CN111893049A - Schizochytrium limacinum genetic engineering strain for over-expressing squalene synthetase gene and construction method and application thereof - Google Patents
Schizochytrium limacinum genetic engineering strain for over-expressing squalene synthetase gene and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a schizochytrium genetic engineering strain for over-expressing squalene synthetase gene, a construction method and application thereof. The invention adopts Schizochytrium sp ATCC1381 as an original strain, constructs an overexpression vector in escherichia coli through a genetic engineering means, selects bleomycin as a screening resistance gene, uses a highly conserved sequence 18S rRNA of a eukaryote as a homology arm, and electrically transfers a linearized overexpression fragment into the Schizochytrium to obtain a genetic engineering strain with high squalene yield, thereby laying a theoretical foundation for producing squalene by microorganisms and realizing product industrialization.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a schizochytrium genetic engineering strain and a construction method and application thereof.
Background
Squalene is a lipid unsaponifiable substance composed of 6 isoprene units, and is widely used in the fields of foods, medical treatment, beauty treatment and the like because it has various effects of protecting against viruses, delaying aging, combating tumors and the like. The main source of squalene in the market at present is shark liver oil, and the content of vegetable oil is also rich. With the increasing market demand of squalene day by day, the natural resource limitation, the complex reaction of chemical synthesis method and the extremely low yield can not meet the demand of squalene, so the mode of producing squalene by using microbial resources has become the development trend in the future. The method draws great attention by virtue of the advantages of short period, simple operation, large production potential and the like. Thraustochytrids, microalgae, yeasts and yeast-like yeasts are the focus of recent research. Reports show that the yield of 18W-13a squalene of thraustochytrid bacteria screened from seawater can reach 1.29 +/-0.13 g/L; the optimized saccharomyces cerevisiae squalene yield reaches 11.00g/L, and is the highest yield of the known genetic engineering strains at present.
Genetic engineering is an important way for modifying strains, and improving the yield of squalene by means of genetic engineering is the center of gravity of current research. Reports show that the squalene yield reaches 52.1mg/L by introducing genes related to the mevalonic acid pathway of fungi into escherichia coli for expression through gene modification, and the major breakthrough of producing squalene by escherichia coli genetic engineering strains is realized; in addition, the overexpression of the gene of the acyltransferase in the saccharomyces cerevisiae improves the oil content and provides more storage space for squalene.
Schizochytrium belongs to the genus Thraustochytrium, a marine fungus, and is well known for its ability to produce unsaturated fatty acids in high yields. The schizochytrium limacinum has the advantages of high growth speed, short fermentation period and mechanical stirring resistance, and is a food safety bacterium. Squalene is an intermediate product in the synthesis of sterol compounds in the schizochytrium mevalonate pathway, and two farnesyl pyrophosphate heads are condensed and reduced by NADPH to squalene, a hydrocarbon containing 30 carbons, under the catalysis of farnesyl transferase or squalene synthase. The oil content of the schizochytrium limacinum accounts for more than 50% of the dry weight of the cells, and a good storage place is provided for squalene. Until now, no method for producing squalene by modifying schizochytrium through genetic engineering technology exists.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a gene engineering strain of schizochytrium limacinum for over-expressing squalene synthetase gene and a construction method and application thereof. The invention takes Schizochytrium sp.ATCC1381 as a receptor strain, and improves the yield of squalene in recombinant bacteria by over-expressing squalene synthetase gene (SQS), which has important significance for the basic theory research and product development of Schizochytrium in the future.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a Schizochytrium limacinum genetic engineering strain for over-expressing squalene synthetase gene is constructed by selecting oil-producing Schizochytrium sp.ATCC1381 as a material, and contains squalene synthetase gene SQS derived from Schizochytrium sp.ATCC1381.
The squalene synthetase gene (SQS) in the strain is cloned in original Schizochytrium sp.ATCC1381, and the strain overexpresses the squalene synthetase gene (SQS), so that the gene copy number is increased, and the squalene yield is improved.
The second technical scheme adopted by the invention for solving the technical problems is as follows:
a construction method of a gene engineering strain of schizochytrium for over-expressing squalene synthetase genes comprises the following steps:
1) cloning a squalene synthetase gene SQS from Schizochytrium sp.ATCC1381, inserting the gene into a pBluzeo-18S plasmid by a gene homologous recombination technology, and constructing an overexpression vector pBluzeo-SQS-18S;
2) electrically transducing the linearized overexpression vector pBluzeo-SQS-18S into Schizochytrium sp.ATCC1381 to obtain the gene engineering strain of the schizochytrium for overexpressing the squalene synthetase gene SQS.
Further, in the step 1), the construction method of the overexpression vector pBluzeo-SQS-18S comprises the following steps: according to the sequence information of a squalene synthetase gene SQS of Schizochytrium sp.ATCC1381, primers shown as SEQ ID No.1 and SEQ ID No.2 are designed, and a squalene synthetase gene SQS fragment is obtained by a PCR method; the squalene synthetase gene SQS fragment and the plasmid pBluzeo-18S are connected and transformed into Escherichia coli Trans110 competent cells, and the overexpression vector pBluzeo-SQS-18S is obtained.
The third technical scheme adopted by the invention for solving the technical problems is as follows:
a method for producing squalene by using a schizochytrium genetic engineering strain which overexpresses a squalene synthetase gene comprises the steps of inoculating the schizochytrium genetic engineering strain overexpressing the squalene synthetase gene into a seed culture medium for activation, and obtaining a strain for fermentation; inoculating the fermentation strain into a fermentation culture medium for fermentation culture; collecting thallus and extracting squalene.
Further, the fermentation strain is obtained by the following method: inoculating the schizochytrium genetic engineering strain for over-expressing squalene synthetase gene into a seed culture medium, and culturing to obtain a first-stage seed; and inoculating the primary seeds into a seed culture medium, and culturing to obtain secondary seeds serving as the fermentation strains.
Wherein the culture conditions are 27-29 ℃ and 150-250 r/min shaking culture.
Further, the method for collecting the thallus and extracting the squalene comprises the following steps:
1) adding 11-13 mol/L hydrochloric acid into the fermentation liquid after the fermentation culture is finished, and carrying out water bath at 64-66 ℃ for 0.5-1.5 h;
2) cooling to room temperature, adding n-hexane for extraction, and collecting an upper organic phase;
3) repeating the step 2) for a plurality of times, combining the organic phases, and volatilizing the solvent to obtain the total oil;
4) adding 9-11% potassium hydroxide ethanol solution into the total grease, and carrying out water bath saponification reaction at 64-66 ℃ for 1-2 h;
5) cooling to room temperature, adding n-hexane for extraction, and collecting an upper organic phase;
6) repeating the step 5) for a plurality of times until the organic phase becomes colorless, and combining the organic phases;
7) adding 9-11% ethanol water solution to elute the organic phase for a plurality of times until the eluent is neutral;
8) and combining the organic phases, and volatilizing the solvent to obtain a total unsaponifiable matter, namely squalene.
Further, the whole period of the fermentation culture may reach 168h, for example, 48h, 60h, 72h, 84h, 96h, 120h, 144h, or 168 h.
Inoculating the Schizochytrium limacinum genetic engineering strain for over-expressing squalene synthetase gene to a plate culture medium, culturing by a resistant plate, selecting a single colony, inoculating the single colony to a seed culture medium, and activating; the pH value of the plate culture medium is 6.4-6.6, and the plate culture medium comprises the following components in a formula ratio of 19-21 g: 9-11 g: 15-20 g: 49-51 mL: 1.5-2.5 mL of glucose, yeast powder, agar, 20 multiplied by component A and 500 multiplied by CaCl2(ii) a Wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as a solvent; the 500 XCaCl2The method comprises the following steps: CaCl2·2H2O84-86 g/L or anhydrous CaCl264-65 g/L, and the solvent is water.
Wherein the pH value of the seed culture medium is 6.4-6.6, and the seed culture medium comprises the following components in a formula ratio of 19-21 g: 9-11 g: 49-51 mL: 1.5-2.5 mL of glucose, yeast powder, 20 multiplied by component A and 500 multiplied by CaCl2(ii) a Wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as a solvent; the 500 XCaCl2The method comprises the following steps: CaCl2·2H2O84-86 g/L or anhydrous CaCl264～65g/L, and the solvent is water.
Wherein, the pH value of the fermentation medium is 6.4-6.6, and the fermentation medium comprises: 90g/L glucose, 5g/L corn starch, 5g/L peptone and 20 × component A50 mL/L; wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as solvent.
The invention adopts Schizochytrium sp ATCC1381 as an original strain, constructs an overexpression vector in escherichia coli through a genetic engineering means, selects bleomycin (Zeocin) as a screening resistance gene, and takes a highly conserved sequence 18S rRNA of eukaryote as a homology arm. The linearized overexpression segment is electrically transferred into schizochytrium to obtain a genetic engineering strain with high squalene yield, and theoretical basis is laid for producing squalene by microorganisms and product industrialization.
The equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.
All ranges recited herein include all point values within the range.
In the present invention,% is mass percentage and ratio is mass ratio, unless the general meaning in the field or the specific description is given. The units of said mass are for example grams, kilograms or tons.
In the invention, the room temperature, namely the normal environment temperature, can be 10-30 ℃.
Compared with the background technology, the technical scheme has the following advantages:
(1) the invention constructs a gene engineering strain of squalene synthetase overexpressed in schizochytrium by adopting an electrotransformation mode on the basis of a schizochytrium transformation system, and the obtained strain has genetic stability of multiple passages.
(2) According to the invention, a key enzyme for squalene synthesis is selected for overexpression on a metabolic pathway of a schizochytrium mevalonate pathway, so that the metabolism of the mevalonate pathway is enhanced, the lipid metabolic pathway is weakened, the accumulation amount of squalene in a growth period of the schizochytrium is increased, and a foundation is provided for industrial synthesis of squalene by using the gene engineering strain.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a schematic diagram of a constructed E.coli squalene synthetase (SQS) overexpression vector.
Figure 2 is a schematic representation of a linear recombinant fragment transduced into Schizochytrium sp.
FIG. 3 is an agarose gel electrophoresis image of recombinant strain identification. Performing agarose gel electrophoresis analysis, wherein M is marker; lane 1 is a schizochytrium wild-type control strain; lane 2 is the recombinant strain transformed with the vector pBluzeo-SQS-18S.
FIG. 4 is a graph comparing the squalene yield of wild type strain of Schizochytrium sp.ATCCC 1381 and engineering strain over expressing squalene synthetase gene (SQS).
FIG. 5 is the result analysis diagram of Schizochytrium sp.ATCCC 1381 wild strain and gene engineering strain squalene synthetase gene (SQS) RT-qPCR.
Detailed Description
The present invention will be described in detail with reference to the following examples:
the media used in the following examples are as follows:
the pH value of the plate culture medium is 6.5, and the plate culture medium is prepared from 20g of glucose, 10g of yeast powder, 15-20 g of agar, 20 multiplied by 50mL of component A and 500 multiplied by CaCl 22 mL.
Per 250mL of 20 × component a the composition is: na (Na)2SO460g，MgSO410g，KH2PO45g，(NH4)2SO45g，K2SO43.25g, KCl 2.5g and water to 250 mL.
500 CaCl per 100mL2The composition of (A) is as follows: CaCl2·2H2O8.5 g or anhydrous CaCl26.418g, the volume of water is 100 mL.
First and secondThe pH of the seed culture medium of the seed-grade liquid is 6.5, and the seed-grade liquid is prepared from 20g of glucose, 10g of yeast powder, 20 × 50mL of component A and 500 × CaCl 22 mL.
The pH of the basal medium (i.e., fermentation medium) was 6.5, and each liter of the fermentation medium contained 90g/L glucose, 5g/L corn steep liquor, 5g/L peptone and 20X 50mL of component A.
Example 1 cloning of Sqs
According to the sequence information of squalene synthetase gene (SQS) of Schizochytrium limacinum, primers P1 and P2 shown as SEQ ID No.1 and SEQ ID No.2 are designed, underlined parts are enzyme cutting sites NheI and XbaI respectively, Schizochytrium sp.ATCC1381 genome is used as a template, and the squalene synthetase is amplified through PCR by using primers P1/P2 and Primerstar Hi-Fi polymerase, so as to obtain a squalene synthetase gene (SQS) fragment. The PCR procedure was: at 94 ℃ for 30s, at 60 ℃ for 30s, at 72 ℃ for 1.5min, for 34 cycles, and the PCR product was purified and verified by electrophoresis on a 1% agarose gel containing the purified product.
SEQ ID No.1 P1(sense)：GCGTCTAGAATGTTCTCCATGCTCACATT
SEQ ID No.2 P2(antisense)：GCGGCTAGCTTAGTCAGAGTGGGTTTGGCC
Example 2 construction of overexpression vector pBluzeo-SQS-18S
1. Enzyme digestion reaction
The overexpression vector plasmid pBluzeo-18S (plasmid source: purchased from Shanghai Bioengineering, Inc.) and the squalene synthetase gene (SQS) fragment of the PCR purified product were double-digested with restriction endonucleases NheI and XbaI, respectively, and gel recovered. Enzyme digestion system (50 μ L): 2 μ L NheI, 2 μ L XbaI, 5 μ L Loading Buffer, 20 μ L plasmid or PCR purified product, 21 μ L ddH2O, performing enzyme digestion in water bath at 37 ℃ for 2 h. And (5) carrying out electrophoresis recovery on 1% agarose gel on the enzyme digestion product.
2. Ligation reaction
The enzyme-cut squalene synthetase gene (SQS) fragment and the vector pBluzeo-18S fragment are connected by T4 ligase, and the connection is carried out for 12h at 16 ℃ to obtain the recombinant over-expression vector pBluzeo-SQS-18S. Ligation system (25 μ L): 2. mu.L of the target Gene fragment, 1. mu.L of the vectorAfter digestion, 2.5. mu.L ligase buffer, 19.5. mu.L ddH2O, 16 ℃ for 12 h.
3. The ligation product was transformed into E.coli Trans110 competent cells by the following method:
(1) taking 100 μ L of competent cells under sterile condition, adding ligation product, mixing, and standing on ice for 30 min.
(2) And (3) thermally shocking for 90s at 42 ℃, and rapidly placing on ice for 3-5 min.
(3) 900 μ L of LB medium was added and incubated at 37 ℃ and 150r/min for 1 h.
(4) 200. mu.L of the suspension was applied to LB plates containing 100. mu.g/mL of ampicillin. The cells were inverted and cultured overnight at 37 ℃.
And (3) selecting positive transformants, extracting plasmids, and obtaining a recombinant over-expression vector pBluzeo-SQS-18S, wherein the sequencing verification result shows that the connection is successful.
Example 3 construction of a Schizochytrium Gene engineering Strain overexpressing the Sqs Gene
1. Preparation of schizochytrium competent cells
(1) The activated Schizochytrium sp.ATCC1381 Schizochytrium single colony on the plate is picked to 50mL of seed culture medium, and is subjected to shake culture at 28 ℃ and 200r/min for 24 h.
(2) Transferring the strain to 50mL of seed culture medium according to the inoculum size of 4%, and performing shake culture at 28 ℃ and 200r/min for 24 h.
(3) 20mL of the bacterial solution was centrifuged at 4000rpm at room temperature for 2min, and the supernatant was discarded.
(4) The cells were resuspended in 25mL of pretreatment (20 mM pH 6.5 phosphate buffer containing 25mL of DTT) and shaken at 150rpm for 30min to loosen the cell walls.
(5) The cells were washed twice with 20mL of pre-chilled sterile water under centrifugation conditions: centrifuging at 4000rpm and 4 ℃ for 2 min.
(6) The thalli is washed twice by using 1M sterile precooled sorbitol solution, and the centrifugation conditions are as follows: centrifuging at 4000rpm and 4 ℃ for 2 min.
(7) The cells were resuspended in 200. mu.L of 1M sterile pre-cooled sorbitol solution and dispensed into 1.5mL sterile centrifuge tubes, 100. mu.L each, on ice for use.
2. Electro-transformation of Schizochytrium limacinum
(1) mu.L of the linearized recombinant overexpression vector pBluzeo-SQS-18S (about 5. mu.g) was added to 100. mu.L of schizochytrium competent cells, mixed well, transferred to a precooled electric rotor, and allowed to stand on ice for 30 min.
(2) Shock, 2KV, one pulse.
(3) Immediately adding 1mL of precooled seed culture medium containing 1M sorbitol into the electric rotor, uniformly mixing, and transferring to the seed culture medium containing 1M sorbitol.
(4) Culturing at 28 ℃ and 200rpm for 2-3 h.
(5) And taking a proper amount of bacterial liquid to coat a plate, and culturing for 2-4 days at 28 ℃.
3. Screening and identification of Schizochytrium limacinum genetic engineering strains overexpressing squalene synthetase gene (SQS)
(1) The colonies were picked and inoculated into seed medium containing 50mg/L bleomycin, and cultured at 28 ℃ for 24 hours at 200 rpm.
(2) The stable inheritance of the overexpression vector is ensured after 7 passages, and the experiment described in the step (1) is repeated in each passage.
(3) The stably inherited strain is a gene engineering strain phenotype of Schizochytrium limacinum which overexpresses squalene synthetase gene (SQS), and is preserved in a refrigerator at-80 ℃.
(4) Extracting Schizochytrium limacinum genome with overexpressed squalene synthetase gene (SQS), designing a pair of primers (shown as SEQ ID No.3 and SEQ ID No. 4) specifically combined with the bleomycin resistance gene for PCR verification:
SEQ ID No.3P3(sense)：CTTCAAAACACCCAAGCACAGCA
SEQ ID No.4P4(antisense)：GTGGACACGACCTCCGACCACTC
the result of agarose gel electrophoresis analysis of recombinant strain identification is shown in FIG. 3, and the electrophoresis result shows that the recombinant over-expression vector pBluzeo-SQS-18S is successfully inserted into the schizochytrium genome.
Example 4 determination of squalene content in Squalene synthetase Gene (SQS) engineered Strain of Schizochytrium limacinum
1. Culturing fermentation seeds: after the genetic engineering strain and the original schizochytrium limacinum strain are subjected to resistance plate culture, a single colony is picked out and inoculated into a 250mL conical flask (containing 50mL of seed culture medium), and the strain is subjected to shake cultivation at 28 ℃ and 200r/min for 24h to form a first-stage seed. Inoculating 2mL of the primary seed culture solution into a 250mL conical flask (containing 50mL of seed culture medium), and performing shake culture at 28 ℃ and 200r/min for 24h to obtain a secondary seed serving as a strain for fermentation.
2. And (3) shake flask fermentation culture: inoculating 4mL of the secondary seed culture solution into a 500mL conical flask (containing 100mL of fermentation medium), performing shake culture at 28 ℃ and 200r/min for 168h, and sampling every 24 h.
3. Collecting thallus to extract squalene, comprising the following steps:
(1) 100mL of 12mol/L hydrochloric acid is added into the fermentation liquor, and water bath is carried out for 1h at 65 ℃.
(2) Cooling to room temperature, adding 50mL of n-hexane, shaking for 10min, standing for 10min, and collecting an upper organic phase.
(3) And (3) repeating the step (2) twice, combining the organic phases, and carrying out rotary evaporation at 65 ℃ for 10min until n-hexane is completely volatilized to obtain the total grease.
(4) 50mL of 10% potassium hydroxide ethanol solution was added, and saponification was performed in a water bath at 65 ℃ for 1.5 hours.
(5) Cooling to room temperature, adding 50mL of n-hexane, shaking for 10min, standing for 10min, and collecting an upper organic phase.
(6) And (5) repeating the step (5) for 3 times until the eluted organic phase is colorless, and combining the organic phases.
(7) Adding 10% ethanol water solution to elute organic phase for 3 times, and determining effluent liquid to be neutral by pH test paper.
(8) And (3) carrying out rotary evaporation on the organic phase at 65 ℃ for 10min until n-hexane is completely volatilized to obtain a total unsaponifiable matter.
(9)5mL of chromatographically pure hexane redissolves unsaponifiables, and the content of squalene is analyzed by gas phase detection.
Example 5 RT-qPCR detection of the transcript level of the squalene synthetase Gene (SQS) in Positive transformants
Designing primers shown as SEQ ID No.5 to SEQ ID No.8 according to a squalene synthetase gene (SQS) sequence and an internal reference Actin sequence:
SEQ ID No.5 SQSF:TGTACTCGAGGAAGACAAGAAAGAC
SEQ ID No.6 SQSR:CACCAAAACCATGCAACGG
SEQ ID No.7 ActinF:TGTCCTCACGCTCAAGTACCCCAT
SEQ ID No.8 ActinR:GAAGGTCTCGAACATGATCTGGGTCAT
1. extracting total RNA of schizochytrium:
(1) the sample was transferred to a pre-cooled mortar, ground thoroughly, the powder transferred to a 1.5mL sterile tube, and 600 μ L of lysate (containing DTT) was added.
(2) Repeatedly blowing with pipette until no obvious precipitate appears, centrifuging at 12,000rpm and 4 deg.C for 5 min.
(3) The supernatant was taken up in 1.5mL RNase Free Tube.
(4) The gDNA Eraserin Column was mounted on a 2mL Collection Tube.
(5) The supernatant was transferred to gDNA Eraserin Column at 12,000rpm and centrifuged at room temperature for 1 min.
(6) Discard gDNA Eraserin Column and keep 2mL of the filtrate in the Collection Tube.
(7) To the filtrate was added one-half volume of anhydrous ethanol, the solution was mixed with a pipette, and the mixture (containing the precipitate) was immediately transferred to an RNAin Column (containing 2mL of Collection Tube) and centrifuged at 12,000rpm at room temperature for 1min, and the filtrate was discarded.
(8) 500. mu.L of Buffer RWA, 12,000rpm, was added and centrifuged at room temperature for 30 s.
(9) 600 μ L of Buffer RWB (with 100% ethanol added), 12,000rpm, centrifuged at room temperature for 30 s.
(10) And (5) repeating the step (9).
(11) The RNAin Column was mounted on a 2mL Collection Tube, 12,000rpm, and centrifuged at room temperature for 2 min.
(12) The RNase Free Collection Tub was placed on the RNase Column at 1.5ml, 50. mu.L of RNase Free water was added, and the mixture was allowed to stand at room temperature for 5min, 12,000rpm, and centrifuged at room temperature for 2 min.
2. RNA reverse transcription:
(1) reaction reagents were added to the PCR tube.
(2) After keeping the temperature at 65 ℃ for 5min, the mixture is rapidly cooled on ice.
(3) In the PCR tube, a reverse transcription reaction solution was prepared in a total amount of 20. mu.L according to Table 1.
(4) Reverse transcription was performed according to the conditions of Table 2.
TABLE 1 reverse transcription System
TABLE 2 reverse transcription conditions
3. Real-time fluorescent quantitative PCR:
qPCR A TransStart Top Green qPCR SuperMix (+ dyeI) was used. The reaction was added in eight tubes as described in Table 3. The reaction was carried out using a PCR apparatus using a relative quantification method, and the reaction procedure is shown in Table 4.
TABLE 3 qPCR reaction System
TABLE 4 qPCR program
The result shows that the gene engineering strain of the schizochytrium limacinum for over-expressing the squalene synthetase gene (SQS) obtained by the method has the genetic stability of multiple passages, and the qPCR result shows that the transcription level of the squalene synthetase gene (SQS) at different time points is obviously improved. The yield of squalene is greatly improved and is increased by 193% compared with a wild strain. The genetic engineering strain constructed by the method and the construction method lay a strong foundation for subsequent theoretical research and industrial production.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
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<213> Artificial Sequence (Artificial Sequence)
<400>1
gcgtctagaa tgttctccat gctcacatt 29
<210>2
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gcggctagct tagtcagagt gggtttggcc 30
<210>3
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cttcaaaaca cccaagcaca gca 23
<210>4
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gtggacacga cctccgacca ctc 23
<210>5
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tgtactcgag gaagacaaga aagac 25
<210>6
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
caccaaaacc atgcaacgg 19
<210>7
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
tgtcctcacg ctcaagtacc ccat 24
<210>8
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gaaggtctcg aacatgatct gggtcat 27
Claims (10)
1. A gene engineering strain of Schizochytrium limacinum for over-expressing squalene synthetase gene is characterized in that: the strain is constructed by selecting Schizochytrium sp.ATCC1381 as a material, and contains a squalene synthetase gene SQS from the Schizochytrium sp.ATCC1381.
2. A method for constructing a gene engineering strain of Schizochytrium limacinum for overexpressing a squalene synthetase gene as set forth in claim 1, wherein: the method comprises the following steps:
1) cloning a squalene synthetase gene SQS from Schizochytrium sp.ATCC1381, inserting the gene into a pBluzeo-18S plasmid, and constructing an overexpression vector pBluzeo-SQS-18S;
2) and (3) transforming Schizochytrium sp.ATCCC 1381 by using the over-expression vector pBluzeo-SQS-18S to obtain the gene engineering strain of the Schizochytrium for over-expressing the squalene synthetase gene SQS.
3. The construction method according to claim 1, characterized in that: in the step 1), the construction method of the overexpression vector pBluzeo-SQS-18S comprises the following steps: designing primers shown as SEQ ID No.1 and SEQ ID No.2 according to the sequence information of a squalene synthetase gene SQS of Schizochytrium sp.ATCC1381, and obtaining a squalene synthetase gene SQS fragment by a PCR method; the squalene synthetase gene SQS fragment and the plasmid pBluzeo-18S are connected and transformed into Escherichia coli Trans110 competent cells, and the overexpression vector pBluzeo-SQS-18S is obtained.
4. A method for producing squalene by using the Schizochytrium limacinum gene engineering strain overexpressing squalene synthetase gene as set forth in claim 1, which is characterized in that: inoculating the schizochytrium genetic engineering strain for over-expressing squalene synthetase gene to a seed culture medium for activation to obtain a strain for fermentation; inoculating the fermentation strain into a fermentation culture medium for fermentation culture; collecting thallus and extracting squalene.
5. The process for producing squalene according to claim 4, wherein: the fermentation strain is obtained by the following method: inoculating the schizochytrium genetic engineering strain for over-expressing squalene synthetase gene into a seed culture medium, and culturing to obtain a first-stage seed; and inoculating the primary seeds into a seed culture medium, and culturing to obtain secondary seeds serving as the fermentation strains.
6. The process for producing squalene according to claim 4 or 5, wherein: the culture conditions are 27-29 ℃ and 150-250 r/min shaking culture.
7. The process for producing squalene according to claim 4, wherein: the method for collecting the thallus and extracting the squalene comprises the following steps:
1) adding 11-13 mol/L hydrochloric acid into the fermentation liquid after the fermentation culture is finished, and carrying out water bath at 64-66 ℃ for 0.5-1.5 h;
2) cooling to room temperature, adding n-hexane for extraction, and collecting an upper organic phase;
3) repeating the step 2) for a plurality of times, combining the organic phases, and volatilizing the solvent to obtain the total oil;
4) adding 9-11% potassium hydroxide ethanol solution into the total grease, and carrying out water bath saponification reaction at 64-66 ℃ for 1-2 h;
5) cooling to room temperature, adding n-hexane for extraction, and collecting an upper organic phase;
6) repeating the step 5) for a plurality of times until the organic phase becomes colorless, and combining the organic phases;
7) adding 9-11% ethanol water solution to elute the organic phase for a plurality of times until the eluent is neutral;
8) and combining the organic phases, and volatilizing the solvent to obtain a total unsaponifiable matter, namely squalene.
8. The process for producing squalene according to claim 4, wherein: the pH value of the seed culture medium is 6.4-6.6, and the seed culture medium comprises the following components in a formula ratio of 19-21 g: 9-11 g: 49-51 mL: 1.5-2.5 mL of glucose, yeast powder, 20 multiplied by component A and 500 multiplied by CaCl2(ii) a Wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as a solvent; the 500 XCaCl2The method comprises the following steps: CaCl2·2H2O84-86 g/L or anhydrous CaCl264-65 g/L, and the solvent is water.
9. The process for producing squalene according to claim 4, wherein: the pH value of the fermentation medium is 6.4-6.6, and the fermentation medium comprises: 90g/L glucose, 5g/L corn starch, 5g/L peptone and 20 × component A50 mL/L; wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as solvent.
10. The process for producing squalene according to claim 4, wherein: inoculating the schizochytrium genetic engineering strain for over-expressing squalene synthetase gene to a plate culture medium, culturing by a resistant plate, selecting a single colony, inoculating the single colony to a seed culture medium, and activating; the pH value of the plate culture medium is 6.4-6.6, and the plate culture medium comprises the following components in a formula ratio of 19-21 g: 9-11 g: 15-20 g: 49-51 mL: 1.5-2.5 mL of glucose, yeast powder, agar, 20 multiplied by component A and 500 multiplied by CaCl2(ii) a Wherein the 20 x component a comprises: na (Na)2SO4238～242g/L，MgSO438～42g/L，KH2PO419～21g/L，(NH4)2SO419～21g/L，K2SO412-14 g/L, KCl 9-11 g/L and water as a solvent; the 500 XCaCl2The method comprises the following steps: CaCl2·2H2O84-86 g/L or anhydrous CaCl264-65 g/L, and the solvent is water.
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| CN114456957A (en) * | 2021-12-22 | 2022-05-10 | 南京师范大学 | Schizochytrium limacinum genetic engineering strain, construction method and application thereof |
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