CN105219649A - A kind of method utilizing metabolic gene engineering to improve diatom fat content - Google Patents

Abstract

The invention provides a kind of method utilizing metabolic gene engineering to improve diatom fat content.Clone ocean produce oil diatom Phaeodactylum tricornutum (Phaeodactylum? tricornutum) GPAT gene cDNA; through authentication function in yeast GPAT gene mutation body; build diatom over-express vector; electrization or particle bombardment is utilized to import Phaeodactylum tricornutum cell; the engineering algae strain of triglyceride level (TAG) content raising is obtained through careless fourth phosphine screening, can through amplification culture for extracting microalgae grease.The target spot that GPAT in diatom regulates and controls as diatom lipid metabolism gene engineering by the present invention, adopts the method for metabolic engineering to improve diatom fat content, for the research of diatom grease provides new thinking and the starting point.

Description

A kind of method utilizing metabolic gene engineering to improve diatom fat content

Technical field

The invention belongs to genetically engineered field, relate to a kind of method utilizing metabolic gene engineering, improving diatom fat content by importing endogenous glycerol-3-phosphate acyltransferase gene.

Background technology

Biofuel (Biodiesel) refers to that the grease (being mainly triglyceride level) from organism forms monoalkyl fatty acid ester with alcohols (methyl alcohol or ethanol) through transesterification.The performance of biofuel is substantially suitable with petrifaction diesel, has the good feature of environmental protection and biological degradability, has become desirable renewable energy source most widely used, with fastest developing speed in the world.

The micro-algae of single celled eukaryotic is that the low of photoautotrophy type waits waterplant, and of a great variety, widely distributed, have photosynthetic efficiency high, growth is quick, the features such as biomass is large.Part is comparatively considerable from micro-algae kind fat content of Chlorophyta and Bacillariophyta, become the high-quality natural matter of preparation biofuel, compared with traditional oil tree (tung oil tree, Cortex jatrophae, Chinese pistache etc.), marine microalgae cultivation does not occupy cultivated land, do not consume fresh water, is the study hotspot of future biological diesel oil development.

At present, the production energy consumption of microalgae biodiesel and sky high cost are the key issues hindering its industrialization.Improve microdisk electrode technology, optimize produce oil condition and micro-algae product separation method, particularly improve the oil-producing capacity of micro-algae self, it is the important channel reducing preparation cost, improve the seed output and quality of biofuel, wherein, very crucial through the high-quality algae kind that screening, mutagenesis or genetically engineered directive breeding acquisition oleaginousness are high.

Micro-algae is substantially identical with the lipid route of synthesis of higher plant.In plastid, the precursor of lipid acid synthesis is acetyl-CoA, and two acetyl-CoA synthesize malonyl--CoA under the katalysis of acetyl-CoA carboxylase.Then; with malonyl--CoA for substrate; under fatty acid synthetase system (FAS) catalysis, carry out continuous print polyreaction, circulate all to increase the mode synthesizing acyl carbochain of two carbon at every turn, under the catalysis of thioesterase, finally generate the saturated fatty acid of 16 to 18 carbon.Free fatty acids enters in kytoplasm by the transhipment of plastid film; mostly exist with acyl-CoA form; catalysis through three kinds of different acyl transferring enzymes final synthetic glycerine three ester; these three kinds of enzymes are all positioned on endoplasmic reticulum; may be defined as triglyceride level assembling enzyme; be GPAT, lysophosphatidate acyltransferase and Diacrylglycerol acyl transferase respectively, they act on Sn-1, Sn-2 and Sn-3 position of glycerol backbone respectively.

Plant lipid route of synthesis resolve to the target spot that lipid metabolism gene engineering provides genetic manipulation.Wherein, the acetyl-CoA carboxylase of initial step and the Diacrylglycerol acyl transferase of final step are modal target spots, adopt strategy to be by genetically engineered, realize the process LAN of homology or allos said gene, the final content increasing triglyceride level.

Acetyl-CoA carboxylase is lipid acid from the beginning biosynthetic crucial rate-limiting enzyme, and controllable carbon flows to into fatty acid biosynthetic pathway.Research in rape and potato shows, channel genes effectively can improve content of triglyceride, but does not succeed in marine diatom.The substrate diacylglycerol of Diacrylglycerol acyl transferase has two outlets: synthetic phospholipid or triglyceride level, and therefore, Diacrylglycerol acyl transferase is also key enzyme and the rate-limiting enzyme of synthetic glycerine three ester.Higher plant and the engineering research of microalgae grease metabolic gene show, import Diacrylglycerol acyl transferase gene and make it process LAN in cell, can cause diacylglycerol more synthetic glycerines three ester.

Because upper diatom of evolving has notable difference compared with higher plant, very likely there is special oil synthesis accumulation regulatory mechanism, such as, recent studies have found that, result in the oil and fat accumulation of frustule in diatom from the carbon stream of cell glycolysis-and branched-amino acid degradation, this and higher plant are obviously different.Therefore, necessary for the enzyme coding gene in diatom fat metabolic approach, find the brand-new target spot being applicable to diatom lipid metabolism gene Engineering operation.

Summary of the invention

For the problems referred to above, the object of this invention is to provide a kind of method utilizing metabolic gene engineering, improving diatom fat content by importing endogenous glycerol-3-phosphate acyltransferase gene.

For achieving the above object; technical solution of the present invention is as follows: a kind of method utilizing metabolic gene engineering to improve diatom fat content; using the target spot that the GPAT in diatom regulates and controls as diatom lipid metabolism gene engineering; clone diatom GPAT gene; construction of expression vector; import frustule again, obtain the conversion algae strain of fat content lifting through screening and inspection.

Described method concrete steps are:

1) clone diatom GPAT gene, utilize cDNA sequence to build Yeast expression carrier;

2) reverse mutation of yeast GPAT mutant is utilized to verify this gene function;

3) build diatom expression vector, import frustule, through the conversion algae strain that screening and inspection acquisition fat content promote.

Described diatom is the produce oil marine diatom such as Phaeodactylum tricornutum (Phaeodactylumtricornutum), little ring algae (Cyclotellasp.).

Described diatom expression vector comprises the gene expression regulation elements such as diatom endogenesis promoter, terminator, and weedicide grass fourth phosphine resistant gene.

Described screening is by the flat board of transformant coating containing doses weedicide grass fourth phosphine, cultivates resistant transformants.

Compared with the conventional method, the present invention proposes a kind of new approaches, there is following beneficial effect:

1. Late Cambrian of the present invention; GPAT in diatom; the effect enzyme of the first step enzymatic reaction be namely combined with glycerine for free fatty acids; final accumulation synthesis for diatom triglyceride level is extremely important, can as the potential novel targets of diatom lipid metabolism gene engineering regulation and control.

2. the present invention is first by diatom GPAT channel genes diatom; successfully transformant can be obtained through screening; detect the integration finding to realize quiding gene; its process LAN can improve the fat content of diatom, proves the new way of the diatom lipid metabolism gene engineering regulation and control of a practical.

The present invention is by importing fat metabolic key gene---the GPAT gene in diatom source; make its process LAN; thus promote the combination of free fatty acids and glycerine; the engineering diatom algae strain that final acquisition content of triglyceride promotes; grease is extracted by carrying out amplification culture to the strain of engineering algae; thus adopt the method for metabolic engineering to improve diatom fat content, for the research of diatom grease provides new thinking and the starting point.

Accompanying drawing explanation

Fig. 1 is the clone (M:Marker of Phaeodactylum tricornutum GPAT gene cDNA sequence; 0: blank).

Fig. 2 is that yeast expression sets out carrier pYES2.

Fig. 3 is Yeast expression carrier pYGPAT1.

Fig. 4 is the PCR qualification result of yeast Y13037 positive colony.Wherein, 1-4 imports the Plasmid samples extracted in the positive strain of pYES2 plasmid in Y13037 bacterial strain; 5-8 imports the Plasmid samples extracted in the positive strain of pYGPAT1 plasmid in Y13037 bacterial strain.

Fig. 5 is the PCR qualification result of yeast Y15983 positive colony.Wherein, 1-8 imports the Plasmid samples extracted in the positive strain of pYGPAT1 plasmid in Y15983 bacterial strain; 9-14 imports the Plasmid samples extracted in the positive strain of pYES2 plasmid in Y13037 bacterial strain.

Fig. 6 is that yeast pYGPAT1 transformant Nile red fluorescent staining is observed.(left: wild-type yeast imports unloaded pYES2; Right: wild-type yeast imports pYGPAT1).

Fig. 7 is yeast pYGPAT1 transformant liquid chromatography TAG quantitative analysis (SY: wild-type yeast imports unloaded pYES2; SG: wild-type yeast imports pGPAT1; Y1-Y: Auxotrophie mutant Y13037 imports unloaded pYES2; Y1-G: Auxotrophie mutant Y13037 imports pGPAT1; Y2-Y: Auxotrophie mutant Y15983 imports unloaded pYES2; Y2-G: Auxotrophie mutant Y15983 imports pGPAT1).

Fig. 8 is diatom nuclear expression skeleton carrier pfcpA-MCS-fcpB-bar.

Fig. 9 is Phaeodactylum tricornutum GPAT gene overexpression carrier pfcpA-gpat1-fcpB-bar.

Figure 10 is the PCR detected result of Phaeodactylum tricornutum GPAT gene overexpression carrier pfcpA-gpat1-fcpB-bar, and band shows the fragment of the expection size amplified.

Figure 11 is that electrization transforms Phaeodactylum tricornutum and to fall (upper left: the blank of unconverted through the resistance algae that the screening of careless fourth phosphine obtains; Upper right: transformed expression vector pfcpA-gpat1-fcpB-bar)

Figure 12 is the result that picking grass fourth phosphine resistance list algae falls to proceeding to 24 orifice plates and carries out spreading cultivation.

Figure 13 is Phaeodactylum tricornutum transformant PCR detected result.

Figure 14 is Phaeodactylum tricornutum transformant liquid chromatography TAG quantitative analysis (blank: wild-type is set out algae strain; Transformant: the engineering algae strain importing pfcpA-gpat1-fcpB-bar).

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read content of the present invention, these equivalent form of values fall within limited range of the present invention equally.

Embodiment 1, the clone of Phaeodactylum tricornutum (Phaeodactylumtricornutum) GPAT gene and functional verification

1. the clone of Phaeodactylum tricornutum GPAT gene cDNA sequence

In the centrifugal 5min of 6000r/min, collect frustule, proceed to precooling mortar and add liquid nitrogen grinding, extract RNA with RNApreppurePlantKit (TaKaRa, Dalian), detect RNA quality through 1.6% agarose gel electrophoresis, and measure OD ₂₃₀/ OD ₂₈₀, OD ₂₆₀/ OD ₂₈₀ratio, detect the concentration of RNA and purity.Use PrimeScript1 ^ststrandcDNASynethesisKit (TaKaRa, Dalian), carries out reverse transcription and prepares cDNA, and arrange reverse transcription system in 42 DEG C of temperature baths 30-60min, 95 DEG C of sex change 5min, 20 DEG C save backup.

Annotated the GPAT gene mRNA sequence (XM_002181618.1) of Phaeodactylum tricornutum in GenBank, but its function not yet carries out experimental verification.According to its nucleotide sequence, design 5 ' primers F the 1:5 '-ATGGCGATGCGAGCCGTCAA-3 ' started from initiator codon ATG and 3 ' primer the R1:5 '-TCACTTGGGTTTGTTTGGAT-3 ' terminating in terminator codon TAG respectively.Get cDNA as template, increase with F1/R1 primer pair, detect the product obtaining about 2kb through agarose gel electrophoresis, clip size meets expection (Fig. 1).Connect TA cloning vector, transformation of E. coli competent cell by after fragment recovery, after amoxicillin screening, picking resistant clones carries out order-checking inspection, under acquisition sequence is shown in:

The GPAT gene group sequence of Phaeodactylum tricornutum is 2302bp; comparison is known; its CDS sequence of cDNA sequence that amplification obtains is 1947bp; align analysis is carried out with genome sequence; find that its gene structure has 5 exons and 4 introns, montage position and size are in table 1.It should be noted that; the CDS sequence of the GPAT gene mRNA sequence (XM_002181618.1) of the Phaeodactylum tricornutum annotated in GenBank is only 1656bp; the sequence that the present invention obtains is than long 291bp, and the two exists obviously difference.

Table 1 Phaeodactylum tricornutum GPAT gene extron splicing site

2. the functional verification of Phaeodactylum tricornutum GPAT gene

2.1 Yeast expression carriers build

The GPAT gene clone carrier obtained with step 1 is for template; according to recording nucleotide sequence; respectively design band restriction enzyme enzyme recognition site 5 ' primers F 2 (5 '-AATCTAGAATGGCGATGCGAGCCGTC-3 '; band XbaI site) and 3 ' primer R2 (5 '-GCGGTACCTCACTTGGGTTTGTTTGGATC-3 '; band KpnI site); pMD18-T cloning vector is inserted through TA clone after pcr amplification; construct plasmid pMGPAT1, prove that sequence is correct through amplification order-checking.

Utilize restriction enzyme XbaI and KpnI digested plasmid pMGPAT1 and Yeast expression carrier pYES2 (Fig. 2) simultaneously; product is through agarose agar-agar electrophoresis; reclaim test kit by glue and reclaim the gene fragment and carrier pYES2 large fragment that enzyme cuts respectively; transformed competence colibacillus cell after connecting, obtains through screening, inspection the yeast expression recombinant plasmid pYGPAT1 (Fig. 3) inserting Phaeodactylum tricornutum GPAT gene C DS sequence.Picked clones, carries out pcr amplification qualification by pYES2 universal primer and checks order, and result shows that the complete CDS sequence of GPAT gene is entirely true.

2.2 yeast mutants bacterial strains transform and qualification

The two strain BY4742 yeast strains that sequence number is respectively Y13037 and Y15983 are bought from EUROSCARF; GPAT gene has two copies in Yeast genome; lay respectively at karyomit(e) YKL011 and YKR067 site; because the GPAT of its coding forms katalaze enzyme important in cell membrane phospholipid acid path; two genes knock out simultaneously and can cause lethal mutation; therefore two strain BY4742 yeast strains only have a gene to be knocked respectively, and in cell, GPAT is active in BY4742 wild-type.SCY62 is wild contrast type yeast, the equal uracil auxotrophy of three Accharomyces cerevisiaes, (0.67% without amino acid whose yeast nitrogen (YeastNitrogenBase) for the synthesis minimum medium (SC-Ura) that cannot lack at uridylic, 2% glucose, 0.074%DoSupplement (-Ura), pH nature, 115 DEG C of sterilizing 30min, solid medium adds agar powder 1.5%) middle growth (referring to table 2).

Used yeast genes type tested by table 2

By lithium acetate transformation method, by recombinant yeast expression vector pYGPAT1 and contrast empty carrier pYES2 transformed saccharomyces cerevisiae bacterial strain wild-type SCY62 and GPAT defective type Y13037 and Y15983 mutant strain respectively, the yeast list bacterium colony that picking SC-Ura solid medium grows, plasmid is extracted respectively after enlarged culturing, with T7 (5 '-TAATACGACTCACTATAGGG-3 ') and Pyes2.R (5 '-TCGGTTAGAGCGGATGTG-3 ') for amplimer, to yeast-positive, clone carries out PCR qualification, obtain and confirm to transform successful positive yeast clone (Fig. 4, Fig. 5).

2.3 yeast mutants positive transformants bacterial strain TAG content detection

Choose positive yeast bacterial strain, by semi-lactosi and the genetic expression of Low-temperature culture induction GPAT, the transgenic yeast of abduction delivering, after Nile red dyeing, uses fluorescent microscope, observes with the exciting light of about 540nm.Visible fluorescent orange after the phosphoric acid ester of visual field inner cell film and protein staining, and send intense fluorescence after the neutral fat dyeing such as TAG, in golden yellow.Observations shows, the visible golden yellow oil droplet of control group SCY62-pYES2, but also shows the orange cells without oil droplet; Become clear in the experimental group SCY62-pYGPAT1 visual field of abduction delivering PtGPAT1, all contain oil droplet in nearly all cell, entirety presents golden yellow (Fig. 6).

Further, the TAG content of liquid chromatography technology to each yeast strain is utilized to carry out quantitative assay.To collect after each yeast strain ferments, grind, take about 20mg in 15mL glass centrifuge tube, add Extraction solvent chloroform-methanol-water (1: 2: 0.8) 1.5mL, cell crushing instrument ultrasonication 2min, add 400 μ L chloroforms, whirlpool concussion 20s, add 400 μ L water whirlpool concussion 20s, centrifugal segregation upper strata, by chloroform layer constant volume in 1mL.Use Yi Lite liquid chromatograph, chromatographic column is silicagel column, 150 × 4.6mm, 5 μm; Mobile phase A is normal hexane-Virahol-glacial acetic acid (97: 3: 0.1v/v/v), and B is Virahol; Light scattering detector is drift tube 60 DEG C, N ₂: 3.5bar; gain: 6; the TAG quantified results of each yeast strain shows; no matter wild type yeast strain; or two strain defective type Wine brewing yeast strain Y13037, Y15983; the transformant importing PtGPAT1 gene improves 36%, 276%, 503% (Fig. 7) respectively compared with the TAG content accounting of control strain; prove that GPAT possesses phospho-glycerol acyltransferase activity, prompting GPAT gene can as the important target gene of Phaeodactylum tricornutum lipid metabolism gene engineering.

Embodiment 2: the electrization of Phaeodactylum tricornutum endogenous glycerol-3-phosphate acyltransferase gene imports and detects

1. Phaeodactylum tricornutum GPAT gene diatom over-express vector builds

With Yeast expression carrier pYGPAT1 for template; the primer pair F3 of design band restriction enzyme SacI recognition site (5 '-CGAGCTCATGGCGATGCGAGCC-3 ') and R3 (5 '-CGAGCTCTCACTTGGGTTTGTTTG-3 '); GPAT gene amplification is got off; insert MCS (multiple clone site) site in self-built diatom nuclear expression carrier pfcpA-MCS-fcpB-bar (Fig. 8) by SacI single endonuclease digestion, thus build diatom over-express vector pfcpA-gpat1-fcpB-bar (Fig. 9).Because be that SacI single endonuclease digestion inserts; in order to determine the direction of inserting; design the primers F 4 (5 '-AACCCCCACACGATTTTT-3 ') that one end is positioned carrier respectively; the other end is positioned the primer R4 (5 '-TTTTAGCCCAACCGCACT-3 ') of GPAT gene, proves that direction of insertion and sequence are all correctly (Figure 10) through amplification order-checking.

2. in Phaeodactylum tricornutum, the electrization of over-express vector imports and detects

Plasmid is little takes out pfcpA-gpat1-fcpB-bar carrier, utilizes the cutting of restriction enzyme NdeI enzyme for linearization plasmid, and after agarose gel electrophoresis reclaims, measuring final concentration is 0.13 μ g/ μ l.Use electrization to transform, unit type is the GenePulserXcell electric exciter that BioRad company produces.

Each electric shock reaction uses linearization plasmid pfcpA-gpat1-fcpB-bar totally 4 μ g and salmon sperm DNA 40 μ g.Shock parameters is: exponential wave, voltage 500V, electric capacity 25 μ F, resistance 400 Ω, and use electric shock cup is 2mm specification.The time that electric exciter exports is at about 5ms.

Phaeodactylum tricornutum with f/2 nutrient solution in 22 DEG C, light intensity 50 μm of olphotonsm ^-2s ^-1, cultivate under light dark period 12h:12h condition, before transforming, algae liquid concentration is adjusted to 4.0 × 10 ⁶individual cell/mL, each conversion reaction uses about 2.0 × 10 ⁹individual cell.Be coated with each solid plate about 6.0 × 10 ⁸individual cell, dull and stereotyped containing screening reagent weedicide grass fourth phosphine 60 μ g/L, flat board is placed in 22 DEG C, light intensity 50 μm of olphotonsm ^-2s ^-1, carry out cultivation screening under light dark period 12h:12h condition, resistance algae can be obtained and fall (Figure 11).The single resistance algae of picking falls access 24 orifice plate respectively, include the f/2 liquid medium adding careless fourth phosphine 40 μ g/L to carry out spread cultivation (Figure 12), the algae liquid spread cultivation is after collected by centrifugation, use sky root Plant Genome to extract test kit and extract genome DNA, design primers F 5 (5 '-AACCCCCACACGATTTTT-3 ') and R5 (5 '-TTTTAGCCCAACCGCACT-3 ') respectively to carry out PCR and detects and obtains positive findings, point out the gene of importing to integrate (Figure 13).Through collected by centrifugation frustule, carry out TAG assay by 2.3 steps of embodiment 1 through liquid chromatography technology, find that the TAG content of transformant algae strain improves about 10% (Figure 14) compared with the contrast algae strain of unconverted.

Be more than the description to the embodiment of the present invention, by the above-mentioned explanation to the disclosed embodiments, professional and technical personnel in the field realized or uses the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiments, General Principle as defined herein can without departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention can not be restricted to these embodiments shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.

Claims (9)

1. the method utilizing metabolic gene engineering to improve diatom fat content; it is characterized in that; using the target spot that the GPAT in diatom regulates and controls as diatom lipid metabolism gene engineering; clone diatom GPAT gene; construction of expression vector; import frustule again, obtain the conversion algae strain of fat content lifting through screening and inspection.

2., by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 1, it is characterized in that, described method concrete steps are:

1) clone diatom GPAT gene, utilize cDNA sequence to build Yeast expression carrier;

2) reverse mutation of yeast GPAT mutant is utilized to verify this gene function;

3) build diatom expression vector, import frustule, through the conversion algae strain that screening and inspection acquisition fat content promote.

3. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 2; it is characterized in that, the primer of described clone diatom GPAT gene is F1:5 '-ATGGCGATGCGAGCCGTCAA-3 ' and R1:5 '-TCACTTGGGTTTGTTTGGAT-3 '.

4. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 2, it is characterized in that, primer during described structure Yeast expression carrier is F2:5 '-AATCTAGAATGGCGATGCGAGCCGTC-3 ', band XbaI site and R2:5 '-GCGGTACCTCACTTGGGTTTGTTTGGATC-3 ', band KpnI site.

5. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 2, it is characterized in that, primer during described structure diatom expression vector is F3:5 '-CGAGCTCATGGCGATGCGAGCC-3 ' and R3:5 '-CGAGCTCTCACTTGGGTTTGTTTG-3 '.

6., by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 2, it is characterized in that, the introduction method of described importing frustule is electrization or particle bombardment.

7. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 2, it is characterized in that, the conversion algae strain of the engineering diatom obtained is for amplification culture and extract grease.

8. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 1, it is characterized in that, described diatom expression vector comprises diatom endogenesis promoter, terminator as gene expression regulation element, and weedicide grass fourth phosphine resistant gene.

9. by the method utilizing metabolic gene engineering to improve diatom fat content according to claim 1, it is characterized in that, described diatom is produce oil marine diatom Phaeodactylum tricornutum (Phaeodactylumtricornutum), little ring algae (Cyclotellasp.).