A recombinant bacterium for producing tetraacetyl phytosphingosine and its construction method and application

Abstract

The invention discloses a recombinant bacterium for producing tetraacetyl phytosphingosine, a construction method and an application thereof, and relates to the field of biotechnology. The invention inserts a key gene for synthesizing tetraacetyl phytosphingosine into the genome of S. wickhamii by homologous recombination, thereby obtaining a recombinant bacterium expressing heterologous sphingosine phosphatase and serine palmitoyl transferase. The production of tetraacetyl phytosphingosine by the recombinant bacterium can increase the yield of tetraacetyl phytosphingosine, shorten the fermentation cycle, and save materials and energy, thereby greatly reducing the production cost. Therefore, the recombinant bacterium and the method for producing tetraacetyl phytosphingosine of the invention have good prospects for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant bacterium for producing tetraacetyl phytosphingosine, a construction method thereof and an application thereof. Background Art

[0002] Tetra-acetyl phytosphingosine (TAPS) is the acetylation product of phytosphingosine. Phytosphingosine is a long-chain amino alcohol widely present in plant cell membranes with unique biological activity and chemical stability. Tetra-acetyl phytosphingosine enhances lipophilicity and stability by introducing acetyl groups on the hydroxyl and amino groups of phytosphingosine. Tetra-acetyl phytosphingosine has biological activities such as anti-inflammatory, antioxidant and regulation of cell signal transduction, and can be used to develop new drugs to treat skin diseases, nervous system diseases and cardiovascular diseases. Tetra-acetyl phytosphingosine has good moisturizing, antioxidant and anti-aging properties, making it a commonly used active ingredient in cosmetics, and can be used in products such as skin creams, essences and facial masks. As a natural antioxidant and moisturizer, tetra-acetyl phytosphingosine can be used in food preservation and nutritional supplements to extend the shelf life of food and improve nutritional value. After deacetylation, tetraacetyl phytosphingosine can be converted into phytosphingosine, which then serves as a precursor for the synthesis of ceramide and plays an important role in the preparation of ceramide.

[0003] As people pursue natural, green and healthy, the market demand for tetraacetyl phytosphingosine is growing. At present, the preparation method of tetraacetyl phytosphingosine mainly includes bacterial strain fermentation method, but the existing bacterial strains have low yields, resulting in high production costs.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant bacterium for producing tetraacetyl phytosphingosine, a construction method and application thereof, and a recombinant bacterium capable of high-yield tetraacetyl phytosphingosine is obtained by screening genes of different sources for key proteins for synthesizing tetraacetyl phytosphingosine.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The synthetic route of TAPS used in the present invention is as follows Figure 1As shown. Based on this TAPS synthesis pathway, sphingosine phosphatase (Lcb3), serine palmitoyltransferase long-chain basic subunit 2 (Lcb2) and serine palmitoyltransferase long-chain basic subunit 1 (Lcb1) from different sources were screened, thereby obtaining a recombinant bacterium with high TAPS production, which can express the key enzymes of this synthesis pathway: Lcb3, Lcb2 and Lcb1, and is used for the production of TAPS, which has a high TAPS yield.

[0008] In the present invention, the starting strain of the recombinant bacteria constructed is S. wickhamii ( Wickerhamomyces cifrii ), the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on May 19, 2023, with the deposit number CGMCC No. 27391. It is an existing strain and has been disclosed in patent CN202410019615.2.

[0009] The above-mentioned S. wickhamii can be used to produce TAPS, but in order to further increase the yield of TAPS, the present invention optimizes the key enzymes Lcb3, Lcb2 and Lcb1 for synthesizing TAPS during the research and development process. The results show that Lcb3, Lcb2 and Lcb1 from different sources have an important influence on the TAPS yield.

[0010] For Lcb3, the present invention screened out 5 enzymes with relatively higher yields from Lcb3 from different sources, namely: Botrytis cinerea Bclcb3 (NCBI Reference Sequence: XM_024696698.1), derived from Fusarium pseudogramineum Fplcb3 (NCBI Reference Sequence: XM_009256063.1), derived from Purpureocillium takamizusanense Ptlcb3 (NCBI Reference Sequence: XM_047987418.1), derived from Podospora pseudocomata Pplcb3 (NCBI Reference Sequence: XM_062888877.1), derived from Metarhizium acridum Malcb3 (NCBI Reference Sequence: XM_007811787.2).

[0011] The nucleotide sequences of the above five Lcb3 after codon optimization are shown in SEQ ID NO. 1 to 5. Through the optimization of the above genes, recombinant bacteria capable of expressing exogenous Lcb3 are constructed, and the obtained recombinant bacteria can increase the TAPS yield based on the starting strain.

[0012] For Lcb2, the present invention screened out 5 enzymes with relatively higher yields from Lcb2 from different sources, specifically: Penicillium cosmopolitanum Pclcb2 (NCBI Reference Sequence: XM_056631772.1), derived from Aspergillus nidulans Anlcb2 (NCBI Reference Sequence: XM_653614.2), derived from Tuber melanosporum Tmlcb2 (NCBI Reference Sequence: XM_002839585.2), derived from Kazachstania barnettii Kblcb2 (NCBI Reference Sequence: XM_041548502.1), derived from Neurospora tetrasperma Ntlcb2 (NCBI Reference Sequence: XM_009852915.1).

[0013] The nucleotide sequences of the above five Lcb2 after codon optimization are shown in SEQ ID NO. 6 to 10. Through the optimization of the above genes, recombinant bacteria capable of expressing exogenous Lcb3 and Lcb2 are constructed, and the obtained recombinant bacteria can further improve the TAPS production based on the above recombinant bacteria.

[0014] For Lcb1, the present invention screened out 5 enzymes with relatively higher yields from Lcb1 from different sources, specifically: Rhizophagus irregularis Rilcb1 (NCBI Reference Sequence: XM_025329453.2), derived from Coprinopsis cinerea Cclcb1 (NCBI Reference Sequence: XM_001837986.2), derived from Bovine pig Sblcb1 (NCBI Reference Sequence: XM_041455992.1), derived from Trametes versicolor Tvlcb1 (NCBI Reference Sequence: XM_008045601.1), derived from Candida albicans Calcb1 (NCBI Reference Sequence: XM_711183.1).

[0015] The nucleotide sequences of the above five Lcb1 after codon optimization are shown in SEQ ID NO. 11 to 15. Through the optimization of the above genes, recombinant bacteria capable of expressing exogenous Lcb3, Lcb2 and Lcb1 were constructed, and the obtained recombinant bacteria had higher TAPS production than the above starting bacteria and recombinant bacteria.

[0016] The method for constructing the above-mentioned recombinant bacteria includes: inserting the exogenous gene into the starting strain in a homologous recombination manner, and selecting the recombinant bacteria by nourseoin resistance.

[0017] In the present invention, the gene inserted into the starting strain is at least one of the exogenous Lcb3, Lcb2 and Lcb1 genes.

[0018] Optionally, the recombinant bacteria include an exogenous Lcb3 gene; or exogenous Lcb3 and Lcb2 genes; or exogenous Lcb3, Lcb2 and Lcb1 genes. More preferably, the recombinant bacteria include exogenous Lcb3, Lcb2 and Lcb1 genes.

[0019] In some embodiments, the recombinant bacteria include genes of Pplcb3, Pclcb2 and Sblcb1. Experiments have shown that the combination of Pplcb3, Pclcb2 and Sblcb1 can maximize the TAPS yield.

[0020] In some embodiments, the above-mentioned recombinant bacteria are prepared by constructing a recombinant suicide vector, transforming the recombinant suicide vector into a starting strain, and then screening the recombinant bacteria for resistance to nourseoin.

[0021] The method for constructing the recombinant suicide vector includes: overlapping PCR amplification of the lig4-UP fragment, the GAPDH promoter fragment, the nourseoinhibin resistance marker NAT1 fragment, and the lig4-DOWN fragment to obtain a lig4-up-GAPDH-NAT1-lig4-down homologous recombination fragment, and then TA cloning the lig4-up-GAPDH-NAT1-lig4-down homologous recombination fragment with the pUC19 linear vector to obtain a pUClig4 recombinant suicide vector, the nucleotide sequence of which is shown in SEQ ID NO.16.

[0022] Furthermore, on this basis, the present invention also provides a whole-cell catalyst, which contains the above-mentioned recombinant bacteria.

[0023] The above-mentioned recombinant bacteria are used as whole-cell catalysts, and the exogenous Lcb3, Lcb2 and Lcb1 expressed by them can efficiently produce TAPS.

[0024] The present invention also provides the use of the above recombinant bacteria in synthesizing TAPS and its downstream products. Based on this, the present invention can use the recombinant bacteria to produce TAPS, and the method is as follows: the above recombinant bacteria is added to the reaction system for whole-cell catalysis to obtain TAPS.

[0025] In some embodiments, the recombinant bacteria are expanded before whole-cell transformation, and the expansion culture process is: the recombinant bacteria are inoculated into YPD liquid culture medium, and cultured at 25-30° C. and 180-220 rpm for 24-60 hours to obtain seed liquid.

[0026] In some embodiments, the seed solution can be inoculated into a shake flask fermentation medium, and the culture conditions are: temperature of 25-30° C., rotation speed of 180-220 rpm, and fermentation culture time of 36-60 h.

[0027] In some embodiments, the above recombinant bacteria can be inoculated into the fermentation medium of the fermenter, and the culture conditions are: temperature of 25-30°C, dissolved oxygen higher than 20%, 80% glycerol feed added, and fermentation culture time of 72-120h.

[0028] The present invention has the following beneficial effects:

[0029] The present invention inserts a key gene for synthesizing TAPS into the genome of Saccharomyces cerevisiae by homologous recombination, thereby obtaining a recombinant bacterium expressing heterologous Lcb3, Lcb2 and Lcb1. The production of TAPS using the recombinant bacterium can increase the yield of TAPS, shorten the fermentation cycle, and save materials and energy, thereby greatly reducing the production cost. Therefore, the recombinant bacterium and the method for producing TAPS of the present invention have good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The following is a synthetic route of TAPS of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0033] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0034] The culture medium formula involved in the embodiment of the present invention is as follows:

[0035] YPD liquid culture medium: yeast powder 10g / L, peptone 20g / L, glucose 20g / L.

[0036] YPD solid culture medium: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 20 g / L.

[0037] Secondary seed culture medium: glycerol 30g / L, peptone 10g / L, yeast powder 5g / L, malt extract 2g / l, serine 5g / L, potassium dihydrogen phosphate 2g / L, ammonium sulfate 0.8g / L, calcium chloride 1.2g / L, nicotinic acid 0.05g / L, p-aminobenzoic acid 0.03g / L, calcium pantothenate 0.03g / L, thiamine hydrochloride 0.05g / L, anhydrous magnesium sulfate 0.8g / L, ferrous sulfate heptahydrate 0.02g / L.

[0038] Shake flask fermentation medium: glycerol 30 g / L, yeast powder 5 g / L, peptone 10 g / L, malt extract 2 g / L, ammonium sulfate 0.8 g / L, dipotassium hydrogen phosphate 1.5 g / L, serine 10 g / L, calcium chloride 1 g / L.

[0039] Fermentation tank fermentation medium: glycerol 25~40g / L, peptone 10~20g / L, yeast powder 1~2g / L, malt extract 2~4g / l, serine 3~6g / L, potassium dihydrogen phosphate 2~3g / L, ammonium sulfate 0.8~1.2g / L, calcium chloride 1~1.2g / L, nicotinic acid 0.03~0.06g / L, p-aminobenzoic acid 0.03~0.06g / L, calcium pantothenate 0.02~0.03g / L, thiamine hydrochloride 0.03~0.12g / L, anhydrous magnesium sulfate 0.8~1.4g / L, ferrous sulfate heptahydrate 0.02~0.04g / L, defoaming agent 0.4~0.8ml / L.

[0040] Example 1

[0041] This example is to construct a recombinant bacterium containing a sphingosine phosphatase gene (lcb3), and the construction method is as follows:

[0042] (1) PCR amplification was used to obtain the upstream homology arm lig4-UP fragment of the lig4 gene, the GAPDH promoter fragment, the nourseoinhibin resistance marker NAT1 fragment, and the downstream homology arm lig4-DOWN fragment of the lig4 gene;

[0043] (2) Overlap PCR amplification of the lig4-UP fragment, GAPDH promoter fragment, NAT1 fragment, and lig4-DOWN fragment obtained the lig4-up-GAPDH-NAT1-lig4-down homologous recombination fragment; TA cloning of the lig4-up-GAPDH-NAT1-lig4-down homologous recombination fragment and the pUC19 linear vector was performed to obtain the pUClig4 recombinant suicide vector, whose nucleotide sequence is shown in SEQ ID NO.16.

[0044] (3) Obtain the source from NCBI Botrytis cinerea , Fusarium pseudogramineum , Purpureocillium takamizusanense , Podospora pseudocomata , Metarhizium acridum The gene sequence of sphingosine phosphatase (lcb3) was codon optimized and then fully synthesized. The synthesized nucleotide sequences are shown in SEQ ID NO.1-5.

[0045] (4) The vector pUClig4 and gene lcb3 were double-digested with XhoI / PstI as restriction sites and ligated to obtain the recombinant plasmids pUClig4-Bclcb3, pUClig4-Fplcb3, pUClig4-Ptlcb3, pUClig4-Pplcb3, and pUClig4-Malcb3.

[0046] (5) The above suicide vector was transformed into Saccharomyces cerevisiae (CGMCC No. 27391), and transformants were obtained after nourseoin resistance screening, and the following recombinant bacteria were obtained: strain 1 (lig4 gene knocked out and NAT1 and Bclcb3 genes were integrated), strain 2 (lig4 gene knocked out and NAT1 and Fplcb3 genes were integrated), strain 3 (lig4 gene knocked out and NAT1 and Ptlcb3 genes were integrated), strain 4 (lig4 gene knocked out and NAT1 and Pplcb3 genes were integrated), strain 5 (lig4 gene knocked out and NAT1 and Malcb3 genes were integrated), and the colony PCR method was used to verify the correctness. At the same time, this embodiment also set up control 1 (starting strain) and control 2 (lig4 gene knocked out and NAT1 gene was integrated).

[0047] Among them, the nourseoin resistance screening is to spread the recombinant yeast on the YPD solid culture medium containing 50 μg / mL nourseoin, culture it at 30°C for 2 days to grow a single colony, and verify the correct strain by PCR, that is, the positive strain of correct homologous recombination.

[0048] Table 1 Genotypes of different recombinant bacteria

[0049]

[0050] Shake flask fermentation was performed to detect the yield of TAPS in the fermentation broth. The fermentation conditions were as follows: the correct transformants were picked into YPD liquid medium and cultured overnight at 30°C and 220 rpm. 5% of the seed solution was inoculated into 30 mL of shake flask fermentation medium and fermented at 30°C and 220 rpm for 48 hours. 0.2 mL of the fermentation broth was taken, 1 mL of methanol was added, and the mixture was shaken and mixed before HPLC detection.

[0051] The test results are shown in Table 2. Comparing the TAPS yields of the recombinant strains, the TAPS yield concentration of the fermentation recombinant strain 4 was the highest, reaching 1.22 g / L.

[0052] Table 2 TAPS production of each recombinant strain

[0053]

[0054] Example 2

[0055] This example is to construct a recombinant engineering bacterium containing the sphingosine phosphatase gene (lcb3) and the serine palmitoyltransferase long chain basic subunit 2 gene (lcb2)

[0056] Obtained from NCBI Penicillium cosmopolitanum , Aspergillus nidulans , Tuber melanosporum , Kazachstania barnettii , Neurospora tetraspermaThe gene sequence of serine palmitoyltransferase long chain basic subunit 2 (lcb2) was synthesized after codon optimization, and the nucleotide sequence is shown in SEQ ID NO.6-10. The suicide vector pUClig4-Pplcb3 with the highest TAPS yield in Example 1 was double-digested with XhoI / PstI as restriction sites and connected with gene lcb2 to obtain recombinant plasmids pUClig4-Pplcb3-Pclcb2, pUClig4-Pplcb3-Anlcb2, pUClig4-Pplcb3-Tmlcb2, pUClig4-Pplcb3-Kblcb2, and pUClig4-Pplcb3-Ntlcb2. The above suicide vectors were transformed into yeast, and transformants were obtained after nourseoin resistance screening. The following recombinant bacteria were obtained: strain 6 (lig4 gene knocked out and NAT1, Pplcb3 and Pclcb2 genes were integrated), strain 7 (lig4 gene knocked out and NAT1, Pplcb3 and Anlcb2 genes were integrated), strain 8 (lig4 gene knocked out and NAT1, Pplcb3 and Tmlcb2 genes were integrated), strain 9 (lig4 gene knocked out and NAT1, Pplcb3 and Kblcb2 genes were integrated), strain 10 (lig4 gene knocked out and NAT1, Pplcb3 and Ntlcb2 genes were integrated), and the correct transformants were verified by colony PCR.

[0057] Among them, the nourseoin resistance screening is to spread the recombinant yeast on the YPD solid culture medium containing 50 μg / mL nourseoin, culture it at 30°C for 2 days to grow a single colony, and verify the correct strain by PCR, that is, the positive strain of correct homologous recombination.

[0058] Table 3 Genotypes of different recombinant bacteria

[0059]

[0060] The fermentation was carried out in shake flasks under the same fermentation conditions as in Example 1. The yield of TAPS in the fermentation broth was detected. The test results are shown in Table 4. By comparing the TAPS yields of the recombinant strains, the TAPS yield concentration of the fermentation recombinant strain 6 was the highest, reaching 1.71 g / L.

[0061] Table 4 TAPS production of each recombinant strain

[0062]

[0063] Example 3

[0064] This example is to construct a recombinant engineering bacterium containing a sphingosine phosphatase gene (lcb3), a serine palmitoyltransferase long-chain basic subunit 2 gene (lcb2), and a serine palmitoyltransferase long-chain basic subunit 1 gene (lcb1).

[0065] Obtained from NCBI Rhizophagus irregularis , Coprinopsis cinerea , Pig bovine , Trametes versicolor , Candida albicans The gene sequence of serine palmitoyltransferase long chain basic subunit 1 (lcb1) was synthesized after codon optimization, and the nucleotide sequences were shown in SEQ ID NO.11-15. The suicide vector pUClig4-Pplcb3Pclcb2 with the highest TAPS yield in Example 2 was double-digested with XhoI / PstI as restriction sites and connected with gene lcb1 to obtain recombinant plasmids pUClig4-Pplcb3-Pclcb2-Rilcb1, pUClig4-Pplcb3-Pclcb2-Cclcb1, pUClig4-Pplcb3-Pclcb2-Sblcb1, pUClig4-Pplcb3-Pclcb2-Tvlcb1, and pUClig4-Pplcb3-Pclcb2-Calcb1. The above suicide vectors were transformed into yeast, and transformants were obtained after nourseoin resistance screening. The following recombinant bacteria were obtained: strain 11 (lig4 gene knocked out and NAT1, Pplcb3, Pclcb2 and Rilcb1 genes were integrated), strain 12 (lig4 gene knocked out and NAT1, Pplcb3, Pclcb2 and Cclcb1 genes were integrated), strain 13 (lig4 gene knocked out and NAT1, Pplcb3, Pclcb2 and Sblcb1 genes were integrated), strain 14 (lig4 gene knocked out and NAT1, Pplcb3, Pclcb2 and Tvlcb1 genes were integrated), strain 15 (lig4 gene knocked out and NAT1, Pplcb3, Pclcb2 and Calcb1 genes were integrated). The correct transformants were verified by colony PCR.

[0066] Among them, the nourseoin resistance screening is to spread the recombinant yeast on the YPD solid culture medium containing 50 μg / mL nourseoin, culture it at 30°C for 2 days to grow a single colony, and verify the correct strain by PCR, that is, the positive strain of correct homologous recombination.

[0067] Table 5 Genotypes of different recombinant bacteria

[0068]

[0069] The fermentation was carried out in shake flasks under the same fermentation conditions as in Example 1. The yield of TAPS in the fermentation broth was detected. The test results are shown in Table 6. By comparing the TAPS yields of the recombinant strains, the TAPS yield concentration of the fermentation recombinant strain 13 was the highest, reaching 2.19 g / L.

[0070] Table 6 TAPS production of each recombinant strain

[0071]

[0072] Example 4

[0073] This example is a fermentation tank fermentation of the strain with the highest TAPS shake flask yield

[0074] The colonies of recombinant strain 13 were picked and placed in YPD liquid medium for 48 hours at 30°C and 220rpm to obtain primary seeds. The primary seeds were inoculated into 200mL of secondary seed medium at 5%, and cultured at 30°C and 220rpm for 24 hours to obtain secondary seeds. The secondary seeds were inoculated into 2L fermentation medium in a fermenter at an inoculation rate of 15%, and fermented in a 5L fermenter, maintaining a temperature of 30°C and dissolved oxygen above 20%. The fermentation was terminated by adding 80% glycerol feed and fermentation culture for 96 hours. The yield of TAPS in the fermentation broth was detected by HPLC. The results showed that the TAPS yield of recombinant strain 13 reached 31.4g / L.

[0075] The above results show that by comparing and selecting the recombinant engineered bacteria obtained from different sources of sphingosine phosphatase genes (lcb3), serine palmitoyltransferase long-chain basic subunit 2 genes (lcb2) and serine palmitoyltransferase long-chain basic subunit 1 genes (lcb1), it was finally confirmed that the recombinant engineered bacteria containing the optimized sphingosine phosphatase gene (lcb3) from Podospora pseudocomata, the optimized serine palmitoyltransferase long-chain basic subunit 2 gene (lcb2) from Penicillium cosmopolitanum, and the optimized serine palmitoyltransferase long-chain basic subunit 1 gene (lcb1) from Suillus bovinus can significantly increase the production of TAPS. The TAPS concentration in the culture obtained after shake flask fermentation of the recombinant bacteria can reach 2.19 g / L, and the fermentation in the fermenter can achieve a maximum TAPS yield of 31.4 g / L, which is significantly higher than the yield of the same type of TAPS.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant bacterium for producing tetraacetyl phytosphingosine, characterized in that: The genome of the recombinant bacterium is integrated with heterologous sphingosine phosphatase and serine palmitoyltransferase genes; The starting strain of the recombinant bacteria is Saccharomyces cerevisiae, and its deposit number is CGMCC No. 27391; The serine palmitoyltransferase comprises serine palmitoyltransferase long-chain basic subunit 1 and serine palmitoyltransferase long-chain basic subunit 2; The sphingosine phosphatase is Pplcb3; the serine palmitoyltransferase long-chain basic subunit 2 is Pclcb2; the serine palmitoyltransferase long-chain basic subunit 1 is selected from any one of Rilcb1, Cclcb1, Sblcb1, Tvlcb1 and Calcb1; Among them, Pplcb3 is derived from Podosporapseudocomata ; Pclcb2 is derived from Penicilliumcosmopolitanum ; Rilcb1 is derived from Rhizophagusirregularis ; Cclcb1 is derived from Coprinopsis a; Sblcb1 is derived from Suillus bovinus ; Tvlcb1 is derived from Trametes versicolor Calcb1 is derived from Candida albicans ; The nucleotide sequences of Pplcb3 in the sphingosine phosphatase are shown as SEQ ID NO.4; the nucleotide sequences of Pclcb2 in the serine palmitoyltransferase long-chain basic subunit 2 are shown as SEQ ID NO.6; the nucleotide sequences of Rilcb1, Cclcb1, Sblcb1, Tvlcb1 and Calcb1 in the serine palmitoyltransferase long-chain basic subunit 1 are shown as SEQ ID NO.11-15.

2. The recombinant bacterium according to claim 1, characterized in that The genes of Pplcb3, Pclcb2 and Sblcb1 are integrated into the genome of the recombinant bacteria.

3. The method for constructing a recombinant bacterium according to claim 1 or 2, characterized in that: include: The genes of sphingosine phosphatase and serine palmitoyl transferase are inserted into the starting strain in a homologous recombination manner, and the recombinant bacteria producing tetraacetyl phytosphingosine are obtained through screening.

4. Use of the recombinant bacteria according to claim 1 or 2 in the synthesis of tetraacetyl phytosphingosine.

5. A whole cell catalyst, characterized in that Contains the recombinant bacteria according to claim 1 or 2.

6. A method for producing tetraacetyl phytosphingosine, characterized in that: The method comprises adding the recombinant bacteria according to claim 1 or 2 into a reaction system for culturing to obtain the tetraacetyl phytosphingosine.

7. The method according to claim 6, characterized in that The culture conditions are: temperature 25-30°C, rotation speed 180-220rpm, and time 24-60h.

Images