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CN-122012259-A - Engineering schizochytrium limacinum capable of producing palmitoleic acid in high yield, and construction method and application thereof

CN122012259ACN 122012259 ACN122012259 ACN 122012259ACN-122012259-A

Abstract

The invention belongs to the technical field of bioengineering and microbial fermentation, and discloses engineering schizochytrium palmitoleic acid, a construction method and application thereof, wherein the engineering schizochytrium is obtained by introducing and expressing a heterologous delta 9 desaturase gene into the schizochytrium by a genetic engineering means, and carrying out gene source screening, promoter optimization and target gene copy number optimization on the heterologous delta 9 desaturase gene so as to enhance the conversion capability of C16:0 to C16:1, thereby remarkably improving the content of palmitic acid in schizochytrium grease. The engineering strain can realize remarkable improvement of the palmitoleic acid content, preferably can reach more than 4%, and further can reach more than 6%. The method has clear technical route and strong operability, and can provide engineering strains and application schemes for the industrial production of the high palmitoleic acid functional lipid raw material.

Inventors

  • QI XIN
  • SUN XIAOMAN
  • JIN QIAOQIAO
  • ZHANG ZIXU
  • MA WANG

Assignees

  • 南京师范大学

Dates

Publication Date
20260512
Application Date
20260413

Claims (9)

  1. 1. The engineering schizochytrium limacinum is characterized in that the engineering schizochytrium limacinum is introduced and expresses a heterologous delta 9 desaturase gene in the schizochytrium limacinum by a genetic engineering means, and the heterologous delta 9 desaturase gene is subjected to gene source screening, promoter optimization and target gene copy number optimization to enhance the capability of converting C16:0 into C16:1, so that the content of palmitic acid in schizochytrium limacinum grease is remarkably improved; The heterologous delta 9 desaturase gene is a gene DSM9DE derived from scenedesmus (Desmodesmus sp.), and the nucleotide sequence of the gene is shown as SEQ ID No. 3; the Schizochytrium is Schizochytrium limacinum bacteria (Schizochytrium sp. HX-308).
  2. 2. The engineered schizochytrium palmitoleic acid of claim 1, wherein the promoter optimization comprises placing the heterologous Δ9 desaturase gene under the control of an endogenous schizochytrium promoter to increase the expression intensity of the target gene in the schizochytrium; the endogenous promoter is schizochytrium endogenous strong promoter P3626, and the nucleotide sequence of the endogenous promoter is shown as SEQ ID No. 6.
  3. 3. The engineered schizochytrium palmitoleic acid of any of claims 1-2, wherein the target gene copy number optimization comprises achieving 2-3 copy integration of the heterologous Δ9 desaturase gene in the schizochytrium genome to enhance gene dose effects and further increase palmitoleic acid content.
  4. 4. The engineered schizochytrium palmitoleic acid of claim 3, wherein 2-3 copies are integrated by a resistance selection marker comprising a G418 resistance gene, a bleomycin Zeocin resistance gene.
  5. 5. The method for constructing high-yield engineering schizochytrium palmitoleic acid according to any one of claims 1 to 4, comprising the following steps: the method comprises the steps of taking a schizochytrium HX-308 genome as a template, amplifying to obtain an endogenous strong promoter P3626, sequentially connecting the endogenous strong promoter P3626 with an artificially synthesized heterologous delta 9 desaturase gene DSM9DE derived from scenedesmus and a terminator element to construct a target gene expression cassette driven by the strong promoter, cloning the expression cassettes into transformation plasmids containing a G418 resistance gene and a bleomycin Zeocin resistance gene respectively, constructing two recombinant expression vectors with different screening markers, transforming schizochytrium competent cells to obtain positive transformants with primarily integrated exogenous DSM9DE, continuously screening the obtained transformants by gradually increasing the screening pressure of antibiotics, detecting the integrated copy number of the target gene DSM9DE in the genome by combining qPCR, and simultaneously, continuously verifying the genetic stability of the target gene expression cassette by 3-4 generations, and finally screening to obtain recombinant engineering strains with integrated copy number of DSM9DE of 2-3 copies and stable genetic characteristics, namely the high-yield oleic acid engineering schizochytrium palmae.
  6. 6. The construction method of the positive transformant of the exogenous DSM9DE according to claim 5, wherein the construction method comprises adding 10-20. Mu.L of recombinant expression vector into 100. Mu.L of schizochytrium competent cells, mixing, adding into a precooled 0.2 cm electric rotating cup, shocking under the conditions of voltage of 0.75 kV, capacitance of 25. Mu.F and resistance of 200Ω, immediately adding 1ml of non-resistant seed culture solution after shocking, resuscitating and culturing for 2 hours under the conditions of 28 ℃ and 150-220 rpm, coating the resuscitated bacterial solution on a screening plate containing G418 and bleomycin Zeocin, culturing for 3-5 d at 28 ℃ in a dark place, picking up a resistant colony, and identifying whether the integration of the resistant marker gene and the target gene DSM9DE is successful or not by genome PCR, thereby obtaining the positive transformant of the exogenous DSM9 DE; wherein the specific formulation of the screening plate is peptone 10 g/L, yeast powder 5 g/L, glucose 50 g/L, sea salt 20 g/L, agar 20 g/L, and water as solvent.
  7. 7. Use of the engineered schizochytrium limacinum with high yield of palmitoleic acid according to any of claims 1 to 4, in the fermentative production of palmitoleic acid.
  8. 8. A method for producing palmitoleic acid by fermentation of the high-yield palmitoleic acid engineering schizochytrium as claimed in any one of claims 1 to 4, comprising the following steps: Inoculating engineering schizochytrium limacinum into a seed culture medium for activation to obtain fermentation strains, inoculating the fermentation strains into the fermentation culture medium for fermentation culture, collecting thalli after fermentation is finished, and extracting grease to obtain palmitoleic acid.
  9. 9. The method according to claim 8, wherein the specific steps are as follows: Inoculating high-yield palmitoleic acid engineering schizochytrium into a seed culture medium, culturing at 28 ℃ for 24 h% to obtain first-stage seeds, inoculating the first-stage seeds into the seed culture medium according to the inoculum size of 10% of the volume of the seed culture medium, culturing again at 28 ℃ for 24 h to obtain second-stage seeds, inoculating the second-stage seeds into the seed culture medium according to the inoculum size of 10% of the volume of the seed culture medium, culturing again at 28 ℃ for 24 h, and performing the activation process to obtain fermentation strains, inoculating the fermentation strains with the OD600 value of 8-10 into the fermentation culture medium according to the inoculum size of 0.8% -1.5%, and fermenting and culturing for 120 h under the conditions of the temperature of 25 ℃ and the rotating speed of 170 rpm; Or the seed culture medium comprises 45 g/L glucose, 2 g/L yeast powder and 15 g/L、MgCl 2 ·7H 2 O 4 g/L、Na 2 SO 4 15 g/L、KCl 1 g/L、NaCl 1 g/L、MgSO 4 ·7H 2 O 5 g/L、KH 2 PO 4 3 g/L, sodium glutamate as water; Or the fermentation medium comprises glucose 90 g/L, yeast powder 4 g/L, sodium glutamate 15 g/L、MgCl 2 ·7H 2 O 4 g/L、Na 2 SO 4 15 g/L、KCl 1 g/L、NaCl 1 g/L、MgSO 4 ·7H 2 O 5 g/L、KH H 2 PO 4 3 g/L and (NH 4 ） 2 SO 4 4 g/L, and water as solvent; or inoculating the strain for fermentation into a fermentation medium according to the inoculation amount of 1% for fermentation culture.

Description

Engineering schizochytrium limacinum capable of producing palmitoleic acid in high yield, and construction method and application thereof Technical Field The invention belongs to the technical field of bioengineering and microbial fermentation, and particularly relates to engineering schizochytrium limacinum with high yield of palmitoleic acid, and a construction method and application thereof. Background Schizochytrium sp is an important industrial oleaginous microorganism, can efficiently accumulate intracellular grease and synthesize various fatty acid components, and has wide application value in the industrial production of functional lipids. Palmitoleic acid (Palmitoleic acid, C16:1) is a monounsaturated fatty acid with important physiological activity, and has higher application potential in the fields related to metabolic regulation and anti-inflammatory, so that the demand for high-content palmitoleic acid grease raw materials is growing. In Schizochytrium sp HX-308 systems in the prior art, lipid metabolism flux naturally and remarkably deviates to the synthesis of polyunsaturated fatty acids such as docosahexaenoic acid (DHA), so that the content of palmitoleic acid in grease is usually extremely low (generally lower than 1.5%), and the production requirement of palmitoleic acid as target functional lipid is difficult to meet. The existing method for improving the grease yield or fatty acid composition of schizochytrium is more, but has obvious technical bottleneck for specific enrichment of palmitoleic acid, particularly, the formation of palmitoleic acid depends on the fact that delta 9 desaturase converts C16:0 into C16:1, and under the endogenous background of schizochytrium, factors such as substrate preference, expression strength, gene dosage effect and the like of exogenous delta 9 desaturase often become keys for limiting the improvement amplitude and stability of palmitoleic acid. Therefore, how to construct the high-palmitoleic acid engineering schizochytrium through the systematic optimization of the gene element level is a technical problem to be solved in the field. Specifically, the following drawbacks exist in the prior art: 1. schizochytrium limacinum lipid metabolism is naturally biased towards synthesis of polyunsaturated fatty acids such as DHA, carbon flow is difficult to redirect to C16:1 effectively, so that the enrichment and promotion amplitude of palmitoleic acid is limited and the stability is insufficient. 2. The existing delta 9 desaturase has various sources but obvious substrate specificity difference, partial enzyme has low conversion efficiency to C16:0 or is not suitable for expression in schizochytrium, and the optimal gene source with high activity and high suitability is difficult to obtain. 3. Lack of systematic optimization strategies in terms of promoter selection and gene dose (copy number), common expression systems are either insufficiently strong or insufficiently copy number to form a significant dose effect, making it difficult to establish a sustained high conversion capacity in cells and to achieve a significant enrichment of palmitoleic acid. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides engineering schizochytrium limacinum with high yield of palmitoleic acid, and a construction method and application thereof. The technical scheme adopted for solving the technical problems is as follows: A high-yield engineering schizochytrium palmitoleic acid is prepared by introducing and expressing a heterologous delta 9 desaturase gene into the schizochytrium by a genetic engineering means, and carrying out gene source screening, promoter optimization and target gene copy number optimization on the heterologous delta 9 desaturase gene to enhance the capability of converting C16:0 into C16:1, thereby remarkably improving the content of palmitoleic acid (Palmitoleic acid, C16:1, POA) in schizochytrium palmitoleic acid grease. Further, the heterologous delta 9 desaturase gene is a gene DSM9DE derived from scenedesmus (Desmodesmus sp.), and the nucleotide sequence of the gene is shown as SEQ ID No. 3; Or the Schizochytrium comprises Schizochytrium Pot bacteria (Schizochytrium sp. HX-308). Further, the promoter optimization includes placing the heterologous Δ9 desaturase gene under the control of schizochytrium endogenous promoters to increase the expression intensity of the target gene in schizochytrium; the endogenous promoter is schizochytrium endogenous strong promoter P3626, and the nucleotide sequence of the endogenous promoter is shown as SEQ ID No. 6. Further, the target gene copy number optimization includes achieving 2-3 copy integration of the heterologous Δ9 desaturase gene in the schizochytrium genome to enhance gene dose effects and further increase palmitoleic acid content. Further, 2-3 copies integration is achieved by a resistance selection marker including the G418 resistance gene, bleomycin Zeocin