD plate containing 5-fluoroorotic acid (1 g / L) and cultured at 30° C. for 2 days to eliminate the plasmid, and the Yarrowia lipolytica recombinant engineering strains YLSQ22 to YLSQ25 were obtained by screening.

[0107] Among them, after the squalene synthase is subjected to protein engineering, the amino acid sequences of the four squalene synthase mutants obtained are:

[0108] (1) The threonine at position 37 of the amino acid sequence shown in SEQ ID No. 3 was replaced with serine, and the result was named SQS1; the corresponding genome integration engineering strain was named YLSQ22;

[0109] (2) The alanine at position 55 of the amino acid sequence shown in SEQ ID No. 3 was replaced with leucine, and the result was named SQS2; the corresponding genome integration engineering strain was named YLSQ23;

[0110] (3) The isoleucine at position 81 of the amino acid sequence shown in SEQ ID No. 3 was replaced with threonine, and the result was named SQS3; the corresponding genome integration engineering strain was named YLSQ24;

[0111] (4) The lysine at position 137 of the amino acid sequence shown in SEQ ID No. 3 was replaced with threonine and named SQS4. The corresponding genome integration engineering strain was named YLSQ25.

[0112] Example 5 Fermentation of recombinant Yarrowia lipolytica strains YLSQ21-YLSQ25 to produce squalene

[0113] Single colonies on the YPD medium plate were picked up with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 20 h. According to the 20% inoculation amount, the inoculation was transferred to a 250 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 4 days. The squalene production in the fermentation broth was detected. The results are shown in Table 3. The recombinant strain YLSQ24 had the highest squalene production.

[0114] Table 3 Squalene production in shake flask fermentation of recombinant Yarrowia lipolytica YLSQ21-25

[0115]

[0116] Example 6 Fermentation of recombinant Yarrowia lipolytica YLSQ24 to produce squalene

[0117] Single colonies on the YPD medium plate were picked up with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 20 h. According to the inoculation amount of 20%, the colonies were transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid medium (pH 7.0) containing 20 g / L sodium lactate, sodium pyruvate, and sodium citrate, respectively, and cultured in a constant temperature shaker at 30°C and 220 rpm for 4 days. The squalene production in the fermentation broth was detected. The results are shown in Table 4. The addition of sodium pyruvate resulted in the highest squalene production.

[0118] Table 4 Squalene production in shake flask fermentation of recombinant Yarrowia lipolytica YLSQ24

[0119]

[0120] Example 7 Fermentation of recombinant Yarrowia lipolytica YLSQ24 to produce squalene

[0121] Single colonies on the YPD medium plate were picked up with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask filled with 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 20 h. According to 20% of the inoculation amount, the colonies were transferred to 250 mL Erlenmeyer flasks filled with 50 mL of YPD liquid medium (pH 7.0) containing 10-50 g / L sodium pyruvate, and cultured in a constant temperature shaker at 30°C and 220 rpm for 4 days. The squalene production in the fermentation broth was detected. The results are shown in Table 5. When 20 g / L sodium pyruvate was added, the squalene production was the highest.

[0122] Table 5 Squalene production in shake flask fermentation of recombinant Yarrowia lipolytica YLSQ24

[0123]

[0124] Example 8 Fermentation of recombinant Yarrowia lipolytica YLSQ24 to produce squalene

[0125] Single colonies on the YPD medium plate were picked up with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 20 h. The inoculation amount was transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid medium (pH 7.0) containing 20 g / L sodium pyruvate, and cultured in a constant temperature shaker at 30°C and 220 rpm for 24 h. 10 mL of n-octane, isooctane, n-decane, dodecane and tetradecane were added respectively, and culture was continued for 3 days. The squalene production in the fermentation broth was detected. The results are shown in Table 6. When dodecane was used as the extractant, the squalene production was the highest.

[0126] Table 6 Squalene production in shake flask fermentation of recombinant Yarrowia lipolytica YLSQ24

[0127]

[0128]

[0129] Example 9 Fermentation of recombinant Yarrowia lipolytica YLSQ24 to produce squalene

[0130] The single colonies on the YPD medium plate were picked up with an inoculation loop, inoculated into a 250 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and cultured in a constant temperature shaker at 30°C and 220 rpm for 20 h. The inoculation amount was transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid medium (pH 7.0) containing 20 g / L sodium pyruvate, and cultured in a constant temperature shaker at 30°C and 220 rpm for 24 h. 2.5-15 mL of dodecane was added, and the culture was continued for 3 days. The squalene production in the fermentation broth was detected. The results are shown in Table 7. When the amount of dodecane added was 7.5% (v / v), the squalene production was the highest.

[0131] Table 7 Squalene production from shake flask fermentation of recombinant Yarrowia lipolytica YLSQ24

[0132]

[0133] Example 10 Fermentation of recombinant Yarrowia lipolytica YLSQ24 to produce squalene

[0134] Prepare 1.8 L fermentation medium (40 g peptone, 20 g yeast extract, 40 g sodium pyruvate) and put it into a 5 L fermentation tank, sterilize it at 121°C for 20 min, and add 200 mL of filtered sterilized glucose solution (40%, w / v) after cooling.

[0135] Use an inoculation loop to pick a single colony on the YPD medium plate, inoculate it into a 500mL Erlenmeyer flask containing 100mL of fresh YPD liquid medium, and shake and culture it in a constant temperature shaker at 30°C and 220rpm for 24h. Inoculate 100mL of seed liquid into a fermenter, calibrate the initial dissolved oxygen to 100%, the ventilation volume to 2vvm, the dissolved oxygen to 25%, the fermentation temperature to 25°C, and the stirring speed range to 300-800rpm, and associate the dissolved oxygen and stirring speed. During the fermentation process, add 5M ammonia or 2M sulfuric acid solution to maintain the pH of the culture medium at 6.5. As the fermentation process progresses, the glucose concentration decreases rapidly. After the glucose is exhausted, 800g / L of glucose solution is added at a feed rate of 3mL / h. After 48h of fermentation, 150mL of sterilized dodecane is added. After 140h of fermentation, the squalene production reaches 42.1g / L.

[0136] After the fermentation was completed, the reaction solution was allowed to stand, the upper organic phase was separated, saturated brine was added and mixed thoroughly, the reaction solution was allowed to stand, the organic phase was separated, and 87.4 g of squalene was obtained by vacuum distillation.

[0137] Example 11 Chemical Reduction of Squalene to Produce Squalane

[0138] 10g of squalene obtained as in Example 7 was dissolved in 100ml of petroleum ether, and then injected into a high-pressure reactor, stirring was started, and 0.5g of Raine nickel catalyst was added under nitrogen protection. The reactor was closed, nitrogen was replaced with hydrogen, and then the hydrogenation reduction reaction was carried out for 8h at 40°C and a pressure of 0.1MPa. After the reaction was completed, the mixture was completely cooled, hydrogen was replaced with nitrogen, and then the catalyst was separated by filtration. The filtrate was subjected to reduced pressure distillation to obtain 8.4g of squalane with a yield of 84%.

[0139] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A Yarrowia lipolytica strain with high squalene production, characterized in that: It is a recombinant Yarrowia lipolytica strain in which the diacylglycerol acyltransferase DGA1 gene is knocked out based on the starting strain YLSQ0, wherein the nucleic acid sequence of the diacylglycerol acyltransferase DGA1 is shown in SEQ ID No. 1; The starting strain YLSQ0 is selected from recombinant Yarrowia lipolytica ATCC MYA-2613, MATA ura3-302, Leu2-270, xpr2-322, axp2-deltaNU49 XPR2::SUC2, Δku70, AXP::ScRAD52+ScRAD59, A08::ERG10+ERG13, POX3::tHMG1+ERG12, POX4::ERG8+ERG19, POX6::IDI+ERG2, CIT2::ACL1, MLS1::ACS L641P ,D17::ERG10-tHMG1,ERG1::P ACL -ERG1 G29S ,ΔERG5,ZETA::P hp4d -Ura3-T XPR2 -P Leu -Leu2-T XPR2 .

2. The method for constructing the high-squalene-producing Yarrowia lipolytica strain according to claim 1, characterized in that: The following steps are involved: 1) In recombinant Yarrowia lipolytica ATCC MYA-2613, MATAura3-302, Leu2-270, xpr2-322, axp2-deltaNU49 XPR2::SUC2, Δku70, AXP::ScRAD52+ScRAD59, A08::ERG10+ERG13, POX3::tHMG1+ERG12, POX4::ERG8+ERG19, POX6::IDI+ERG 2, CIT2::ACL1, MLS1::ACS L641P ,D17::ERG10-tHMG1,ERG1::P ACL -ERG1 G29S ,ΔERG5,ZETA::P hp4d -Ura3-T XPR2 -P Leu -Leu2-T XPR2 Based on the extracted genome as a template, the left and right homology arms for knocking out DGA1 were amplified with primers DGA1-LH-F / DGA1-LH-R and DGA1-RH-F / DGA1-RH-R, respectively. Then, the left and right homology arms of DGA1 were connected with the pHR plasmid backbone amplified with primers HR-DGA1-XF / HR-DGA1-XR by seamless cloning. After sequencing verification, the recombinant plasmid pHR_ΔDGA1 was obtained. 2) Construction of plasmid pCRISPRyl_ΔDGA1: Using plasmid pCRISPRyl as a template, reverse PCR amplification was performed with primers DGA1-N20-F / DGA1-N20-R. After amplification, the amplified fragment was digested with Dpn I and transformed into Escherichia coli. After successful screening and sequencing verification, the plasmid pCRISPRyl_ΔDGA1 for gene knockout was obtained. 3) Construction of the Yarrowia lipolytica engineered strain YLSQ10: The constructed pCRISPRyl_ΔDGA1 and pHR_ΔDGA1 plasmids were co-transformed into recombinant Yarrowia lipolytica YLSQ0 cells by chemical transformation, and the recombinant Yarrowia lipolytica strain YLSQ10-1 with the DGA1 gene knocked out was obtained after sequencing verification; Then, strain YLSQ10-1 was streaked on a YPD plate containing 5-fluoroorotic acid to eliminate plasmids pCRISPRyl_ΔDGA1 and pHR_ΔDGA1, and the recombinant engineered strain YLSQ10 was obtained; The sequence of primer DGA1-LH-F is shown as SEQ ID No.4, the sequence of primer DGA1-LH-R is shown as SEQ ID No.5, the sequence of primer DGA1-RH-F is shown as SEQ ID No.6, the sequence of primer DGA1-RH-R is shown as SEQ ID No.7, the sequence of primer HR-DGA1-XF is shown as SEQ ID No.8, the sequence of primer HR-DGA1-XR is shown as SEQ ID No.9, the sequence of primer DGA1-N20-F is shown as SEQ ID No.10, and the sequence of primer DGA1-N20-R is shown as SEQ ID No.

11.

3. A recombinant engineered strain of Yarrowia lipolytica with squalene synthase gene integrated into its genome, characterized in that: Integrating a squalene synthase gene into the genome of the high-squalene-producing Yarrowia lipolytica strain of claim 1; The squalene synthase is selected from squalene synthase SQS or a squalene synthase SQS mutant, wherein the amino acid sequence of squalene synthase SQS is as shown in SEQ ID No. 3, and the squalene synthase SQS mutant is a protein having the following amino acid sequence: (1) replacing the threonine at position 37 of the amino acid sequence shown in SEQ ID No. 3 with serine; (2) replacing the 55th alanine of the amino acid sequence shown in SEQ ID No. 3 with leucine; (3) replacing the isoleucine at position 81 of the amino acid sequence shown in SEQ ID No. 3 with threonine; (4) The lysine at position 137 of the amino acid sequence shown in SEQ ID No. 3 is replaced by threonine.

4. A method for constructing a recombinant engineering strain of Yarrowia lipolytica with squalene synthase gene integrated into its genome as claimed in claim 3, characterized in that: The following steps are involved: 1) Construction of plasmid pHR_A3: First, the genome of the high-squalene-producing Yarrowia lipolytica strain according to claim 1, i.e., the recombinant Yarrowia lipolytica strain YLSQ10, was used as a template, and the left and right homology arms of the genomic A3 insertion site were amplified using primers A3-LH-F / A3-LH-R and A3-RH-F / A3-RH-R, respectively, and the left and right homology arms of the genomic A3 insertion site were amplified using primers TEFin-F / TEFin-R and primers CYC1-F / CYC1-R, respectively. TEFin Promoter and T CYC1 terminator, and connected them with the pHR plasmid backbone amplified by primers HR-A3-XF / HR-A3-XR in a seamless cloning manner, transformed into Escherichia coli, and after screening and sequencing verification, the plasmid pHR_A3 with genome integration at the A3 site was obtained; 2) Construction of plasmid pHR_A3_SQS0: Using the genome of Yarrowia lipolytica YLSQ10 as a template, the upstream and downstream primers SQS-F / SQS-R of squalene synthase were used to amplify the open reading frame of natural squalene synthase SQS, and the plasmid pHR_A3 was linearized with primers HR-SQS-XF / HR-SQS-XR. After seamless cloning, the plasmid was connected and transferred into Escherichia coli. After successful sequencing verification, the recombinant plasmid pHR_A3_SQS0 was obtained; 3) Construction of recombinant plasmid pCRISPRyl_A3: Using plasmid pCRISPRyl as a template, pCRISPRyl was amplified by reverse PCR using primers gA3-F / gA3-R, and then digested with Dpn I. The plasmid was then transformed into Escherichia coli. After successful sequencing verification, the Cas9 plasmid pCRISPRyl_A3 that can be used for integration at the A3 site was obtained. 4) Construction of an engineered Yarrowia lipolytica strain YLSQ21: The plasmids pCRISPRyl_A3 and pHR_A3_SQS0 were co-transformed into the high-squalene-producing Yarrowia lipolytica strain of claim 1 by chemical transformation, and after gene integration, the recombinant Yarrowia lipolytica strain YLSQ21-1 was obtained; Then strain YLSQ21-1 was streaked on YPD plates containing 5-fluoroorotic acid to eliminate plasmids pCRISPRyl_A3 and pHR_A3_SQS0, and the recombinant engineered strain YLSQ1021 was obtained; In the same way, the squalene synthase SQS mutant gene is integrated into the high-squalene-producing Yarrowia lipolytica strain of claim 1 to obtain recombinant Yarrowia lipolytica strains YLSQ22 to YLSQ25; Wherein, the sequence of the primer A3-LH-F is shown in SEQ ID No.12, the sequence of the primer A3-LH-R is shown in SEQ ID No.13, the sequence of the primer A3-RH-F is shown in SEQ ID No.14, the sequence of the primer A3-RH-R is shown in SEQ ID No.15, the sequence of the primer TEFin-F is shown in SEQ ID No.16, the sequence of the primer TEFin-R is shown in SEQ ID No.17, the sequence of the primer CYC1-F is shown in SEQ ID No.18, the sequence of the primer CYC1-R is shown in SEQ ID No.19, the sequence of the primer HR-A3-XF is shown in SEQ ID No.20, the sequence of the primer HR-A3-XR is shown in SEQ ID No.21, the sequence of the primer SQS-F is shown in SEQ ID No.22, the sequence of SQS-R is shown in SEQ ID No.23, the sequence of HR-SQS-XF is shown in SEQ ID The sequence of primer CRISPR-A3-F is shown in SEQ ID No.26, and the sequence of primer CRISPR-A3-R is shown in SEQ ID No.

27.

5. A method for preparing squalene, characterized in that: Cultivate the high-squalene-producing Yarrowia lipolytica strain of claim 1 or the recombinant engineered Yarrowia lipolytica strain of claim 3, and separate squalene from the culture solution.

6. The method for preparing squalene according to claim 5, characterized in that: Lactic acid, pyruvic acid, and citrate are added to the culture medium at a rate of 1-5% (w / v) to enhance tricarboxylic acid cycle metabolism.

7. The method for preparing squalene according to claim 5, characterized in that: During the culture process, alkanes with a carbon chain length of 8-14 are added in an amount of 5-30% (v / v) and the addition time is 1-4 days after inoculation to promote the timely excretion of squalene accumulated in the cells.

8. The method for preparing squalene according to claim 5, characterized in that: After the cultivation is completed, the reaction solution is extracted with a solvent, or the organic phase containing squalene is separated and then distilled under reduced pressure to obtain purified squalene.

9. A method for preparing squalane, characterized in that: The squalene obtained in claim 5 is subjected to hydrogenation reduction using Raney nickel as a catalyst to obtain the saturated alkane squalane.

10. The method for preparing squalane according to claim 9, characterized in that: Under nitrogen protection, squalene is dissolved in an organic solvent, stirring is started, 1-10% (w / w) Raney nickel is added, and then nitrogen is replaced with hydrogen, and hydrogenation reduction reaction is carried out at 30-50°C for 6-12h, cooled, replaced with nitrogen, the catalyst is filtered out, and squalane is obtained by reduced pressure distillation.

Images