Recombinant yarrowia lipolytica strain with high yield of tetraacetyl phytosphingosine and use thereof

Abstract

The present invention provides a recombinant Yarrowia lipolytica strain with a high yield of tetraacetyl phytosphingosine and use thereof, and belongs to the field of bioengineering. The recombinant Yarrowia lipolytica strain with a high yield of tetraacetyl phytosphingosine constructed in the present invention is named a Yarrowia lipolytica strain XJ-TAPS. The construction method for the recombinant Yarrowia lipolytica strain with a high yield of tetraacetyl phytosphingosine comprises: integrating a Wickerhamomyces ciferrii-derived phytosphingosine acetyltransferase gene expression cassette, a phosphorylase gene knockout cassette, a serine palmitoyltransferase gene expression cassette, a 3-ketodihydrosphingosine reductase gene expression cassette, and a C-4 hydroxylase gene expression cassette on a Yarrowia lipolytica genome by means of homologous recombination. The construction method for the recombinant Yarrowia lipolytica strain of the present invention has simple operation, and tetraacetyl phytosphingosine can be efficiently synthesized.

Description

Recombinant yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and in particular to a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and application thereof. BACKGROUND

[0002] Tetraacetyl phytosphingosine (TAPS) is a long-chain sphingosine carrying three acetylated hydroxyl groups and one acetylated amine, which has the functions of moisturizing, antioxidant, whitening and freckle lightening. The deacetylated phytosphingosine can be used as a precursor to synthesize moisturizing skin care products such as ceramides. Therefore, TAPS has high application value in the skin care and cosmetic industry.

[0003] Currently, TAPS is mainly obtained by microbial synthesis. The unconventional yeast Wickerhamomyces ciferrii is the only microorganism known to naturally secrete TAPS. It acetylates three hydroxyl groups and one amino group of phytosphingosine to produce TAPS, and secretes it outside the cell at high concentrations to resist the cytotoxicity of phytosphingosine. In addition, with the development of synthetic biology, using microorganisms with superior performance to build a cell factory has also become a popular choice. Yarrowia lipolytica is a recognized safe unconventional oleaginous 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 of phytosphingosine and its derivatives due to its strong robustness in resisting harsh environments such as low acid and high salt. Therefore, Yarrowia lipolytica has the potential to be developed as a cell factory for efficient synthesis of TAPS. SUMMARY

[0004] The present application aims to solve the technical problems of the prior art and provides a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and application thereof.

[0005] To solve the above technical problems, the present application discloses a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine and application thereof. The following technical solutions are adopted:

[0006] The application provides a recombinant Yarrowia lipolytica strain with high tetracetylated phytosphingosine yield, wherein the recombinant Yarrowia lipolytica strain simultaneously expresses O- and N-acetyl transferase gene WcSLI1 and O-acetyl transferase gene WcATF2, and the O- and N-acetyl transferase gene WcSLI1 and the O-acetyl transferase gene WcATF2 are derived from Wickerhamomyces ciferrii.

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

[0008] The recombinant Yarrowia lipolytica strain further adopts one or any combination of the following three modes:

[0009] (1) knocking out a phosphorylase gene;

[0010] (2) expressing a serine palmitoyl transferase gene;

[0011] (3) simultaneously expressing a 3-ketodihydrosphingosine reductase gene and a C-4 hydroxylase gene.

[0012] The phosphorylase gene is an endogenous LCB4 gene of Yarrowia lipolytica.

[0013] The nucleotide sequence of the LCB4 gene is shown in SEQ ID No. 7.

[0014] The serine palmitoyl transferase gene is any one or a combination of two of WcLCB1 and WcLCB2 genes, and the WcLCB1 and WcLCB2 genes are coding genes derived from Wickerhamomyces ciferrii serine palmitoyl transferase.

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

[0016] The 3-ketodihydrosphingosine reductase gene is derived from WcTSC10 gene of Wickerhamomyces ciferrii, and the C-4 hydroxylase gene is derived from WcSYR2 gene of Wickerhamomyces ciferrii.

[0017] 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 of SEQ ID No. 1-6 are codon-optimized sequences. 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, 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 application provides a recombinant Yarrowia lipolytica strain with high yield of tetraacetyl phytosphingosine, which simultaneously expresses O- and N-acetyltransferase gene 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 gene WcSLI1, O-acetyltransferase gene WcATF2, serine palmitoyltransferase genes WcLCB1 and WcLCB2, 3-ketodihydrosphingosine reductase gene WcTSC10 and C-4 hydroxylase gene WcSYR2 are derived from Wickerhamiella sp. and are codon-optimized; 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 sequences of the serine palmitoyltransferase genes WcLCB1 and WcLCB2 are shown in SEQ ID Nos. 3-4; the nucleotide sequence of the 3-ketodihydrosphingosine reductase gene WcTSC10 is shown in SEQ ID No. 5; and the nucleotide sequence of the C-4 hydroxylase gene WcSYR2 is shown in SEQ ID No. 6. The recombinant Yarrowia lipolytica strain, which is classified as Yarrowia lipolytica, strain number XJ-TAPS, has been preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2024780, a preservation date of April 24, 2024, and a preservation address of Wuhan, China.

[0020] Preferably, the construction method of the recombinant Yarrowia lipolytica strain XJ-TAPS is as follows: an exogenous acetyltransferase gene expression cassette, a serine palmitoyltransferase gene expression cassette, a 3-ketodihydrosphingosine reductase gene expression cassette, a C-4 hydroxylase expression cassette are inserted into the genome of the original Yarrowia lipolytica strain, and the intermediate product phosphorylase gene expression cassette is knocked out. Specifically, the phosphorylase gene LCB4 is knocked out, the exogenous acetyltransferase gene SLI1 and ATF2 expression cassettes, the serine palmitoyltransferase gene LCB1 and LCB2 expression cassettes, the 3-ketodihydrosphingosine 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 on the Yarrowia lipolytica genome through homologous recombination.

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

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

[0023] The construction method of the recombinant Yarrowia lipolytica strain described above is efficient and simple to operate.

[0024] In a second aspect, the present application provides the use of the recombinant Yarrowia lipolytica strain of the first aspect in the fermentation production of tetraacetyl phytosphingosine.

[0025] Preferably, the recombinant Yarrowia lipolytica strain is inoculated into a fermentation medium for fermentation culture. The fermentation culture time is 5-7 days. Beneficial effects:

[0026] The present application is based on Yarrowia lipolytica with the KU70 gene responsible for encoding non-homologous recombination gene knocked out, so that the homologous recombination ability of Yarrowia lipolytica is enhanced. The integration of genes is realized through the homologous recombination function of Yarrowia lipolytica itself, which can greatly improve the genetic stability of the introduced genes, and a recombinant Yarrowia lipolytica strain with high yield of tetraacetyl phytosphingosine (TAPS) is constructed. The recombinant Yarrowia lipolytica strain constructed in the present application realizes the synthesis of TAPS in Yarrowia lipolytica from scratch through the insertion of the exogenous acetyltransferase gene SLI1 and ATF2 expression cassette. The Yarrowia lipolytica XJ-TAPS strain constructed in the present application knocks out the phosphorylase gene LCB4, inserts the exogenous acetyltransferase gene SLI1 and ATF2 expression cassette, the serine palmitoyltransferase genes LCB1 and LCB2 expression cassette, the 3-ketodihydro sphingosine reductase gene TSC10 expression cassette, and the C-4 hydroxylase gene SYR2 expression cassette. Experiments have proved that the recombinant Yarrowia lipolytica can efficiently ferment and produce TAPS (TAPS yield is 5.28 g / L in a 5L fermenter), realizing the efficient synthesis of TAPS naturally produced by Wickerhamiella sp. in Yarrowia lipolytica. The strain XJ-TAPS has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2024780 and the preservation time of April 24, 2024. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings in which:

[0028] Figure 1 is a diagram of TAPS synthesis strategy provided by the present application. 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.

[0029] Figure 2 is a diagram of the structure of recombinant plasmid pUC-HUH-IntD-WcSLI1, wherein IntD-up represents the upstream homologous arm of the IntD site, IntD-dm represents the downstream homologous arm of the IntD site, TEFin represents the promoter P TEFin , mig1t represents the terminator T mig1t , URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , the terminator T xpr2t ), and WcSLI1 is the O- and N-acetyltransferase-encoding gene.

[0030] Figure 3 is a diagram of the structure of recombinant plasmid pUC-HUH-IntD-WcSLI1-WcATF2, wherein IntD-up represents the upstream homologous arm of the IntD site, IntD-dm represents the downstream homologous arm of the IntD site, FBAin represents the promoter P FBAin , xpr2t represents the terminator T xpr2t , URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , the terminator T xpr2t ), WcSLI1 is the O- and N-acetyltransferase-encoding gene, and WcATF2 is the O-acetyltransferase-encoding gene.

[0031] Figure 4 is a diagram of the structure of recombinant plasmid pUC-HUH-LCB4, wherein LCB4-up represents the upstream homologous arm of the LCB4 site, LCB4-dm represents the downstream homologous arm of the LCB4 site, URA represents the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette, and LCB4 is the phosphorylase gene.

[0032] Figure 5 is a structural diagram of the recombinant plasmid pUC-HUH-LCB4-WcLCB1-WcLCB2, wherein LCB4-up represents the homologous arm upstream of the LCB4 site, LCB4-dm represents the homologous arm downstream of the LCB4 site, TEF in represents the promoter P TEFin , FBAin represents the promoter P FBAin , cyc1t and xpr2t represent terminators T cyc1t and T xpr2t , URA represents an 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.

[0033] Figure 6 is a structural diagram of the recombinant plasmid pUC-HUH-IntA-WcTSC10-WcSYR2, wherein IntA-up represents the homologous arm upstream of the IntA site, IntA-dm represents the homologous arm downstream of the IntA site, TEF and EXP represent the promoters P TEF and P EXP , mig1t and cyc1t represent terminators T mig1t and T cyc1t , URA represents an orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (containing the Yarrowia lipolytica endogenous promoter P TEFin , terminator T xpr2t ), TSC10 is a 3-ketodihydrosphingosine reductase-encoding gene, and SYR2 is a C-4 hydroxylase-encoding gene.

[0034] Figure 7 is an HPLC detection diagram of TAPS provided by the embodiment of the present application. DETAILED DESCRIPTION

[0035] The present application is further described below through specific examples. In the following examples, the experimental methods used are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0036] The Yarrowia lipolytica Po1f described in the following examples was purchased from the American Type Culture Collection, and the number is ATCC MYA-2613.

[0037] Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::hisG, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1fΔku70, described in the following example. Yarrowia lipolytica Po1fΔku70 was constructed by knocking out the KU70 gene, which is responsible for non-homologous recombination, from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2): 63-72).

[0038] The IntD site integration plasmid described in the following example is obtained by inserting a sequence of 1443 bp in size upstream of the start codon (upstream homology arm) and a sequence of 1189 bp in size downstream of the stop codon (downstream homology arm) of the IntD site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-hisG-ura-hisG vector (see Example 1 for construction method), with the two hisG tag encoding genes between the upstream and downstream homology arms of the IntD site.

[0039] The LCB4 site integration plasmid described in the following example is obtained by inserting a sequence of 1003 bp in size upstream of the start codon (upstream homology arm) and a sequence of 934 bp in size downstream of the stop codon (downstream homology arm) of the LCB4 site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-hisG-ura-hisG vector (see Example 1 for construction method), with the two hisG tag encoding genes between the upstream and downstream homology arms of the LCB4 site.

[0040] The IntA site integration plasmid described in the following example is obtained by inserting a sequence of 1633 bp in size upstream of the start codon (upstream homology arm) and a sequence of 1621 bp in size downstream of the stop codon (downstream homology arm) of the IntA site on the chromosome in the Yarrowia lipolytica Po1fΔku70 genome into the pUC57-hisG-ura-hisG vector (see Example 1 for construction method), with the two hisG tag encoding genes between the upstream and downstream homology arms of the IntA site.

[0041] YPD liquid medium described in the following examples, the formula is: 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose. The screening medium SD-Ura described, the formula is: SD-Ura (purchased from Wuhan Pu Yinteng Biological Engineering Co., Ltd.) 48 g / L, agar powder 12 g / L, solvent is water. The YPD plate containing 5-fluoroorotic acid (5-FOA), the formula is: YPD liquid medium added 25 g / L of agar, and 1 g / L of 5-FOA.

[0042] The TAPS synthesis strategy provided by the embodiments of the present application is shown in Figure 1.

[0043] Example 1 Amplification of gene elements and preparation of target plasmid

[0044] According to the nucleotide sequence of the O- and N-acetyltransferase Sli1 encoding gene (GenBank accession number: XM_011273126.1) from Wickerhamomyces ciferrii provided on NCBI, after codon optimization, the optimized O- and N-acetyltransferase Sli1 encoding gene WcSLI1 (SEQ ID No. 1) was synthesized by Anhui General Biological Co., Ltd. and inserted into plasmid pUC57 to obtain plasmid pUC57-WcSLI1.

[0045] According to the nucleotide sequence of the O-acetyltransferase Atf2 encoding gene (GenBank accession number: XM_011272738.1) from Wickerhamomyces ciferrii provided on NCBI, after codon optimization, the optimized O-acetyltransferase Atf2 encoding gene WcATF2 (SEQ ID No. 2) was synthesized by Anhui General Biological Co., Ltd. and inserted into plasmid pUC57 to obtain plasmid pUC57-WcATF2.

[0046] According to the nucleotide sequence of the serine palmitoyltransferase encoding gene (GenBank accession number: JN645676.1, AF053456.1) from Wickerhamomyces ciferrii provided on NCBI, after codon optimization, the optimized serine palmitoyltransferase encoding gene WcLCB1, WcLCB2 (SEQ ID No. 3, SEQ ID No. 4) was synthesized by Anhui General Biological Co., Ltd. and inserted into plasmid pUC57 to obtain plasmid pUC57-WcLCB1, pUC57-WcLCB2.

[0047] The target genes were prepared according to the nucleotide sequences of the 3-ketodihydrosphingosine reductase-encoding gene (GenBank Accession No. JN645686.1) and the C-4 hydroxylase-encoding gene (GenBank Accession No. HQ166122.1) from Wickerhamomyces ciferrii provided on NCBI, and after codon optimization, the optimized 3-ketodihydrosphingosine reductase-encoding gene WcTSC10 (SEQ ID No. 5) and the C-4 hydroxylase-encoding gene WcSYR2 (SEQ ID No. 6) were synthesized by Anhui General Biotech Co., Ltd. and inserted into plasmid pUC57 to obtain plasmids pUC57-WcTSC10 and pUC57-WcSYR2.

[0048] According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase-encoding gene URA (GenBank Accession No. AJ306421.1) and the hisG tag (GenBank Accession No. AF324729.1) of Yarrowia lipolytica provided on NCBI, the two hisG tag-encoding gene sequences were synthesized by Anhui General Biotech Co., Ltd. and inserted into plasmid pUC57, and the orotidine-5'-phosphate decarboxylase-encoding gene URA expression cassette was inserted between the two hisG tag-encoding gene sequences (P TEFin -URA-T xpr2t ) so as to realize Ura selection marker recovery, to obtain plasmid pUC57-hisG-ura-hisG.

[0049] The upper homologous arm LCB4-up of the phosphorylase-encoding gene LCB4 was amplified using Yarrowia lipolytica Po1fΔku70 genomic DNA as the template and LCB4::LCB4-up-F and LCB4::LCB4-up-R as the primers, and the lower homologous arm LCB4-dm of the phosphorylase-encoding gene LCB4 was amplified using LCB4::LCB4-dm-F and LCB4::LCB4-dm-R as the primers (GenBank Accession No. of LCB4: XM_002142977.1, SEQ ID No. 7).

[0050] The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P TEFin is shown in SEQ ID No. 8; the nucleotide sequence of the Yarrowia lipolytica endogenous promoter P GPDin is shown in SEQ ID No. 9; the nucleotide sequence of the Yarrowia lipolytica endogenous promoter P TEFThe nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11. FBAin The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11.

[0051] The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11. mig1t The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11. cyc1t The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11. lip2t The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11. xpr2t The nucleotide sequence of the Yarrowia lipolytica endogenous promoter P is shown as SEQ ID No. 10; the nucleotide sequence of the Yarrowia lipolytica endogenous terminator T is shown as SEQ ID No. 11.

[0052] (II) Construction of recombinant plasmids

[0053] The structure of the recombinant plasmids is shown in Table 3 and Figures 2-6; the primers used for constructing the recombinant plasmids are shown in Table 4.

[0054] 1. Construction of recombinant integration plasmids:

[0055] The recombinant integration plasmids pUC-HUH-IntD-WcSLI1, pUC-HUH-IntD-WcSLI1-WcATF2, pUC-HUH-LCB4, pUC-HUH-LCB4-WcLCB1-WcLCB2, pUC-HUH-IntA-WcTSC10-WcSYR2 are constructed with pUC57-hisG-ura-hisG as the backbone, inserting the IntD site, LCB4 site, homologous arm upstream of the initiation codon and homologous arm downstream of the termination codon in Yarrowia lipolytica PolfAku70, and inserting the gene expression cassette between the upstream and downstream homologous arms. They are respectively: 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 (P TEFin- URA-T xpr2t ); LCB4-up, LCB4-dm, WcLCB1 expression cassette (P TEFin - WcLCB1-T xpr2t ), URA expression cassette (P TEFin - URA-T xpr2t );

[0056] 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 WcSLIl expression cassette (P TEFin - WcSLIl-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 ) were amplified from the promoter, coding gene and terminator fragments, respectively, using the primers described in Table 4. These are: IntD::P TEFin -F, IntD::P TEFin -R, IntD::WcSLIl-F, IntD::WcSLIl-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 is exemplified by recombinant plasmid pUC-HUH-IntD-WcSLI1. The recombinant plasmid pUC-HUH-IntD-WcSLI1 is based on pUC57-hisG-ura-hisG, and the homologous arm IntD-up upstream of the start codon and the homologous arm IntD-dm downstream of the stop codon of the IntD site in Yarrowia lipolytica PolfΔku70 are inserted, and the WcSLI1 expression cassette (P TEFin -WcSLI1-T mig1t ) is also inserted between the upstream and downstream homologous arms. TEFin , the terminator T xpr2t ) is also inserted between the upstream and downstream homologous arms.

[0059] Yarrowia lipolytica Po1fΔku70 genomic DNA as template, IntD::P TEFin -F and IntD::P TEFin -R as primers to amplify the WcSLI1 expression cassette promoter P TEFin . IntD::T mig1t -F and IntD::T mig1t -R as primers to amplify the WcSLI1 expression cassette terminator T mig1t .

[0060] The plasmid pUC57-WcSLI1 as template, IntD::WcSLI1-F and IntD::WcSLI1-R as primers to amplify the WcSLI1 gene with the promoter P TEFin and terminator T mig1t homologous arms at both ends.

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

[0062] Table 1 PCR amplification system

[0063] Wherein, PrimerSTAR Max Premix is purchased from Baodi Medical Biotechnology (Beijing) Co., Ltd.

[0064] The above PCR program is as follows: denaturation at 98℃ for 10s, annealing at 55℃ for 5s, extension at 72℃ (extension time = target fragment length / 1kb, unit min), repeated for 34 cycles.

[0065] Each fragment is purified and recovered by using TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Baisheng Biotechnology Co., Ltd.).

[0066] The IntD site integration plasmid is linearized by Hind III restriction enzyme of NEB company, and the linearized IntD site integration plasmid is recovered by agarose gel electrophoresis.

[0067] The linearized IntD site integration plasmid and each element (promoter P TEFin , gene WcSLI1 and terminator T mig1t in the WcSLI1 gene expression cassette constructed in this embodiment are ligated.) One-step cloning was realized by using ClonExpress MultiS One Step Cloning Kit of Nanjing Nvwaibio Co., Ltd. to insert the WcSLI1 gene expression cassette into the upstream and downstream homologous arms of the IntD site integration plasmid, and the two hisG tag coding genes were on the same side of the WcSLI1 gene expression cassette. The one-step cloning reaction system is shown in Table 2. After incubation of the reaction system at 37°C for 30 min, the circular recombinant vector was obtained.

[0068] Table 2 One-step cloning system

[0069] Wherein, the usage amount of linearized vector (x) and insert fragment (y) can be obtained by the following formula: optimal usage amount of each fragment or linearized vector = [0.02 x base pair number of the fragment or linearized vector] ng.

[0070] The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin-resistant LB plates, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-HUH-IntD-WcSLI1.

[0071] After the plasmid pUC-HUH-IntD-WcSLI1 was digested by the restriction endonuclease Not I of NEB company, the linearized pUC-HUH-IntD-WcSLI1 plasmid was recovered by agarose gel electrophoresis.

[0072] 2. Construction of recombinant knockout plasmid pUC-HUH-LCB4:

[0073] The recombinant knockout plasmid pUC-HUH-LCB4 is based on the pUC57-hisG-ura-hisG backbone, and the LCB4-up homologous arm upstream of the start codon and the LCB4-dm homologous arm downstream of the stop codon in the LCB4 site of Yarrowia lipolytica Po1fΔku70 are inserted. The above-mentioned fragments are respectively amplified by the primers in Table 4.

[0074] The recombinant plasmid pUC-HUH-LCB4 is based on the pUC57-hisG-ura-hisG backbone, and the LCB4-up homologous arm of 1003 bp upstream of the start codon and the LCB4-dm homologous arm of 934 bp downstream of the stop codon in the LCB4 site of Yarrowia lipolytica Po1fΔku70 are inserted, and the orotidine-5'-phosphate decarboxylase coding gene URA expression cassette (containing the endogenous promoter P TEFin , terminator T xpr2t) also between the upstream and downstream homology arms.

[0075] LCB4-up-F and LCB4-up-R as primers, Yarrowia lipolytica Po1fΔku70 genomic DNA as template, LCB4 site homology arm LCB4-up upstream of the start codon was amplified. After linearized pUC57-hisG-ura-hisG plasmid was recovered by agarose gel electrophoresis, linearized pUC57-hisG-ura-hisG plasmid and LCB4 site homology arm LCB4-up (both ends with pUC57-hisG-ura-hisG homologous arm sequence) constructed in this embodiment were ligated by NEBuilder® Hi-SFI DNA Assembly Kit of Nanjing Novozyme BioTech Co., Ltd. to obtain recombinant plasmid pUC-HUH-LCB4-up. IIOne Step Cloning Kit to achieve one-step cloning, and a circular recombinant vector was obtained. The circular recombinant vector was transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-LCB4-up was obtained through LB plate screening of ampicillin resistance, colony PCR and sequencing verification.

[0076] LCB4-dm-F and LCB4-dm-R as primers, Yarrowia lipolytica Po1fΔku70 genomic DNA as template, LCB4 site homology arm LCB4-dm downstream of the stop codon was amplified. After linearized pUC-HUH-LCB4-up plasmid was recovered by agarose gel electrophoresis, linearized pUC-HUH-LCB4-up plasmid and LCB4 site homology arm LCB4-dm (both ends with pUC-HUH-LCB4-up homologous arm sequence) constructed in this embodiment were ligated by NEBuilder® Hi-SFI DNA Assembly Kit of Nanjing Novozyme BioTech Co., Ltd. to obtain recombinant plasmid pUC-HUH-LCB4. IIOne Step Cloning Kit to achieve one-step cloning, and a circular recombinant vector was obtained. The circular recombinant vector was transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-LCB4-up was obtained through LB plate screening of ampicillin resistance, colony PCR and sequencing verification.

[0077] Table 3 Inserted sequences in each recombinant plasmid

[0078] Table 4 Primer sequences

[0079] Example 2 Construction of recombinant Yarrowia lipolytica

[0080] (I) Construction of Recombinant Bacterium 1 (Expression of WcSLI1)

[0081] The recombinant plasmid pUC-HUH-IntD-WcSLI1 was introduced into Yarrowia lipolytica Po1fAkU70, and the WcSLI1 expression cassette was integrated into the genomic IntD site by homologous recombination, so as to express O- and N-acetyltransferase genes, and then after losing a hisG tag and a Ura screening marker under the screening pressure of 5-fluoroorotic acid, the recombinant bacterium 1 was obtained.

[0082] The specific method is as follows:

[0083] Yarrowia lipolytica Po1fAkU70 was inoculated in YPD liquid medium and cultured overnight at 30°C to prepare competent cells and introduce the recombinant plasmid: the recombinant plasmid pUC-HUH-IntD-WcSLI1 was transformed into Yarrowia lipolytica Po1fAkU70 by using Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation, homologous recombination was performed, positive clones were screened by using a screening medium SD-Ura, and then PCR identification was performed.

[0084] The positive clones correctly 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. Single colonies that could grow on YPD plates containing 5-fluoroorotic acid but could not grow on SD-Ura plates were selected, and were named as recombinant bacterium 1.

[0085] (II) Construction of Recombinant Bacterium 2 (Expression of WcSLI1 and WcATF2)

[0086] The recombinant plasmid pUC-HUH-IntD-WcSLI1-WcATF2 was introduced into Yarrowia lipolytica Po1fAkU70, and the WcSLI1 expression cassette and the WcATF2 expression cassette were integrated into the genomic IntD site by homologous recombination, so as to express O- and N-acetyltransferase genes and O-acetyltransferase genes, and then after losing a hisG tag and a Ura screening marker under the screening pressure of 5-fluoroorotic acid, the recombinant bacterium 2 was obtained. The specific construction method is the same as that of the recombinant bacterium 1.

[0087] (III) Construction of Recombinant Bacterium 3 (Expression of WcSLI1 and WcATF2, Knockout of LCB4)

[0088] The recombinant plasmid pUC-HUH-LCB4 is introduced into the recombinant bacteria 2, and the hisG-ura-hisG fragment is integrated into the genomic LCB4 site by homologous recombination to knock out the phosphorylase gene, and then after losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, the recombinant bacteria 3 is obtained. The specific construction method is the same as that of the recombinant bacteria 1.

[0089] (Four) Construction of recombinant bacteria 4 (expressing WcSLI1, WcATF2, knocking out LCB4, expressing WcLCB1, WcLCB2)

[0090] The recombinant plasmid pUC-HUH-LCB4-WcLCB1-WcLCB2 is introduced into the recombinant bacteria 3, and the WcLCB1, WcLCB2 expression cassette is integrated into the genomic LCB4 site by homologous recombination, and then after losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, the recombinant bacteria 4 is obtained. The specific construction method is the same as that of the recombinant bacteria 1.

[0091] (Five) Construction of recombinant bacteria 5 (expressing WcSLI1, WcATF2, knocking out LCB4, expressing WcLCB1, WcLCB2, expressing WcTSC10, WcSYR2)

[0092] The recombinant plasmid pUC-HUH-IntA-WcTSC10-WcSYR2 is introduced into the recombinant bacteria 4, and the WcTSC10, WcSYR2 expression cassette is integrated into the genomic IntA site by homologous recombination, and then after losing one hisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, the recombinant bacteria 5 is obtained. The specific construction method is the same as that of the recombinant bacteria 1.

[0093] The recombinant bacteria 5 is named Yarrowia lipolytica XJ-TAPS strain. The Yarrowia lipolytica XJ-TAPS has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2024780, the preservation time is April 24, 2024, and the preservation address is Wuhan University, Wuhan, China.

[0094] Example 3 Application of recombinant Yarrowia lipolytica bacteria in the production of TAPS

[0095] (One) Cultivation of engineering bacteria

[0096] TAPS was produced by initial strain Yarrowia lipolytica Po1fΔku70 and recombinant strains 1-5 in Example 2, respectively. The specific method is as follows: the initial strain and the recombinant strains were activated and cultured in YPD liquid medium at 30°C, 200 rpm for 12 h to obtain seed liquid. The seed liquid was inoculated into 50 mL shake flask fermentation medium at an initial OD value of 0.1, and cultured at 28°C, 200 rpm for 7 days. After fermentation, the fermentation liquid was transferred to a 1.5 mL centrifuge tube. 600

[0097] The shake flask fermentation medium formula is as follows: 200 g / L glycerol, 10 g / L yeast extract, 2 g / L magnesium sulfate heptahydrate, 1 g / L potassium dihydrogen phosphate, 0.67 g / L yeast nitrogen base (without amino acids), 10 g / L calcium carbonate, 2.5 g / L ammonium sulfate, 5 g / L L-serine, and 0.8 g / L leucine.

[0098] (II) Qualitative and quantitative analysis of TAPS

[0099] 1. Extraction of TAPS

[0100] 0.5 mL of fermentation liquid was taken into a centrifuge tube, 500 μL of acetonitrile was added, vortexed at 1000 rpm for 10 min at 37°C, and then centrifuged at 5000 g for 10 min. The supernatant was taken for high performance liquid chromatography analysis.

[0101] 2. Detection of TAPS

[0102] The detection conditions are as follows: a Sepax GP-C18 chromatographic column (Sephadex, 4x6 mmx250 mm, USA), a UV detector (200 nm), a column temperature of 40°C, a sample injection volume of 10 μL. The mobile phase consists of 99.95% acetonitrile and 0.05% trifluoroacetic acid, and the flow rate is 1 mL / min, and each run is 10 min. Under the above conditions, the peak time of TAPS detected by HPLC is about 6.5 min (Figure 7).

[0103] As shown in Table 5, after 7 days of fermentation, the TAPS yield of recombinant strain 5 was the highest, and the highest yield was reached the fastest (5 days of fermentation), reaching 342 mg / L, i.e. 342 mg of TAPS was produced per liter of fermentation liquid. The initial strain and recombinant strain 1 cannot synthesize TAPS, and the TAPS yields of recombinant strains 2-4 after 7 days of fermentation are 30.65 mg / L, 70 mg / L and 75 mg / L, respectively.

[0104] Table 5 TAPS yield of initial strain and recombinant strains 1-5

[0105] ​The recombinant bacteria 5 is inoculated into 50 mL YPD liquid medium and cultured at 30℃ for 24 h, then inoculated into 5 L fermenter containing 2.5 L fermentation medium at 5% v / v, the fermentation temperature is 28℃, and the fermentation time is 6 days. The dissolved oxygen is controlled at 20% for 0-48 h of fermentation, and at 0-5% for >48 h of fermentation. The pH value is constantly controlled at 5.5 during the fermentation until the end of the fermentation. At 40 h and 96 h of fermentation, 800 g / L glucose aqueous solution is supplemented to make the glucose concentration of the fermentation broth reach 80 g / L. The composition of the fermentation medium is as follows: glucose 150 g / L, ammonium sulfate 11 g / L, yeast extract (Sigma-Aldrich, USA) 3 g / L, corn peptone 0.1 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 2 g / L, calcium sulfate 0.8 g / L, sodium chloride 0.4 g / L, ammonium chloride hydrochloride 12 mg / L, biotin 1 mg / L, sodium molybdate 160 mg / L, copper sulfate 0.2 mg / L, boric acid 40 mg / L, manganese sulfate 180 mg / L, ferrous chloride 75 mg / L. After 5 days of fermentation, the TAPS yield of the recombinant bacteria 5 is 5.28 g / L. That is, 5.28 g of TAPS is produced per liter of fermentation broth, realizing the efficient accumulation of TAPS in Yarrowia lipolytica.

[0106] The present application provides a recombinant Yarrowia lipolytica with high yield of tetraacetyl phytosphingosine, and a thought and method for application thereof. There are many methods and approaches to realize the technical solution, and the above description is only the preferred embodiment of the present application. It should be pointed out that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principle of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by the existing technology.

Claims

1. A recombinant Yarrowia lipolytica strain producing high levels of tetraacetyl phytosphingosine, characterized in that, The recombinant Yarrowia lipolytica expresses O- and N-acetyltransferase gene 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 phosphokinase gene LCB4; The O- and N-acetyltransferase gene WcSLI1, O-acetyltransferase gene WcATF2, serine palmitoyltransferase genes WcLCB1 and WcLCB2, 3-ketodihydrosphingosine reductase gene WcTSC10 and C-4 hydroxylase gene WcSYR2 are derived from Wickerhamomyces ciferrii; The phosphokinase gene LCB4 is derived from Yarrowia lipolytica; The nucleotide sequence of the O- and N-acetyltransferase gene WcSLI1 is shown as SEQ ID No. 1; The nucleotide sequence of the O-acetyltransferase gene WcATF2 is shown as SEQ ID No. 2; The nucleotide sequence of the phosphokinase gene LCB4 is shown as SEQ ID No. 7; The nucleotide sequences of the serine palmitoyltransferase genes WcLCB1 and WcLCB2 are shown as SEQ ID No. 3-4; The nucleotide sequence of the 3-ketodihydrosphingosine reductase gene WcTSC10 is shown as SEQ ID No. 5; The nucleotide sequence of the C-4 hydroxylase gene WcSYR2 is shown as SEQ ID No.

6.

2. The recombinant Yarrowia lipolytica of claim 1, wherein, The classification name is Yarrowia lipolytica, the strain number is XJ-TAPS, which has been preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2024780, and the preservation time is April 24, 2024.

3. The recombinant Yarrowia lipolytica in any one of claims 1-2 for use in the fermentation production of tetraacetyl phytosphingosine.

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