CN117778217A

CN117778217A - Yarrowia lipolytica capable of producing nervonic acid and construction method and application thereof

Info

Publication number: CN117778217A
Application number: CN202311822032.5A
Authority: CN
Inventors: 刘延峰; 陈坚; 谷胜博; 李江华; 刘龙; 吕雪芹
Original assignee: Jiaxing Future Food Research Institute; Jiangnan University
Current assignee: Jiaxing Future Food Research Institute; Jiangnan University
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-03-29

Abstract

The invention discloses yarrowia lipolytica capable of producing nervonic acid, and a construction method and application thereof, and belongs to the technical fields of metabolic engineering and genetic engineering. According to the invention, through heterologously expressing a plurality of 3-ketoacyl-CoA synthase KCS and a plurality of esterifying enzymes in yarrowia lipolytica, and intensively expressing omega-9 desaturase, ATP-citrate lyase, malate dehydrogenase, malate enzyme, acetyl-CoA carboxylase and endoplasmic reticulum regulating factors, peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene, AMP-protein kinase gene and delta 12 desaturase gene, the yield of nervonic acid reaches 0.22g/L under a shake flask condition; the yield of the nervonic acid after fed-batch fermentation can reach 8g/L, and the proportion of the nervonic acid to the total oil is 12 percent. The method provided by the invention lays a foundation for the artificial efficient synthesis of the nervonic acid, and has a wide industrial application prospect.

Description

Yarrowia lipolytica capable of producing nervonic acid and construction method and application thereof

Technical Field

The invention relates to yarrowia lipolytica capable of producing nervonic acid, and a construction method and application thereof, and belongs to the technical fields of metabolic engineering and genetic engineering.

Background

Nervonic acid (cis-15-tetracosenoic acid, NA) was found early in the shark brain and is therefore also known as shark acid, an ultralong chain monounsaturated fatty acid (Very long chain fatty acid, VLCMFA). Nervonic acid can promote brain development, promote proliferation and differentiation of brain cells, and regulate and improve the composition, structure and function of the biomembrane; regulate the level of neurotransmitter in brain, promote the recovery and regeneration of damaged nerve fiber, restore nerve ending activity, improve brain function, concentrate attention and strengthen memory. Meanwhile, the nervonic acid is widely applied to the treatment of nervous system diseases such as Alzheimer's disease, parkinsonism and the like.

At present, the main source of the nervonic acid is animal and plant extraction, but the method has long extraction period and high cost. Therefore, the production of the nervonic acid by the microorganism gradually enters the sight of a plurality of scientific researchers, and the biological method for producing the nervonic acid has wide application prospect. The Japanese scholars can select a strain of microalgae producing nervonic acid by collecting microalgae and fungi in natural environment and further screening, but the yield is lower than 1g/L, and the industrial application is difficult.

Yarrowia lipolytica (Yarrowia lipolytica) is a binary, obligate aerobic, unconventional oleaginous yeast belonging to the semi-ascomycete family, considered to be the GRAS (Generally Recognized as Safe) strain. Yarrowia lipolytica has very strong lipid metabolism capacity, and is a superior host for converting low-value carbon into high-value secondary metabolites and lipid derivatives due to its unique powerful acetyl-coa supply pattern, broad substrate spectrum, multiple carbon source utilization pathways, and excellent lipid accumulation capacity. However, the selection of yarrowia lipolytica for the production of nervonic acid has been less studied, mainly due to the lack of 3-ketoacyl-CoA synthase elongase and lipid accumulation problems with preference for nervonic acid synthesis. The present invention has been made accordingly.

Disclosure of Invention

In order to solve the problems, the invention provides a method for producing the nervonic acid by using yarrowia lipolytica engineering bacteria through a biological fermentation method, which reaches 0.22g/L under the shaking condition; the yield of the nervonic acid after fed-batch fermentation can reach 8g/L, and the proportion of the nervonic acid to the total oil is 12 percent.

It is a first object of the present invention to provide a recombinant yarrowia lipolytica producing a neuraminic acid, which has inserted into its genome a heterologous 3-ketoacyl-CoA synthase expression cassette, an esterifying enzyme expression cassette, a heterologous omega-9 desaturase expression cassette, an ATP-citrate lyase expression cassette, a malate dehydrogenase expression cassette, a malate enzyme expression cassette, an acetyl CoA carboxylase expression cassette and an endoplasmic reticulum regulatory factor expression cassette, knocked out the peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene, AMP-protein kinase gene and Δ12 desaturase gene;

wherein the heterologous 3-ketoacyl-CoA synthase expression cassette contains a CgKCS encoding gene and a LaKCS encoding gene, the esterifying enzyme expression cassette contains a DGAT encoding gene, a GPAT encoding gene and a LAPPT encoding gene, the heterologous omega-9 desaturase expression cassette contains a delta 15D encoding gene, the ATP-citrate lyase expression cassette contains an ACL encoding gene, the malate dehydrogenase expression cassette contains an MDH encoding gene, the malate enzyme expression cassette contains an RTME encoding gene, the acetyl-CoA carboxylase expression cassette contains an ACC1 encoding gene, and the endoplasmic reticulum regulating factor expression cassette contains an INO2 encoding gene.

Further, the above expression cassette is integrated into the genome by a non-homologous end joining method, the integration site being the Zeta site, and the auxotrophic complementary selection markers being uracil and leucine.

Further, the nucleotide sequence of the CgKCS coding gene is shown in SEQ ID NO.1, the nucleotide sequence of the LaKCS coding gene is shown in SEQ ID NO.2, the nucleotide sequence of the delta 15D coding gene is shown in SEQ ID NO.3, the nucleotide sequence of the DGAT coding gene is shown in SEQ ID NO.4, the nucleotide sequence of the GPAT coding gene is shown in SEQ ID NO.5, the nucleotide sequence of the LAPPT coding gene is shown in SEQ ID NO.6, the nucleotide sequence of the ACL coding gene is shown in SEQ ID NO.7, the nucleotide sequence of the MDH coding gene is shown in SEQ ID NO.8, the nucleotide sequence of the RTME coding gene is shown in SEQ ID NO.9, the nucleotide sequence of the ACC1 coding gene is shown in SEQ ID NO.10, and the nucleotide sequence of the INO2 coding gene is shown in SEQ ID NO.12.

Further, the peroxisome biogenesis factor 10 gene NCBI gene No. 2909503, triacylglycerol lipase 4 gene NCBI gene No. 2908993, Δ12 desaturase gene NCBI gene No. 2906773, and AMPK has the nucleotide sequence shown in SEQ ID NO.11.

Further, the promoter of each gene in the recombinant yarrowia lipolytica expression cassette is selected from the group consisting of P _TEF8uas 、P _TEFin 、P _YAT1 Any one of the terminators is selected from T _lip2t 、T _pRC1 、T _pRL3 Any one of the following.

Further, promoter P _YAT1 The nucleotide sequence of (a) is shown as SEQ ID NO.13, and a terminator T _pRL3 The nucleotide sequence of the promoter P is shown in SEQ ID NO.14 _TEFin The nucleotide sequence of (a) is shown as SEQ ID NO.15, and a terminator T _pRC1 The nucleotide sequence of the promoter is shown in SEQ ID NO.16 and the promoter P _TEF8uas The nucleotide sequence of the polypeptide is shown in SEQ ID NO.17 and terminator T _lip2t The nucleotide sequence of (C) is shown as SEQ ID NO.18.

Further, po1 f-. DELTA.ku70 (MatA,. DELTA.ku70:: hisG, leu2-270, ura3-302, xpr2-322, axp 1-2) was used as the starting strain.

Further, pUC linearization plasmid was used as an expression vector.

The first object of the present invention is to provide a construction method of the recombinant yarrowia lipolytica, comprising the following steps:

s1, linearizing plasmid by non-homologous end joining method

Integrating vector-CgKCS-LaKCS-delta 15D into the genome of a yarrowia lipolytica original strain to obtain recombinant bacteria Yali 1;

s1, integrating a linearization plasmid vector-DGAT-GPAT-LAPPT onto a recombinant bacterium Yali 1 genome by a non-homologous end connection method to obtain a recombinant bacterium Yali2;

s3, knocking out a peroxisome biogenesis factor 10 gene, a triacylglycerol lipase 4 gene and a delta 12 desaturase gene in the recombinant bacteria Yali2 to obtain recombinant bacteria Yali 3;

s4, integrating the linearization plasmid vector-ACL-MDH-RTME onto a recombinant bacteria Yali 3 genome by a non-homologous end connection method to obtain recombinant bacteria Yali 4;

s5, knocking out AMP-protein kinase in the recombinant bacteria Yali 4, and integrating linearization plasmid vector-ACC1-INO2 onto a recombinant bacteria Yali 4 genome by a non-homologous end-joining method to obtain the recombinant yarrowia lipolytica.

Further, the linearized plasmid vector-CgKCS-LaKCS-Delta15D is pUC-Zeta-CgKCS-LaKCS-Delta15D, vector-DGAT-GPAT-LAPPT is pUC-Zeta-DGAT-GPAT-LAPPT, vector-ACL-MDH-RTME is pUC-Zeta-CgKCS-LaKCS-Delta15D

pUC-Zeta-ACL-MDH-RTME, vector-ACC1-INO2 is pUC-Zeta-ACC1-INO2.

Further, the construction method of the linearization plasmid comprises the following steps:

a) pUC-Zeta-CgKCS-LaKCS-Delta15D: pUC19 is a plasmid skeleton, and comprises a Zeta integrated fragment, a 3-ketoester acyl-CoA synthase (KCS) gene expression cassette and a heterologous omega-9 desaturase expression cassette, and is obtained by cutting a circular plasmid through Not I enzyme;

b) pUC-Zeta-DGAT-GPAT-LAPPT: pUC19 is a plasmid skeleton, and comprises a Zeta integrated fragment and an esterifying enzyme expression cassette, and is obtained by cutting a circular plasmid through Not I enzyme;

c) pUC-Zeta-ACL-MDH-RTME: pUC19 is a plasmid skeleton, and comprises a Zeta integrated fragment, an ATP-citrate lyase expression cassette, a malate dehydrogenase expression cassette and a malate enzyme expression cassette, and is obtained by cutting a circular plasmid through Not I enzyme;

d) pUC-Zeta-ACC1-INO2: pUC19 is a plasmid backbone comprising a Zeta-integrated fragment, an acetyl-CoA carboxylase expression cassette and an endoplasmic reticulum regulatory factor expression cassette, and is obtained by cleavage of a circular plasmid by Not I.

It is a third object of the present invention to provide the use of said recombinant yarrowia lipolytica in the food or biological field.

Further, the method is used for preparing food or biological products containing the nervonic acid.

It is a fourth object of the present invention to provide a method for producing a neural acid, using said recombinant yarrowia lipolytica for fermentation production.

Further, the fermentation process is to inoculate the strain into a fermentation culture medium according to the inoculum size of 5-15%, and ferment under the conditions that the temperature is 28-36 ℃, the rotating speed is 200-1000rpm and the ventilation is 0.2-10 vvm.

Further, the fermentation medium comprises 5-15g/L glucose, (NH) ₄ ) ₂ SO ₄ 5-15g/L, 0.1-2g/L zinc sulfate, 10-20g/L potassium dihydrogen phosphate, 1-5g/L magnesium sulfate, and optionally small amounts of trace elements and vitamins.

Further, the fermentation is fed-batch fermentation.

Further, in the fed-batch fermentation, a constant pH of 5.5 was used.

Further, in the fed-batch fermentation, an exponential feeding mode is adopted. The method comprises the following steps: after inoculation, a control mode of combining dissolved oxygen and stirring is started, the dissolved oxygen is set to be 35%, and the rotating speed reducing function is closed, so that the rotating speed can only be increased but not reduced. When the on-line dissolved oxygen detector detects that the dissolved oxygen in the fermentation tank is rapidly increased, namely after the carbon source in the fermentation tank is consumed, the functions of index feeding and rotation speed reduction are started, so that the dissolved oxygen is maintained to be 35% while the index feeding is performed, wherein the index feeding parameters are calculated by combining the growth condition of recombinant bacteria in the fermentation tank with an index feeding method, the flow rate is increased exponentially, the specific growth rate is a constant value, and the thallus density is increased exponentially.

Further, the specific growth rate in the exponential feed equation is 0.08-0.20.

The invention has the beneficial effects that:

the invention heterologously expresses 3-ketoacyl-CoA synthase KCS in yarrowia lipolytica by metabolic engineering means, realizes the synthesis of nervonic acid in yarrowia lipolytica, knocks out related genes related to fatty acid degradation and overexpresses related genes related to nervonic acid accumulation to promote fatty acid accumulation. In particular, the present invention also expresses malate and malate dehydrogenase, enhances the supply of the precursors acetyl-coa and cofactor NADPH, enhances the expression of acetyl-coa carboxylase and endoplasmic reticulum modulator, enhances the supply of propionyl-coa and enlarges the area of the endoplasmic reticulum, thereby further improving the yield of neuraminic acid. The invention lays a solid foundation for further improving the yield of the nervonic acid in yarrowia lipolytica and the industrialized application.

Drawings

FIG. 1 is a schematic diagram of the construction of a linear plasmid exemplified by pUC-Zeta-CgKCS-LaKCS-Delta15D.

FIG. 2 is a schematic diagram of the Golden Gate assembly principle.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The scheme of the invention is as follows:

constructing a yarrowia lipolytica gene cloning kit based on Golden Gate assembly, constructing a 3-ketoacyl-CoA synthase (KCS) gene expression cassette and a heterologous omega-9 desaturase expression cassette by using the kit, and integrating the kit into a yarrowia lipolytica genome by a non-homologous end-joining method to obtain a yarrowia lipolytica recombinant strain Yali 1 producing nervonic acid; integrating the esterifying enzyme gene expression cassette into a yarrowia lipolytica recombinant strain Yali 1 genome by a non-homologous end joining method to obtain a yarrowia lipolytica recombinant strain Yali2; further, the peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene and delta 12 desaturase gene in the recombinant bacteria Yali2 are knocked out to obtain the yarrowia lipolytica recombinant bacteria Yali 3. The malate dehydrogenase expression cassette, ATP-citrate lyase and malate enzyme expression cassette are then integrated into the yarrowia lipolytica recombinant Yali 3 genome by non-homologous end joining to yield yarrowia lipolytica recombinant Yali 4. Finally, the AMP-protein kinase in the recombinant bacteria Yali 4 is knocked out, and simultaneously, an endoplasmic reticulum regulating factor and an acetyl-CoA carboxylase expression cassette are integrated on a genome to obtain the recombinant bacteria Yali 5.

Wherein, the yarrowia lipolytica Yali 5 is a genome into which a heterologous 3-ketoacyl-CoA synthase expression cassette, an esterifying enzyme expression cassette, a heterologous omega-9 desaturase expression cassette, an acetyl-CoA carboxylase expression cassette, an ATP-citrate lyase expression cassette, a malate dehydrogenase expression cassette, an endoplasmic reticulum regulatory factor expression cassette and a malate enzyme expression cassette are inserted, and the peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene, gene encoding AMP-protein kinase and delta 12 desaturase gene are knocked out.

The materials and methods involved in the following examples are as follows:

the yarrowia lipolytica starting strain is Po1 f-Deltaku 70 (MatA, deltaku 70:: hisG, leu2-270, ura3-302, xpr2-322, axp 1-2);

yarrowia lipolytica seed medium:

YPD Medium (g/L): yeast powder 10, peptone 20 and glucose 20.

Yarrowia lipolytica shake flask medium: 120g/L glucose, 6g/LYNB,12g/L ammonium sulfate.

Yarrowia lipolytica fermentation medium: 10g/L glucose, 10g/L ammonium sulfate, 0.6g/L zinc sulfate, 14.4g/L monopotassium phosphate and 3g/L magnesium sulfate.

Example 1 introduction of yarrowia lipolytica neurogenic pathway

The linear plasmid construction method comprises the following steps:

according to the nucleotide sequences disclosed on NCBI encoding 3-ketoacyl-CoA synthase, esterifying enzyme, omega-9 desaturase, acetyl-CoA carboxylase, ATP-citrate lyase, malate dehydrogenase, endoplasmic reticulum modulator and malate dehydrogenase, the nucleotide sequences are modified by codonsAnd optimizing and delivering the gene synthesized by a third party company, wherein the optimized sequence is shown in SEQ ID NO.1-12. By usingGolden Gate Assembly Kit the construction of pUC19 plasmid with Zeta skeleton integration site and intermediate plasmid containing the gene expression frame and screening label is shown in figure 1. After the plasmid construction is completed, the plasmid is transferred into escherichia coli to amplify the plasmid, and the plasmid is extracted and then digested with Not I restriction enzyme to obtain the linear plasmid.

Yarrowia lipolytica transformation method:

yarrowia lipolytica preserved in glycerol tubes was inoculated to YPD solid plates at 30℃for growth. When colonies were large, appropriate colonies were picked, washed with 0.1M lithium acetate solution, and finally resuspended in 50. Mu.l of 0.1M lithium acetate. 450 microliters of 50% PEG4000, 25 microliters of 2MDTT, and 25 microliters of 2M lithium acetate were mixed. The resuspended yeast cells were added to the mixture and 5. Mu.l salmon sperm and 20. Mu.l of the linear plasmid to be transformed were added and mixed by tapping. After incubation at 28 degrees celsius for 30 minutes, heat shock at 42 degrees celsius for 10 minutes. And finally, centrifugally collecting the thalli for coating.

The linear plasmid pUC-Zeta-CgKCS-LaKCS-delta 15D is transformed into yarrowia lipolytica to obtain engineering bacteria Yali 1, and colony is picked and subjected to shake flask fermentation. Wherein, the plasmid map is shown in FIG. 1, wherein insert sequence is used as promoter, in this example, P is used _YAT1 、T _pRL3 Initiation and termination of LaKCS expression using P _TEFin 、T _pRC1 Initiation and termination of Δ15d expression using P _TEF8uas 、T _lip2t Start and stop CgKCS expression. The nucleotide sequence of CgKCS is shown in SEQ ID NO.1, the nucleotide sequence of LaKCS is shown in SEQ ID NO.2, and the nucleotide sequence of delta 15D is shown in SEQ ID NO.3.

Example 2 promotion of accumulation of nervonic acid in yarrowia lipolytica

Yarrowia lipolytica gene knockout method:

and designing homologous arm fragments of about 1000bp before and after the gene to be knocked out, respectively obtaining left and right arms and auxotroph tags, and constructing a knocking-out frame through fusion PCR.

The linearized plasmid pUC-Zeta-DGAT-GPAT-LAPPT is transformed into engineering bacteria on the yarrowia lipolytica Yali 1 genome by a non-homologous end joining method to obtain engineering bacteria Yali 2. In the present embodiment, P is used _YAT1 、T _pRL3 Initiation and termination of DGAT expression using P _TEFin 、T _pRC1 Initiation and termination of GPAT expression using P _TEF8uas 、T _lip2t Initiation and termination of LAPPT expression. Wherein, the nucleotide sequence of DGAT is shown in SEQ ID NO.4, the nucleotide sequence of GPAT is shown in SEQ ID NO.5, and the nucleotide sequence of LAPPT is shown in SEQ ID NO.6.

Knocking out peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene and delta 12 desaturase gene in recombinant bacteria Yali2 according to a yarrowia lipolytica gene knocking-out method to obtain yarrowia lipolytica recombinant bacteria Yali 3. Wherein, the NCBI gene of the peroxisome biogenesis factor 10 gene is 2909503, the NCBI gene of the triacylglycerol lipase 4 gene is 2908993, and the NCBI gene of the delta 12 desaturase gene is 2906773.

The linearized plasmid pUC-Zeta-ACL-MDH-RTME was integrated into the engineering bacteria on the yarrowia lipolytica Yali 3 genome by a non-homologous end joining method to give Yali 4. In the present embodiment, P is used _YAT1 、T _pRL3 Initiation and termination of ACL expression, employing P _TEFin 、T _pRC1 Initiation and termination of MDH expression using P _TEF8uas 、T _lip2t Start and terminate RTME expression. Wherein, the nucleotide sequence of ACL is shown in SEQ ID NO.7, the nucleotide sequence of MDH is shown in SEQ ID NO.8, and the nucleotide sequence of RTME is shown in SEQ ID NO.9.

Knocking out AMP-protein kinase in recombinant bacteria Yali 4 according to a yarrowia lipolytica gene knocking-out method, and then converting a linearization plasmid pUC-Zeta-ACC1-INO2 into engineering bacteria on a yarrowia lipolytica Yali 4 genome subjected to knocking-out operation by a non-homologous end joining method to obtain Yali 5. In the present embodiment, P is used _YAT1 、T _pRL3 Initiation and termination of ACC1 expression, using P _TEFin 、T _pRC1 Initiation and termination of INO2 expression. Wherein the nucleotide sequence of ACC1 is shown inThe nucleotide sequence of the AMPK is shown in SEQ ID NO.10, the nucleotide sequence of the AMPK is shown in SEQ ID NO.11, and the nucleotide sequence of the INO2 is shown in SEQ ID NO.12.

Above, promoter P _YAT1 The nucleotide sequence of (a) is shown as SEQ ID NO.13, and a terminator T _pRL3 The nucleotide sequence of the promoter P is shown in SEQ ID NO.14 _TEFin The nucleotide sequence of (a) is shown as SEQ ID NO.15, and a terminator T _pRC1 The nucleotide sequence of the promoter is shown in SEQ ID NO.16 and the promoter P _TEF8uas The nucleotide sequence of the polypeptide is shown in SEQ ID NO.17 and terminator T _lip2t The nucleotide sequence of (C) is shown as SEQ ID NO.18.

EXAMPLE 3 fermentation of yarrowia lipolytica

(1) Shake flask fermentation of yarrowia lipolytica:

and (3) streaking engineering bacteria in the glycerol pipe to a YPD flat plate at 30 ℃ for culture, and after colonies grow, picking single colonies to 5ml YPD liquid culture medium of a 50ml centrifuge tube for culture at 30 ℃ for 24 hours. The seed solution was inoculated into a 250ml Erlenmeyer flask at 30℃and 220rpm for 120 hours, and the flask medium was 120g/L glucose, 6g/L YNB,12g/L ammonium sulfate.

Table 1: shake flask yield of recombinant bacteria

Recombinant strains	Nervonic acid yield (g/L)
		Yali 1	0.05
Yali 2	0.10
		Yali 3	0.13
Yali 4	0.19
		Yali 5	0.22

Because good control of fermentation process conditions is difficult to achieve in shake flasks, the yield of yarrowia lipolytica in shake flasks is not high, but Yali 5 is greatly improved in shake flask level yield relative to recombinant bacteria Yali 1-4.

(2) 3-L fermenter fermentation of yarrowia lipolytica Yali 5:

and (3) picking a single colony, inoculating the single colony to a 5ml YPD liquid culture medium of a 50ml centrifuge tube, and culturing the engineering bacteria streaked in the glycerol pipe to the YPD flat plate at 30 ℃, and after the colony grows, picking the single colony to the YPD liquid culture medium at 30 ℃ for 24 hours. The primary seed solution was inoculated into a 250ml Erlenmeyer flask at 30℃and 220rpm for 24 hours, and the collected cells were inoculated into a fermenter. The method adopts an index feeding mode to ferment, and after inoculation, a control mode of combining dissolved oxygen and stirring is started, the dissolved oxygen is set to be 35%, and the rotating speed is closed to reduce the rotating speed, so that the rotating speed can only be increased but not reduced. When the on-line dissolved oxygen detector detects that the dissolved oxygen in the fermentation tank is rapidly increased, namely after the carbon source in the fermentation tank is consumed, the index feeding is started, and the rotation speed reducing function is started, so that the dissolved oxygen is maintained to be 35% while the index feeding is performed, wherein the specific growth rate in the index feeding parameters is 0.12, the temperature is controlled to be 30 ℃, the ventilation amount is 2vvm, and the feeding bottle is 500g/L glucose. The index feed equation is: f=3e ^0.12△t Wherein F is the feed flow rate, and Δt is the duration of the exponential feed.

Fermenting for 120 hours, the biomass of yarrowia lipolytica reaches 130g/L DCW, the yield of nervonic acid is 8g/L, and the proportion of nervonic acid to total oil reaches 12%.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims

1. Recombinant yarrowia lipolytica producing neuroacid, characterized in that it comprises a heterologous 3-ketoacyl-CoA synthase expression cassette, esterifying enzyme expression cassette, heterologous omega-9 desaturase expression cassette, ATP-citrate lyase expression cassette, malate dehydrogenase expression cassette, malate enzyme expression cassette, acetyl CoA carboxylase expression cassette and endoplasmic reticulum regulatory factor expression cassette, deleted peroxisome biogenesis factor 10 gene, triacylglycerol lipase 4 gene, AMP-protein kinase gene and delta 12 desaturase gene inserted into the genome starting strain Po1 f-Deltaku 70 (MatA, deltaku 70::: hisG, leu2-270, ura3-302, xpr2-322, axp 1-2);

2. The recombinant yarrowia lipolytica of claim 1, wherein the genomic integration site is a Zeta site.

3. The recombinant yarrowia lipolytica according to claim 1, wherein the nucleotide sequence of the CgKCS encoding gene is shown in SEQ ID No.1, the nucleotide sequence of the LaKCS encoding gene is shown in SEQ ID No.2, the nucleotide sequence of the Δ15d encoding gene is shown in SEQ ID No.3, the nucleotide sequence of the DGAT encoding gene is shown in SEQ ID No.4, the nucleotide sequence of the GPAT encoding gene is shown in SEQ ID No.5, the nucleotide sequence of the LAPPT encoding gene is shown in SEQ ID No.6, the nucleotide sequence of the ACL encoding gene is shown in SEQ ID No.7, the nucleotide sequence of the MDH encoding gene is shown in SEQ ID No.8, the nucleotide sequence of the RTME encoding gene is shown in SEQ ID No.9, the nucleotide sequence of the ACC1 encoding gene is shown in SEQ ID No.10, and the nucleotide sequence of the INO2 encoding gene is shown in SEQ ID No.12; the peroxisome biogenesis factor 10 gene NCBI gene No. 2909503, triacylglycerol lipase 4 gene NCBI gene No. 2908993, delta 12 desaturase gene NCBI gene No. 2906773, and AMPK has the nucleotide sequence shown in SEQ ID NO.11.

4. The recombinant yarrowia lipolytica of claim 1 wherein the promoter of each gene in the recombinant yarrowia lipolytica expression cassette is selected from the group consisting of P _TEF8uas 、P _TEFin 、P _YAT1 Any one of the terminators is selected from T _lip2t 、T _pRC1 、T _pRL3 Any one of the following.

5. The method for constructing recombinant yarrowia lipolytica as claimed in any one of claims 1-4, comprising the steps of:

s1, linearizing plasmid by non-homologous end joining method

6. Use of the recombinant yarrowia lipolytica of any one of claims 1-4 in the food or biological arts.

7. A method for producing a nervonic acid, characterized by fermentation production using the recombinant yarrowia lipolytica of any one of claims 1-4.

8. The method according to claim 7, wherein the fermentation is carried out by inoculating the strain into the fermentation medium at a temperature of 28-36 ℃ and a rotational speed of 200-1000rpm with a ventilation of 0.2-10 vvm.

9. The method of claim 8, wherein the fermentation is a fed-batch fermentation.

10. The method of claim 9, wherein the fed-batch fermentation is performed by an exponential feed regime: the control mode of combining dissolved oxygen and stirring is used, the dissolved oxygen is set to be 35%, wherein the exponential feed supplement parameter is calculated by combining the growth condition of recombinant bacteria in a fermentation tank with the exponential feed supplement method, the flow rate is increased exponentially, the specific growth rate is a constant value, the thallus density is increased exponentially, and the specific growth rate in the exponential feed supplement equation is 0.08-0.20.