Recombinant Yarrowia lipolytica for high production of tetraacetylphytosphingosine and its application

Abstract

The present invention provides a recombinant Yarrowia lipolytica of high-yield tetraacetyl phytosphingosine and its application, belonging to the field of bioengineering. The recombinant Yarrowia lipolytica of high-yield tetraacetyl phytosphingosine constructed by the present invention is named as Yarrowia lipolytica (Yarrowia lipolytica) XJ TAPS strain, and the construction method of the recombinant Yarrowia lipolytica of high-yield tetraacetyl phytosphingosine, including Wickerhamomyces ciferrii (Wickerhamomyces ciferrii) phytosphingosine acetyltransferase enzyme gene expression cassette, phosphorylase gene knockout cassette, serine palmitoyltransferase gene expression cassette, 3 keto dihydrosphingosine reductase gene expression cassette, C 4 hydroxylase gene expression cassette, integrated in the Yarrowia lipolytica genome by homologous recombination. The construction method of the recombinant Yarrowia lipolytica of the present invention is simple to operate, and tetraacetyl phytosphingosine can be efficiently synthesized.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a recombinant Yarrowia lipolytica capable of producing high amounts of tetraacetyl phytosphingosine and an application thereof. Background Art

[0002] Tetraacetyl phytosphingosine (TAPS) is a long-chain sphingosine with three acetylated hydroxyl groups and one acetylated amine. It has moisturizing, anti-oxidation, whitening and spot-lightening effects. The phytosphingosine generated after deacetylation can be used as a precursor to synthesize ceramides in moisturizing skin care products. Therefore, TAPS has a high application value in the skin care and cosmetics industries.

[0003] At present, TAPS is mainly obtained through microbial synthesis. The unconventional yeast Wickerhamomyces ciferrii is the only known microorganism that can naturally secrete TAPS. It produces TAPS by acetylation of three hydroxyl groups and one amino group of phytosphingosine, and secretes it outside the cell at high concentrations to counteract the cytotoxicity of phytosphingosine. In addition, with the development of synthetic biology, the use of microorganisms with superior performance to build cell factories has also become a popular choice. Yarrowia lipolytica is a recognized safe unconventional oil-producing yeast that can provide sufficient acetyl-CoA for the synthesis of fatty acids, among which palmitic acid can be used as a precursor for the synthesis of TAPS. In addition, Yarrowia lipolytica has the ability to resist the cytotoxicity caused by phytosphingosine and its derivatives because of its strong robustness in tolerating harsh environments such as low acid and high salt. Therefore, Yarrowia lipolytica has the potential to be developed as a cell factory for the efficient synthesis of TAPS. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and its application in view of the shortcomings of the prior art.

[0005] In order to solve the above technical problems, the present invention discloses a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and its application. The following technical scheme is adopted:

[0006] The present invention provides a recombinant Yarrowia lipolytica strain that produces high yield of tetraacetyl phytosphingosine, wherein the recombinant Yarrowia lipolytica strain simultaneously expresses an O- and N-acetyltransferase (O-, N-Acetyl transferase) gene WcSLI1 and an O-acetyltransferase (O-Acetyl transferase) gene WcATF2. The O- and N-acetyltransferase gene WcSLI1 and the O-acetyltransferase gene WcATF2 are derived from Wickerhamomyces ciferrii.

[0007] The nucleotide sequence of the O- and N-acetyltransferase gene WcSLI1 is shown in SEQ ID No.1; the nucleotide sequence of the O-acetyltransferase gene WcATF2 is shown in SEQ ID No.2.

[0008] Wherein, the recombinant Yarrowia lipolytica also adopts one of the following three methods or any combination thereof:

[0009] (1) Knockout of phosphorylase gene;

[0010] (2) expression of serine palmitoyltransferase gene;

[0011] (3) Simultaneous expression of 3-ketodihydrosphingosine reductase gene and C-4 hydroxylase (C4-hydroxylase) gene.

[0012] Wherein, the phosphorylase gene is the endogenous LCB4 gene of Yarrowia lipolytica.

[0013] Wherein, the nucleotide sequence of the LCB4 gene is shown as SEQ ID No.7.

[0014] The serine palmitoyltransferase gene is any one of the WcLCB1 gene and the WcLCB2 gene or a combination of both of them, and the WcLCB1 gene and the WcLCB2 gene are coding genes of serine palmitoyltransferase from Saccharomyces cerevisiae.

[0015] The nucleotide sequence of the WcLCB1 gene is shown in SEQ ID No.3; the nucleotide sequence of the WcLCB2 gene is shown in SEQ ID No.4.

[0016] The 3-ketodihydrosphingosine reductase gene is derived from the WcTSC10 gene of Saccharomyces cerevisiae; and the C-4 hydroxylase gene is derived from the WcSYR2 gene of Saccharomyces cerevisiae.

[0017] Among them, the nucleotide sequence of the WcTSC10 gene is shown as SEQ ID No.5; the nucleotide sequence of the WcSYR2 gene is shown as SEQ ID No.6.

[0018] Preferably, the nucleotide sequences described in the above SEQ ID No.1-6 are sequences after codon optimization. The original nucleotide sequences are as follows: The GenBank accession number of the O- and N-acetyltransferase encoding gene is XM_011273126.1. The GenBank accession number of the O-acetyltransferase encoding gene is XM_011272738.1. The GenBank accession number of the serine palmitoyltransferase gene is JN645676.1 and AF053456.1. The GenBank accession number of the 3-ketodihydrosphingosine reductase gene is JN645686.1, and the GenBank accession number of the C-4 hydroxylase gene is HQ166122.1.

[0019] Preferably, the present invention provides a recombinant Yarrowia lipolytica that produces high levels of tetraacetylphytosphingosine, wherein the recombinant Yarrowia lipolytica simultaneously expresses O- and N-acetyltransferase genes WcSLI1, O-acetyltransferase gene WcATF2, serine palmitoyltransferase genes WcLCB1 and WcLCB2, 3-ketodihydrosphingosine reductase gene WcTSC10 and C-4 hydroxylase gene WcSYR2, and knocks out the phosphorylase gene LCB4. The O- and N-acetyltransferase genes WcSLI1, O-acetyltransferase gene WcATF2, serine palmitoyltransferase genes WcLCB1 and WcLCB2, 3-ketodihydrosphinganine reductase gene WcTSC10 and C-4 hydroxylase gene WcSYR2 are derived from Wickham syringa and obtained by codon optimization; the phosphorylase gene LCB4 is derived from Yarrowia lipolytica; the nucleotide sequence of the O- and N-acetyltransferase gene WcSLI1 is shown in SEQ ID No.1; the nucleotide sequence of the O-acetyltransferase gene WcATF2 is shown in SEQ ID No.2; the nucleotide sequence of the phosphorylase gene LCB4 is shown in SEQ ID No.7; the nucleotide sequence of the serine palmitoyltransferase genes WcLCB1 and WcLCB2 is shown in SEQ ID No.3-4; the nucleotide sequence of the 3-ketodihydrosphinganine reductase gene WcTSC10 is shown in SEQ ID No.5; the nucleotide sequence of the C-4 hydroxylase gene WcSYR2 is shown in SEQ ID No.6. The recombinant Yarrowia lipolytica is classified and named as Yarrowia lipolytica, strain number XJ-TAPS, and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO:M 2024780, a deposit date of April 24, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0020] Preferably, the construction method of the recombinant Yarrowia lipolytica XJ-TAPS is as follows: inserting an exogenous acetyltransferase gene expression cassette, a serine palmitoyltransferase gene expression cassette, a 3-ketodihydrosphinganine reductase gene expression cassette, and a C-4 hydroxylase expression cassette into the genome of the original strain of Yarrowia lipolytica and knocking out the intermediate product phosphorylase gene expression cassette. Specifically, knocking out the phosphorylase gene LCB4, inserting the exogenous acetylase gene SLI1 and ATF2 expression cassettes, the serine palmitoyltransferase gene LCB1 and LCB2 expression cassettes, the 3-ketodihydrosphinganine reductase gene TSC10 expression cassette, and the C-4 hydroxylase gene SYR2 expression cassette are introduced into Yarrowia lipolytica in the form of a plasmid, and then integrated into the Yarrowia lipolytica genome by homologous recombination.

[0021] More preferably, the promoter of the expression cassette is any one of the TEF promoter, TEFin promoter, EXP promoter, FBAin promoter or GPDin promoter of Yarrowia lipolytica; the terminator of the expression cassette is any one of the xpr2t terminator, mig1t terminator, lip2t terminator or cyc1t terminator of Yarrowia lipolytica; the integration site of the expression cassette is any one of the IntD site, LCB4 site or IntA site of Yarrowia lipolytica.

[0022] More preferably, the original strain of Yarrowia lipolytica is the Yarrowia lipolytica strain Po1f in which the KU70 gene is knocked out (see Kretzschmar A, et al., Current Genetics, 2013, 59(1-2): 63-72).

[0023] The above-mentioned method for constructing the recombinant Yarrowia lipolytica is efficient and easy to operate.

[0024] In a second aspect, the present invention provides use of the recombinant Yarrowia lipolytica described in the first aspect in the fermentation production of tetraacetylphytosphingosine.

[0025] Preferably, the recombinant Yarrowia lipolytica is inoculated into a fermentation medium for fermentation culture for 5 to 7 days.

[0026] Beneficial effects:

[0027] The present invention is based on knocking out the Yarrowia lipolytica responsible for encoding the non-homologous recombination gene KU70, so that its homologous recombination ability is enhanced, and the integration of genes is achieved through the homologous recombination function of the Yarrowia lipolytica itself, which can greatly improve the genetic stability of the introduced gene, and construct a recombinant Yarrowia lipolytica with high yield of tetraacetylphytosphingosine (TAPS). The recombinant Yarrowia lipolytica constructed by the present invention realizes the synthesis of TAPS from scratch in Yarrowia lipolytica by inserting exogenous acetyltransferase gene SLI1 and ATF2 expression cassettes. The Yarrowia lipolytica XJ-TAPS strain constructed by the present invention knocks out the phosphorylase gene LCB4, inserts the exogenous acetyltransferase gene SLI1 and ATF2 expression cassettes, the serine palmitoyltransferase gene LCB1 and LCB2 expression cassettes, the 3-ketodihydrosphinganine reductase gene TSC10 expression cassette, and the C-4 hydroxylase gene SYR2 expression cassette, and experiments prove that the recombinant Yarrowia lipolytica can efficiently ferment and produce TAPS (the TAPS yield at the 5L fermentation tank level is 5.28g / L), and realizes the efficient synthesis of TAPS that can be naturally produced by Wickham's yeast in Yarrowia lipolytica. The strain XJ-TAPS has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO:M 2024780, and a deposit date of April 24, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0029] Figure 1 TAPS synthesis strategy diagram provided for the implementation of the present invention. Sli1: O- and N-acetyltransferase; Atf2: O-acetyltransferase; Lcb4: phosphorylase; Lcb1: serine palmitoyltransferase 1; Lcb2: serine palmitoyltransferase 2; Tsc10: 3-ketodihydrosphingosine reductase; Syr2: C-4 hydroxylase.

[0030] Figure 2 is the structure diagram of the recombinant plasmid pUC-HUH-IntD-WcSLI1, where IntD-up represents the upstream homology arm of the IntD site, IntD-dm represents the downstream homology arm of the IntD site, and TEFin represents the promoter P TEFin , mig1t indicates the terminator T mig1t , URA indicates the orotidine 5'-phosphate decarboxylase encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ), WcSLI1 is a gene encoding O- and N-acetyltransferase.

[0031] Figure 3 The structure of the recombinant plasmid pUC-HUH-IntD-WcSLI1-WcATF2, where IntD-up represents the upstream homology arm of the IntD site, IntD-dm represents the downstream homology arm of the IntD site, and FBAin represents the promoter P FBAin , xpr2t indicates the terminator T xpr2t , URA indicates the orotidine 5'-phosphate decarboxylase encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ), WcSLI1 is an O- and N-acetyltransferase encoding gene, and WcATF2 is an O-acetyltransferase encoding gene.

[0032] Figure 4 It is a structural diagram of the recombinant plasmid pUC-HUH-LCB4, wherein LCB4-up represents the upstream homology arm of the LCB4 site, LCB4-dm represents the downstream homology arm of the LCB4 site, URA represents the expression cassette of the gene encoding orotidine-5'-phosphate decarboxylase, and LCB4 is the phosphorylase gene.

[0033] Figure 5 is the structure diagram of the recombinant plasmid pUC-HUH-LCB4-WcLCB1-WcLCB2, where LCB4-up represents the upstream homology arm of the LCB4 site, LCB4-dm represents the downstream homology arm of the LCB4 site, and TEFin represents the promoter P TEFin , FBAin indicates promoter P FBAin , cyc1t and xpr2t represent the terminator T cyc1t and T xpr2t , URA indicates the orotidine 5'-phosphate decarboxylase encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ), LCB4 is a phosphorylase gene, and LCB1 and LCB2 are serine palmitoyltransferase genes.

[0034] Figure 6 The structure of the recombinant plasmid pUC-HUH-IntA-WcTSC10-WcSYR2, where IntA-up represents the upstream homology arm of the IntA site, IntA-dm represents the downstream homology arm of the IntA site, TEF and EXP represent the promoter P TEF and P EXP , mig1t and cyc1t represent the terminator T mig1t and T cyc1t , URA indicates the orotidine 5'-phosphate decarboxylase encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ), TSC10 is the gene encoding 3-ketodihydrosphingosine reductase, and SYR2 is the gene encoding C-4 hydroxylase.

[0035] Figure 7 This is a HPLC detection chart of TAPS provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below by specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0037] The Yarrowia lipolytica Po1f described in the following examples was purchased from the American Type Culture Collection and is numbered ATCC MYA-2613.

[0038] The Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::hisG, leu2-270, ura3-302, xpr2-322, axp1-2) described in the following examples is abbreviated as Yarrowia lipolytica Po1fΔku70. Yarrowia lipolytica Po1fΔku70 was constructed by knocking out the gene KU70 encoding non-homologous recombination in Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2):63-72).

[0039] The IntD site integration plasmid described in the following embodiment is obtained by inserting a sequence of 1443 bp upstream of the start codon of the IntD site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome (upstream homology arm) and a sequence of 1189 bp downstream of the stop codon (downstream homology arm) into a pUC57-hisG-ura-hisG vector (the construction method is shown in Example 1), and the two hisG tag encoding genes are located between the upstream and downstream homology arms of the IntD site.

[0040] The LCB4 site integration plasmid described in the following example is obtained by inserting a 1003 bp sequence (upstream homology arm) upstream of the start codon of the LCB4 site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome and a 934 bp sequence (downstream homology arm) downstream of the stop codon into a pUC57-hisG-ura-hisG vector (the construction method is shown in Example 1), and two hisG tag encoding genes are located between the upstream and downstream homology arms of the LCB4 site.

[0041] The IntA site integration plasmid described in the following embodiment is obtained by inserting a sequence of 1633 bp upstream of the start codon of the IntA site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome (upstream homology arm) and a sequence of 1621 bp downstream of the stop codon (downstream homology arm) into a pUC57-hisG-ura-hisG vector (the construction method is shown in Example 1), and the two hisG tag encoding genes are between the upstream and downstream homology arms of the IntA site.

[0042] The YPD liquid medium described in the following examples has a formula of 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose. The screening medium SD-Ura has a formula of 48 g / L SD-Ura (purchased from Wuhan Puinte Bioengineering Co., Ltd.), 12 g / L agar powder, and water as solvent. The YPD plate containing 5-fluoroorotic acid (5-FOA) has a formula of 25 g / L agar and 1 g / L 5-FOA added to the YPD liquid medium.

[0043] The TAPS synthesis strategy diagram provided by the embodiment of the present invention is shown in Figure 1 .

[0044] Example 1 Amplification of genetic elements and preparation of target plasmids

[0045] (I) Preparation of target gene According to the nucleotide sequence of the O- and N-acetyltransferase Sli1 encoding gene from Wickerhamomyces ciferrii (GenBank accession number: XM_011273126.1) provided by NCBI, after codon optimization, Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the optimized O- and N-acetyltransferase Sli1 encoding gene WcSLI1 (SEQ ID No. 1) and insert it into plasmid pUC57 to obtain plasmid pUC57-WcSLI1.

[0046] Preparation of the target gene According to the nucleotide sequence of the O-acetyltransferase Atf2 encoding gene from Wickerhamomyces ciferrii (GenBank accession number: XM_011272738.1) provided by NCBI, after codon optimization, Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the optimized O-acetyltransferase Atf2 encoding gene WcATF2 (SEQ ID No. 2) and insert it into plasmid pUC57 to obtain plasmid pUC57-WcATF2.

[0047] Preparation of target genes According to the nucleotide sequence of the serine palmitoyltransferase encoding gene from Wickerhamomyces ciferrii (GenBank accession numbers: JN645676.1, AF053456.1) provided by NCBI, after codon optimization, Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the optimized serine palmitoyltransferase encoding genes WcLCB1 and WcLCB2 (SEQ ID No.3, SEQ ID No.4) and insert them into plasmid pUC57 to obtain plasmids pUC57-WcLCB1 and pUC57-WcLCB2.

[0048] Preparation of target genes According to the nucleotide sequences of the 3-ketodihydrosphinganine reductase encoding gene (GenBank accession number: JN645686.1) and the C-4 hydroxylase encoding gene (GenBank accession number: HQ166122.1) from Wickerhamomyces ciferrii provided by NCBI, after codon optimization, Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the optimized 3-ketodihydrosphinganine reductase encoding gene WcTSC10 (SEQ ID No. 5) and C-4 hydroxylase encoding gene WcSYR2 (SEQ ID No. 6), and inserted into plasmid pUC57 to obtain plasmids pUC57-WcTSC10 and pUC57-WcSYR2.

[0049] According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase encoding gene URA of Yarrowia lipolytica provided by NCBI (GenBank accession number: AJ306421.1) and hisG tag (GenBank accession number: AF324729.1), Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the two hisG tag encoding gene sequences into plasmid pUC57, and the orotidine-5'-phosphate decarboxylase encoding gene URA expression cassette (P TEFin -URA-T xpr2t ) to achieve recovery of the Ura selection marker and obtain the plasmid pUC57-hisG-ura-hisG.

[0050] Using Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, LCB4::LCB4-up-F and LCB4::LCB4-up-R were used as primers to amplify the upper homologous arm LCB4-up of the phosphorylase encoding gene LCB4, and LCB4::LCB4-dm-F and LCB4::LCB4-dm-R were used as primers to amplify the lower homologous arm LCB4-dm of the phosphorylase encoding gene LCB4 (GenBank accession number of LCB4: XM_002142977.1, SEQ ID No.7).

[0051] Yarrowia lipolytica endogenous promoter P TEFin The nucleotide sequence of is shown in SEQ ID No.8; the endogenous promoter P of Yarrowia lipolytica GPDin The nucleotide sequence is shown in SEQ ID No.9; the endogenous promoter P TEFThe nucleotide sequence of is shown in SEQ ID No.10; the endogenous promoter P of Yarrowia lipolytica FBAin The nucleotide sequence is shown in SEQ ID No.11;

[0052] Yarrowia lipolytica endogenous terminator T mig1t The nucleotide sequence is shown in SEQ ID No.12; the terminator T endogenous to Yarrowia lipolytica cyc1t The nucleotide sequence is shown in SEQ ID No.13; the terminator T endogenous to Yarrowialipolytica lip2t The nucleotide sequence is shown in SEQ ID No.14; the terminator T endogenous to Yarrowia lipolytica xpr2t The nucleotide sequence is shown as SEQ ID No.15.

[0053] (II) Construction of recombinant plasmid

[0054] The structures of the recombinant plasmids are shown in Tables 3 and Figure 2-6 ; The primers used to construct the recombinant plasmid are shown in Table 4.

[0055] 1. Construction of recombinant integration plasmid:

[0056] The recombinant integration plasmids pUC-HUH-IntD-WcSLI1, pUC-HUH-IntD-WcSLI1-WcATF2, pUC-HUH-LCB4, pUC-HUH-LCB4-WcLCB1-WcLCB2, and pUC-HUH-IntA-WcTSC10-WcSYR2 were based on pUC57-hisG-ura-hisG, and inserted with the homology arms upstream of the start codon and downstream of the stop codon of the IntD site, LCB4 site, and IntA site in Yarrowia lipolytica Po1fΔku70, and gene expression cassettes were inserted between the upstream and downstream homology arms. They are: IntD-up, IntD-dm, WcSLI1 expression cassette (P TEFin -WcSLI1-T mig1t )、URA expression cassette (P TEFin -URA-T xpr2t ); IntD-up, IntD-dm, WcATF2 expression cassette (P FBAin -WcATF2-T xpr2t )、URA expression cassette (P TEFin -URA-T xpr2t ); LCB4-up, LCB4-dm, URA expression cassette (PTEFin -URA-T xpr2t ); LCB4-up, LCB4-dm, WcLCB1 expression cassette (P TEFin -WcLCB1-T xpr2t )、URA expression cassette (P TEFin -URA-T xpr2t ); LCB4-up, LCB4-dm, WcLCB2 expression cassette (P FBAin -WcLCB2-T cyc1t )、URA expression cassette (P TEFin -URA-T xpr2t ); IntA-up, IntA-dm, WcTSC10 expression cassette (P EXP -WcTSC10-T cyc1t )、URA expression cassette (P TEFin -URA-T xpr2t ); IntA-up, IntA-dm, WcSYR2 expression cassette (P TEF -WcSYR2-T mig1t )、URA expression cassette (P TEFin -URA-T xpr2t );

[0057] The WcSLI1 expression cassette (P TEFin -WcSLI1-T mig1t )、WcATF2 expression cassette (P FBAin -WcATF2-T xpr2t )、LCB4-up、LCB4-dm、WcLCB1 expression cassette (P TEFin -WcLCB1-T xpr2t )、WcLCB2 expression cassette (P FBAin -WcLCB2-T cyc1t )、WcTSC10 expression cassette (P EXP -WcTSC10-T cyc1t )、WcSYR2 expression cassette (P TEF -WcSYR2-T mig1t ) The promoter, coding gene and terminator fragments were amplified by the primers described in Table 4. They are: IntD::P TEFin -F, IntD::P TEFin -R, IntD::WcSLI1-F, IntD::WcSLI1-R, IntD::T mig1t -F, IntD::T mig1t -R, IntD::P FBAin -F, IntD::P FBAin-R, IntD::WcATF2-F, IntD::WcATF2-R, IntD::T xpr2t -F, IntD::T xpr2t -R, LCB4::LCB4-up-F, LCB4::LCB4-up-R, LCB4::LCB4-dm-F, LCB4::LCB4-dm-R, LCB4::P TEFin -F, LCB4::P TEFin -R, LCB4::WcLCB1-F, LCB4::WcLCB1-R, LCB4:T xpr2t -F, LCB4:T xpr2t -R, LCB4::P TEFin -F, LCB4::P TEFin -R, LCB4::WcLCB2-F, LCB4::WcLCB2-R, LCB4:T cyc1t -F, LCB4:T cyc1t -R, IntA::P EXP -F, IntA::P EXP -R, IntA::WcTSC10-F, IntA::WcTSC10-R, IntA::T cyc1t -F, IntA::T cyc1t -R, IntA::P TEF -F, IntA::P TEF -R, IntA::WcSYR2-F, IntA::WcSYR2-R, IntA::T mig1t -F, IntA::T mig1t -R.

[0058] The specific construction method takes the recombinant plasmid pUC-HUH-IntD-WcSLI1 as an example. The recombinant plasmid pUC-HUH-IntD-WcSLI1 is based on pUC57-hisG-ura-hisG as the backbone, and the homology arm IntD-up upstream of the start codon and the homology arm IntD-dm downstream of the stop codon of the IntD site in Yarrowia lipolytica Po1fΔku70 are inserted. The WcSLI1 expression cassette (P TEFin -WcSLI1-T mig1t ), orotidine-5'-phosphate decarboxylase encoding gene URA expression cassette (containing Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ) also between the upstream and downstream homology arms.

[0059] The genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as template and the IntD::P TEFin -F and IntD::P TEFin -R is a primer to amplify the promoter P of the WcSLI1 expression cassette TEFin . With IntD::T mig1t -F and IntD::T mig1t -R is a primer to amplify the terminator T of the WcSLI1 expression cassette mig1t .

[0060] Plasmid pUC57-WcSLI1 was used as template and primers IntD::WcSLI1-F and IntD::WcSLI1-R were used to amplify the gene with promoter P at both ends. TEFin and terminator T mig1t Homologous arms of the WcSLI1 gene.

[0061] The above PCR amplification system is shown in Table 1:

[0062] Table 1 PCR amplification system

[0063]

[0064] Among them, PrimerSTAR Max Premix was purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.

[0065] The PCR program was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C (extension time = target fragment length / 1 kb, unit: min), and repeated for 34 cycles.

[0066] Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Bioshine Biotechnology Co., Ltd.).

[0067] The IntD site integration plasmid was digested with restriction endonuclease HindⅢ from NEB Company, and the linearized IntD site integration plasmid was recovered by agarose gel electrophoresis.

[0068] The linearized IntD site was integrated into the plasmid and the various elements (promoter P TEFin , gene WcSLI1 and terminator T mig1t) One-step cloning was achieved using the ClonExpressMultiS One Step Cloning Kit of Nanjing Novozyme Biotechnology Co., Ltd. The WcSLI1 gene expression cassette was inserted between the upstream and downstream homology arms of the IntD site integration plasmid, and the two hisG tag encoding genes were on the same side of the WcSLI1 gene expression cassette. The one-step cloning reaction system is shown in Table 2. After the reaction system was incubated at 37°C for 30 minutes, a circular recombinant vector was obtained.

[0069] Table 2 One-step cloning system

[0070]

[0071] The usage of the linearized vector (x) and the inserted fragment (y) can be calculated by the following formula: The optimal usage of each fragment or linearized vector = [0.02 × the number of base pairs of the fragment or linearized vector] ng.

[0072] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened on LB plates for ampicillin resistance and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-IntD-WcSLI1.

[0073] The plasmid pUC-HUH-IntD-WcSLI1 was digested with restriction endonuclease NotⅠ from NEB Company, and the linearized pUC-HUH-IntD-WcSLI1 plasmid was recovered by agarose gel electrophoresis.

[0074] 2. Construction of recombinant knockout plasmid pUC-HUH-LCB4:

[0075] The recombinant knockout plasmid pUC-HUH-LCB4 is based on pUC57-hisG-ura-hisG, and the homology arm LCB4-up upstream of the start codon and the homology arm LCB4-dm downstream of the stop codon of the LCB4 site in Yarrowialipolytica Po1fΔku70 are inserted. The above fragments are amplified by the primers described in Table 4 to amplify the upstream and downstream homology arms of the site.

[0076] The recombinant plasmid pUC-HUH-LCB4 is based on pUC57-hisG-ura-hisG, with a homology arm of 1003 bp upstream of the start codon of the LCB4 locus in Yarrowia lipolytica Po1fΔku70 (LCB4-up) and a homology arm of 934 bp downstream of the stop codon (LCB4-dm), and an expression cassette of the orotidine-5'-phosphate decarboxylase encoding gene URA (containing the endogenous promoter P TEFin, terminator T xpr2t ) also between the upstream and downstream homology arms.

[0077] Using LCB4::LCB4-up-F and LCB4::LCB4-up-R as primers and Yarrowia lipolytica Po1fΔku70 genomic DNA as template, the homology arm LCB4-up upstream of the start codon of the LCB4 site was amplified. The plasmid pUC57-hisG-ura-hisG was digested with restriction endonuclease EcoRⅠ from NEB, and the linearized pUC57-hisG-ura-hisG plasmid was recovered by agarose gel electrophoresis. The linearized pUC57-hisG-ura-hisG plasmid and the homology arm LCB4-up upstream of the start codon of the LCB4 site constructed in this example (with pUC57-hisG-ura-hisG homology arm sequences at both ends) were cloned and cloned using the DNA sequence of Nanjing Novozyme Biotechnology Co., Ltd. IIOne Step Cloning Kit achieves one-step cloning to obtain a circular recombinant vector. The circular recombinant vector was transformed into E. coli DH5α competent cells, screened on LB plates for ampicillin resistance, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-LCB4-up.

[0078] Using LCB4::LCB4-dm-F and LCB4::LCB4-dm-R as primers and Yarrowia lipolytica Po1fΔku70 genomic DNA as a template, the homology arm LCB4-dm downstream of the LCB4 site stop codon was amplified. The plasmid pUC-HUH-LCB4-up was digested with the restriction endonuclease HindⅢ from NEB, and the linearized pUC-HUH-LCB4-up plasmid was recovered by agarose gel electrophoresis. The linearized pUC-HUH-LCB4-up plasmid and the homology arm LCB4-dm downstream of the LCB4 site stop codon constructed in this example (with pUC-HUH-LCB4-up homology arm sequences at both ends) were amplified using the IIOne Step Cloning Kit was used to achieve one-step cloning and obtain the recombinant plasmid pUC-HUH-LCB4.

[0079] Table 3 Insertion sequences in each recombinant plasmid

[0080]

[0081] Table 4 Primer sequences

[0082]

[0083]

[0084]

[0085] Example 2 Construction of recombinant Yarrowia lipolytica (I) Construction of recombinant strain 1 (expressing WcSLI1)

[0086] The recombinant plasmid pUC-HUH-IntD-WcSLI1 was introduced into Yarrowia lipolytica Po1fΔku70, and the WcSLI1 expression cassette was integrated into the genomic IntD site by homologous recombination, thereby expressing the O- and N-acetyltransferase genes, and then the recombinant bacteria 1 were obtained after losing a hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure.

[0087] The specific method is as follows:

[0088] Yarrowia lipolytica Po1fΔku70 was inoculated into YPD liquid medium and cultured overnight at 30°C to prepare competent cells and transfer the recombinant plasmid: the recombinant plasmid pUC-HUH-IntD-WcSLI1 was transformed into Yarrowialipolytica Po1fΔku70 using Zymogen Frozen EZYeast Transformation Kit II from Zymo Research Corporation for homologous recombination, and positive clones were screened using screening medium SD-Ura, followed by PCR identification.

[0089] The positive clones identified by PCR were spread on YPD plates containing 5-fluoroorotic acid, and single colonies were streaked on YPD plates containing 5-fluoroorotic acid and SD-Ura plates at the same time. The single colony that could grow on the YPD plate containing 5-fluoroorotic acid but could not grow on the SD-Ura plate was selected and named as recombinant bacteria 1.

[0090] (II) Construction of recombinant strain 2 (expressing WcSLI1 and WcATF2)

[0091] The recombinant plasmid pUC-HUH-IntD-WcSLI1-WcATF2 was introduced into Yarrowia lipolytica Po1fΔku70, and the WcSLI1 expression cassette and the WcATF2 expression cassette were integrated into the genomic IntD site by homologous recombination, thereby expressing the O- and N-acetyltransferase genes and the O-acetyltransferase gene, and then after losing a hisG tag and the Ura selection marker under the selection pressure of 5-fluoroorotic acid, the recombinant bacteria 2 was obtained. The specific construction method is the same as that of the recombinant bacteria 1.

[0092] (III) Construction of recombinant strain 3 (expressing WcSLI1 and WcATF2, knocking out LCB4)

[0093] The recombinant plasmid pUC-HUH-LCB4 was introduced into the recombinant bacteria 2, and the hisG-ura-hisG fragment was integrated into the genomic LCB4 site by homologous recombination, thereby knocking out the phosphorylase gene, and then a hisG tag and Ura selection marker were lost under the selection pressure of 5-fluoroorotic acid to obtain recombinant bacteria 3. The specific construction method is the same as that of recombinant bacteria 1.

[0094] (IV) Construction of recombinant strain 4 (expressing WcSLI1, WcATF2, knocking out LCB4, expressing WcLCB1, WcLCB2)

[0095] The recombinant plasmid pUC-HUH-LCB4-WcLCB1-WcLCB2 was introduced into recombinant bacteria 3, and the WcLCB1 and WcLCB2 expression cassettes were integrated into the genomic LCB4 site by homologous recombination, and then a hisG tag and Ura selection marker were lost under the selection pressure of 5-fluoroorotic acid to obtain recombinant bacteria 4. The specific construction method is the same as that of recombinant bacteria 1.

[0096] (V) Construction of recombinant bacteria 5 (expressing WcSLI1, WcATF2, knocking out LCB4, expressing WcLCB1, WcLCB2, expressing WcTSC10, WcSYR2)

[0097] The recombinant plasmid pUC-HUH-IntA-WcTSC10-WcSYR2 was introduced into recombinant bacteria 4, and the WcTSC10 and WcSYR2 expression cassettes were integrated into the genomic IntA site by homologous recombination, and then a hisG tag and Ura selection marker were lost under the selection pressure of 5-fluoroorotic acid to obtain recombinant bacteria 5. The specific construction method is the same as that of recombinant bacteria 1.

[0098] The recombinant bacteria 5 was named as Yarrowia lipolytica XJ-TAPS strain. The Yarrowia lipolytica XJ-TAPS has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2024780, a deposit date of April 24, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0099] Example 3 Application of recombinant Yarrowia lipolytica in the production of TAPS

[0100] (I) Cultivation of engineered bacteria

[0101] The initial bacteria Yarrowia lipolytica Po1fΔku70 and the recombinant bacteria 1-5 in Example 2 were used to produce TAPS. The specific method is as follows: the initial bacteria and the recombinant bacteria were activated and cultured in YPD liquid medium at 30°C and 200rpm for 12h to obtain seed solution. The seed solution was heated to 400 ℃ and heated to 100 ℃ at an initial OD 600 The inoculum with a value of 0.1 was inoculated into 50 mL shake flask fermentation medium and cultured at 28° C. and 200 rpm for 7 days. After the fermentation was completed, the fermentation broth was transferred to a 1.5 mL centrifuge tube.

[0102] The shake flask fermentation medium formula is: 200g / L glycerol, 10g / L yeast extract, 2g / L magnesium sulfate heptahydrate, 1g / L potassium dihydrogen phosphate, 0.67g / L yeast nitrogen base (without amino acids), 10g / L calcium carbonate, 2.5g / L ammonium sulfate, 5g / L L-serine, and 0.8g / L leucine.

[0103] (II) Qualitative and quantitative analysis of TAPS

[0104] 1. Extraction of TAPS

[0105] 0.5 mL of fermentation broth was taken into a centrifuge tube, and 500 μL of acetonitrile was added. The mixture was vortexed at 1000 rpm for 10 min at 37°C. After vortexing, the mixture was centrifuged at 5000 g for 10 min. The supernatant was taken for HPLC analysis.

[0106] 2. TAPS detection

[0107] Detection conditions: Column: Sepax GP-C18 column (Sepax Technology, 4×6mm×250mm, USA), UV detector (200nm), column temperature 40℃, injection volume 10μL. Mobile phase consists of 99.95% acetonitrile and 0.05% trifluoroacetic acid, flow rate 1mL / min, each run 10min. Under the above conditions, the TAPS peak time detected by HPLC is about 6.5min ( Figure 7 ).

[0108] As shown in Table 5, after 7 days of fermentation, the TAPS production of recombinant bacteria 5 was the highest, and the highest production was achieved the fastest (5 days of fermentation), reaching 342 mg / L, that is, 342 mg of TAPS per liter of fermentation liquid. The initial bacteria and recombinant bacteria 1 could not synthesize TAPS, and the TAPS production of recombinant bacteria 2-4 after 7 days of fermentation was 30.65 mg / L, 70 mg / L and 75 mg / L respectively.

[0109] Table 5 TAPS production of initial bacteria and recombinant bacteria 1-5

[0110]

[0111] The recombinant bacteria 5 was inoculated into 50mL YPD liquid medium and cultured at 30℃ for 24h, then inoculated into a 5L fermenter containing 2.5L fermentation medium at an inoculum of 5% v / v, the fermentation temperature was 28℃, and the fermentation time was 6 days. During the fermentation 0-48h, the dissolved oxygen was controlled to 20%; when the fermentation time was >48h, the dissolved oxygen was controlled to 0-5%. During the fermentation process, the pH value was constantly controlled at 5.5 until the fermentation was completed. At the fermentation time of 40h and 96h, a glucose aqueous solution with a volume concentration of 800g / L was added to make the glucose concentration of the fermentation liquid reach 80g / L. Among them, the components of the fermentation medium are: glucose 150g / L, ammonium sulfate 11g / L, yeast extract (Sigma-Aldrich, USA) 3g / L, corn peptone 0.1g / L, potassium dihydrogen phosphate 4g / L, magnesium sulfate 2g / L, calcium sulfate 0.8g / L, sodium chloride 0.4g / L, ammonium sulfate hydrochloride 12mg / L, biotin 1mg / L, sodium molybdate 160mg / L, copper sulfate 0.2mg / L, boric acid 40mg / L, manganese sulfate 180mg / L, ferrous chloride 75mg / L. After 5 days of fermentation, the TAPS production of recombinant bacteria 5 was 5.28g / L. That is, 5.28g of TAPS was produced per liter of fermentation liquid, achieving efficient accumulation of TAPS in Yarrowia lipolytica.

[0112] The present invention provides a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and a method and idea for its application. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant Yarrowia lipolytica strain that produces high yield of tetraacetylphytosphingosine, characterized in that: The recombinant Yarrowia lipolytica simultaneously expresses O- and N-acetyltransferase genes WLnSLI , O-acetyltransferase gene WCtF2 , serine palmitoyltransferase gene WlqB and WLnB , 3-ketodihydrosphingosine reductase gene WcTSC10 and C-4 hydroxylase gene Wlq , and knocked out the phosphorylase gene LCB4 ; The O- and N-acetyltransferase genes WLn , O-acetyltransferase gene WCtF2 , serine palmitoyltransferase gene WlqB and WLnB , 3-ketodihydrosphingosine reductase gene WcTSC10 and C-4 hydroxylase gene Wlq From Sylvestris wickhamii Wickerhamomyces ciferrii ; Phosphorylase gene LCB4 From Yarrowia lipolytica Yarrowia lipolytica ; The O- and N-acetyltransferase genes WLnSLI The nucleotide sequence is shown in SEQ ID No. 1; The O-acetyltransferase gene WCtF2 The nucleotide sequence is shown in SEQ ID No.2; Phosphorylase gene LCB4 The nucleotide sequence is shown in SEQ ID No.7; The serine palmitoyltransferase gene WlqB and WLnB The nucleotide sequence is shown in SEQ ID No.3-4; The 3-ketodihydrosphinganine reductase gene WcTSC10 The nucleotide sequence is shown in SEQ ID No.5; The C-4 hydroxylase gene Wlq The nucleotide sequence is shown in SEQ ID No.

6.

2. The recombinant Yarrowia lipolytica according to claim 1, characterized in that Yarrowia lipolytica Yarrowia lipolytica , strain number XJ-TAPS, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024780, and the deposit date is April 24, 2024.

3. Use of the recombinant Yarrowia lipolytica according to any one of claims 1 to 2 in the fermentation production of tetraacetylphytosphingosine.

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