CN104031902A - Oleaginous yeast ATP citrate lyase (ACL) and application thereof - Google Patents

Abstract

The invention relates to ACL separated from Rhodosporidium toruloides and a coding nucleotide sequence and a recombinant vector thereof. According to the invention, Rhodosporidium toruloides transcriptome sequencing results are used, the total DNA of a Rhodosporidium toruloides strain CGMCC2.1389 is utilized as a template, RT-PCR is employed, and Rt-ACL is separated; the cDNA sequence of Rt-ACL is shown in SEQ ID No. 1, and the amino acid sequence of Rt-ACL coding proteins is shown in SEQ ID No. 3. Through expression of the genomic DNA sequence of Rt-ACL in rhodotorula, oil content in cells of oleaginous yeast can be increased; and accumulation of oil content is realized through construction of the genomic DNA sequence of Rt-ACL in oleaginous bacteria or microalgae and expression of the cDNA originated from Rt-ACL in the oleaginous bacteria or microalgae. The Rt-ACL gene provided by the invention can be used for construction of microbial oil producing cells.

Description

A kind of oleaginous yeast ATP citrate lyase and its application

technical field

The invention relates to ATP citrate lyase ACL, specifically the cloning of the ATP citrate lyase gene isolated from Rhodosporidium toruloides, the construction of a recombinant vector and its application in genetic engineering research.

Background technique

International oil prices hit new highs repeatedly, and the era of low oil prices is gone forever. In order to ensure national oil security, ease the crisis of over-reliance on oil imports, and effectively reduce motor vehicle pollutant emissions, my country is vigorously promoting the development of biodiesel. Biodiesel mainly refers to fatty acid esters made from animal and vegetable oils through lipid exchange with methanol or ethanol under the action of a catalyst. However, the use of animal and vegetable oils and fats as raw materials has the problem of competing with people for food and land. Microorganisms have shown their outstanding characteristics this year because of their fast growth, basically independent of arable land, continuous production, and easy transformation.[ Zhao Zongbao. China Biotechnology Journal 2005, 25(2), 8-11]. Therefore, the use of microorganisms to produce oil to provide raw materials for biodiesel is one of the available alternatives today.

Some microorganisms in nature can accumulate oil exceeding 20% of their dry cell weight in the cells under extreme conditions (such as nitrogen source deficiency), mainly triglycerides, and these microorganisms are called oleaginous microorganisms. Mainly including bacteria, yeast, mold, algae and so on. The oleaginous yeasts include some strains in the genera Rhodotorula, Candida, Cryptococcus, Rhizopus, Trichosporon and Yarrowia [Ratledge C, Wynn J P. Adv Appl Microbiol 2002, 51, 1-51]. Oleaginous bacteria include certain strains in the genera Colwellia, Shewanella, Alteromonas, Pseudoalteromonas and Ferrimonas [Xiang Guangming. Grain and Oil 2008(6), 7-11]. Oleaginous microalgae include certain strains in the genera Chlorella, Nannoch l and Phaeodactylums [Zheng Hongli, Chinese Journal of Biotechnology 2009, 29, 110-116. Wang Lizhu, Microbiology Bulletin 2010, 37, 336-347. ]

The research on oleaginous yeast, especially Rhodotorula, is relatively scarce, and the mechanism of oil accumulation under specific conditions has not been elucidated at the molecular level. It is only biochemically clarified that oleaginous yeast may play a role in the regulation of lipid synthesis and metabolism. ATP citrate lyase (ACL) and malate dehydrogenase (ME) play key roles [Evans, C.T. and C. Ratledge. Canadian Journal of Microbiology 1985, 31:1000-1005.]. ACL exists in the cytoplasm and catalyzes the cleavage of citrate to acetyl-CoA and oxaloacetate. It is widely found in oleaginous yeasts and molds. This enzyme is absent in non-oleaginous yeasts (ie accumulating lipids not exceeding 20% of dry cell weight). In recent years, studies have shown that the activity of the ACL gene in Phaffia rhodozyme increases with the reduction of nitrogen sources under nitrogen-limited conditions, and the increase in its activity contributes to the accumulation of astaxanthin [Cipriano Chávez-Cabrera, Zoila R , et al. Appl Microbiol Biotechnol 2010, 85: 1953-1960.]. Another study showed that the expression of the ACL gene derived from itself in Mortierella alpina can greatly increase the fatty acid content [Patent No.: 200880113428.X].

However, so far no one has isolated the ACL gene of oleaginous yeast and identified its gene function, and no one has used this gene for oil production.

Contents of the invention

The purpose of the present invention is to provide an oleaginous yeast ATP citrate lyase protein, its coding nucleotide sequence and application. Specifically, it is the cloning and biological function analysis of a new ATP citrate lyase gene Rt-ACL derived from Rhodosporidium toruloides CGMCC2.1389, and the application of this gene in the field of constructing recombinant vectors and constructing genetically engineered strains .

For experimenting above-mentioned purpose, the technical scheme that the present invention adopts is:

First, using the results of Rhodosporidium toruloides transcriptome sequencing, primers for cDNA amplification were designed, and the total RNA of Rhodosporidium toruloides CGMCC2.1389 was used as a template to obtain the ATP citrate lyase gene Rt-ACL by RT-PCR. cDNA has the sequence shown in SEQ ID NO: 1 in the sequence table, its genomic DNA has the sequence shown in SEQ ID NO: 2 in the sequence table, and its amino acid has the sequence shown in SEQ ID NO: 3 in the sequence table. Then by introducing it in Saccharomyces cerevisiae, since there is no ACL gene in Saccharomyces cerevisiae, the activity of ACL can be detected [Beth L.Fatland, Jinshan Ke, et al.PlantPhysiology2002, 130:740-756. ]. Finally, by overexpressing the functionally determined gene in Rhodotorula, other oleaginous yeasts, other oleaginous bacteria, and microalgae, a strain with a large increase in oil content was obtained.

The oleaginous yeast ATP citrate lyase provided by the invention is called Rt-ACL (R.toruloides ATP-citrate lyase, Rhodosporidium toruloides ATP citrate lyase), derived from Rhodosporidium toruloides CGMCC2 .1389. The oleaginous yeast ATP citrate lyase is composed of the following (a) or (b) protein:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;

(b) The amino acid of SEQ ID NO: 3 undergoes one or several amino acid substitutions and/or deletions and/or additions, and is related to a protein derived from SEQ ID NO: 3 with activities related to lipid storage and transportation and metabolic regulation.

In order to facilitate the purification of the protein in (a), the amino-terminus or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 can be linked with the tags shown in Table 1.

Table 1 Tags and their sequences

Label Number of amino acid residues sequence Poly-Arg 5-6 (usually 5) RRRRR FLAG 8 DYKDDDDK Poly-His 2-10 (usually 6) HHHHHH C-myc 10 EQKLISEEDL Strep-tag II 8 WSHPQFEK Poly-Phe 11 FFFFFFFFFFFF

The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then conventional protein expression can be performed. The coding gene of the protein in the above (b) may be obtained by deleting one or several codons encoding amino acids in the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and/or carrying out one or several bases Base pair missense mutations, and/or link the coding sequences of the tags shown in Table 1 at its 5' end and 3' end.

The amino acid sequence and coding nucleotide sequence (rtacl, RHTO_03915.t1) of the oleaginous yeast ATP citrate lyase Rt-ACL both belong to the protection scope of the present invention. Preferably, the nucleotide sequence encoding the protein is the DNA molecule shown in SEQ ID NO: 1 in the sequence listing.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant strains containing the nucleotide sequence all belong to the protection scope of the present invention.

Therefore, the present invention also provides a recombinant vector, preferably a recombinant expression vector, comprising a nucleotide sequence encoding the oleaginous yeast ATP citrate lyase of the present invention; Recombinant cells with recombinant vectors.

Those skilled in the art should understand that the nucleotide sequence encoding oleaginous yeast ATP citrate lyase or the recombinant vector comprising the nucleotide sequence encoding oleaginous yeast ATP citrate lyase can be introduced into host cells according to the routine in the art Techniques are performed, for example, by conventional techniques such as transformation, transfection (eg, Agrobacterium-mediated transfection), or electroporation.

The invention also protects primers for amplifying said genes.

The existing expression vectors of oleaginous yeast, oleaginous bacteria and oleaginous microalgae can be used to construct the recombinant expression vector containing the coding gene (rtacl). Said also includes the gene recombination vector mediated by Agrobacterium tumefaciens and the linear vector which can be used for gene knockout and the like. The expression vector also includes the 3' untranslated region of the foreign gene, that is, includes the polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), torusporidium toruloides gene (such as the G3PDH gene of R. toruloides) the untranslated regions transcribed at the 3' end have similar functions. In order to facilitate the identification and screening of transgenic cell lines or recombinant strains, the vectors used can be modified, such as introducing genes encoding color-changing enzymes (such as green fluorescent protein) or luminescent compounds (such as GUS gene) that can be expressed in cells. , luciferase gene, etc.), antibiotic marker genes with resistance (such as kanamycin marker gene, bleomycin marker gene, hygromycin marker gene) or chemical resistance marker genes (such as herbicide resistance gene ), and nutritional screening marker genes (such as LEU2, URA3, URA3, LYS2, LYS5, MET15), etc.

Another object of the present invention is to provide a method for constructing a recombinant cell for oil production, the method comprising: introducing the nucleotide sequence encoding the oleaginous yeast ATP citrate lyase of the present invention into a host cell to obtain a recombinant cell for oil production . Using any vector capable of promoting the expression of foreign genes in oleaginous yeast, the rtacl provided by the present invention is introduced into oleaginous yeast cells to obtain oil-producing recombinant yeast. Or use any vector that can promote the expression of foreign genes in oleaginous bacteria, and introduce the rtacl provided by the present invention into oleaginous bacteria cells to obtain oil-producing recombinant bacteria. . Or use any vector that can promote the expression of exogenous genes in oleaginous microalgae, introduce the rtacl provided by the present invention into oleaginous microalgae, and obtain oil-producing recombinant microalgae strains.

Experiments have proved that the oleaginous yeast ATP citrate lyase provided by the invention can significantly increase the oil content of recombinant cells.

In summary, the present invention provides the following:

1. ATP citrate lyase, described ATP citrate lyase is by following (a) or (b) protein:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;

(b) The amino acid of SEQ ID NO: 3 undergoes one or several amino acid substitutions and/or deletions and/or additions, and is related to a protein derived from SEQ ID NO: 3 with activities related to lipid storage and transportation and metabolic regulation.

2. A nucleotide sequence encoding the ATP citrate lyase described in item 1.

3. The nucleotide sequence according to item 2, which is SEQ ID NO: 1.

4. A recombinant vector comprising the nucleotide sequence described in item 2 or item 3.

5. A recombinant cell, which is introduced with the recombinant vector described in Item 4.

6. A method for constructing a recombinant cell for oil production, the method comprising: introducing the nucleotide sequence described in item 2 or item 3 into a host cell to obtain a recombinant cell for oil production.

7. The method according to item 6, wherein the nucleotide sequence described in item 2 or 3 is introduced by transforming a host cell with the recombinant vector described in item 4.

8. The method according to item 6, wherein the host cell is selected from yeast, microalgae, Rhodococcus or Escherichia coli.

9. The method according to item 8, wherein the yeast is selected from Saccharomyces cerevisiae or R. toruloides, the microalgae are selected from Chlamydomonas or Synechococcus, the Rhodococcus is R. opacus.

10. The method according to item 9, wherein the Chlamydomonas is C. reinhardtii.

Description of sequence listing

SEQ ID NO: 1 ATP citrate lyase gene Rt-ACL (cDNA sequence)

SEQ ID NO: Genomic DNA sequence of 2ATP citrate lyase gene

SEQ ID NO: Amino acid sequence of 3ATP citrate lyase

Description of drawings

Figure 1 is the RT-PCR result of the amplified Rt-ACL cDNA, the amplified band is about 3.5kb. M is the molecular weight standard, and lane 1 is the amplification product of R.toruloides Rt-ACL cDNA.

Figure 2 shows the analysis results of multiple alignments between Rt-ACL cDNA and homologous sequences of ATP citrate lyases from other species.

Figure 3 is the RT-PCR result of the amplified Rt-ACL gDNA, the amplified band is about 4.3kb. M is the molecular weight standard, and lane 1 is the amplified product of R.toruloides Rt-ACL gDNA.

Fig. 4 is the construction of Rt-ACL cDNA expression vector pYX212-Rt-ACL in Saccharomyces cerevisiae.

Figure 5 is the result of measuring rtaclp activity in Saccharomyces cerevisiae. Each set of data is the average value of three recombinant strains. pYX212 is a strain of Saccharomyces cerevisiae expressing only pYX212 empty vector, and pYX212-ACL is a strain of Saccharomyces cerevisiae expressing pYX212-ACL.

Detailed ways

The following examples will help those of ordinary skill in the art to further understand the present invention, but do not limit the present invention in any form. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.

Rhodosporidium toruloides (R.toruloides) CGMCC2.1389: purchased from China General Microorganism Culture Collection Center (CGMCC, preservation number is CGMCC2.1389), from the Institute of Applied Microbiology, University of Tokyo (IFO8766), from IFO0559 and IFO0880 The diploid formed by mating is equivalent to CBS6016 or NBRC8766 or NRRL Y-6987.

Rhodosporidium toruloides (R. toruloides) ATCC10788: purchased from American Type Culture Collection (ATCC), derived from CBS-KNAW fungal biodiversity centre, equivalent to IFO0559 or JCM3792 or CCRC20306 or DBVPG6740 or IAM13469 or IGC4416 or MUCL30249 or NCYC921 Or NRRL Y-1091 or VKM Y-334 or PYCC4416.

The culture medium formulation and purposes involved in the following examples are as follows:

(1) YEPD medium: Yeast powder 10g/L, peptone 10g/L, glucose 20g/L, pH6.0, solid medium then add agar powder 15g/L; used for strain activation culture, seed liquid preparation and Short-term storage of strains.

(2) Nitrogen-limited medium: glucose 70g/L, yeast powder 0.75g/L, (NH4)2SO40.1g/L, KH2PO41.0g/L, MgSO4·7H2O1.5g/L, pH5.6, and supplemented 1% (V/V) trace element liquid (4.0g/L CaCl2 2H2O, 0.55g/L FeSO4 7H2O, 0.52g/L citric acid H2O, 0.10g/L ZnSO4 7H2O, 0.076g/L MnSO4·H2O and 100μl 18MH2SO4), used for strain oil accumulation and culture of lipid-rich bacteria.

Example 1: Multi-omics study of Rhodosporidium toruloides

Whole-genome de novo sequencing of Rhodosporidium toruloides (R.toruloides): R.toruloides ATCC10788 was cultured in YEPD medium at a temperature of 30°C, a rotational speed of 200 rpm, and a culture time of 48 hours. Collect bacteria, prepare genomic DNA according to the standard genomic DNA extraction method [Refined Molecular Biology Experiment Guide (Fourth Edition), Osper et al., Yan Ziying et al., Translated by Science Press], and entrust BGI to use solexa sequencing The instrument was analyzed, and finally 94 scaffolds (genome framework) were obtained, with a total genome length of 20.2Mb.

Transcriptome sequencing of Rhodosporidium toruloides (R. toruloides): culture R. toruloidesATCC10788 in a 2L fermenter (initial medium volume 1.7L), temperature 30°C, pH 5.6, and aeration rate 100L /h, the dissolved oxygen is maintained at 73% saturation (dissolved oxygen and stirring linkage), the YEPD medium flow acceleration rate is 0.828L/h (dilution rate is 0.53) (the working volume of the reactor is 1.7L). After about 10 working volumes, samples were taken, the bacteria were collected by centrifugation, and immediately frozen in liquid nitrogen, stored at -70°C, stored on dry ice and transported to Huada Gene (full name: Beijing Liuhe Huada Gene Technology Co., Ltd. Shenzhen Branch) . Total RNA was extracted by liquid nitrogen grinding-RNAiso method, and the mRNA-Seq8 Sample Preparation Kit (purchased from Illumina, San Diego, CA, USA) was used to prepare the RNA-seq sample library. For specific steps, refer to the product instructions. Afterwards, a 90bp paired-end sequence was obtained by Illumina HiSeq2000 sequencing.

Based on the whole genome de novo sequencing and transcriptome sequencing data of R. toruloides, combined with de novo gene prediction, a total of 8171 protein-coding genes were annotated. The R.toruloides local genome database and proteome database were established using Bioedit software. The naming rules of genes and proteins are as follows: according to the scaffold (genome framework) where the annotated genes are located, they are coded from large to small, and the first two letters of the two words of R.toruloides are uniformly added before the number, and .t1 is added after transcripts and proteins. and .t1p suffix. For example, the first coding gene from the 5' end of scaffold1 is named RHTO_00001, the corresponding transcript is RHTO_00001.t1, and the corresponding encoded protein sequence is RHTO_00001.t1p. The database contains 94 scaffolds (genome framework), 8171 coding genes and corresponding 8171 proteins.

Embodiment 2: Discovery of Rhodosporidium toruloides ACL gene

R. toruloides ATCC10788 was revived on YEPD solid medium, cultured upside down at 30°C for 48 hours, picked a single colony and inoculated in 50ml YEPD liquid medium (filled in a 250ml Erlenmeyer flask), and cultured at 30°C, 200rpm for 28h. The culture medium was transferred to a 2L continuous fermenter (working volume 1.7L) with automatic feeding at a ratio of 1:50 (V/V), and the medium was MM and MM-N respectively. The temperature of the chemostat culture is 30°C, the pH is automatically controlled at 5.6 by dropping 10.0M NaOH or 2M HCl, the rotation speed is maintained at 600rpm, the ventilation rate is 100L/h (about 0.98VVM), the dissolved oxygen is maintained at 85% saturation, and the dilution rate is 0.085. After about 15 working volumes, samples were collected. The samples were collected by centrifugation at 4°C and 8000rpm for 5 minutes. About 0.7g of wet bacteria could be collected per 45ml of culture. Both MM and MM-N bacteria samples were immediately frozen in liquid nitrogen, -70 Store at ℃. Bacterial samples were used for digital gene expression profile analysis and intracellular lipid content analysis, and culture supernatant was used for residual nitrogen analysis [Shen Hongwei, Jin Guojie, Hu Cuimin, Gong Zhiwei, Bai Fengwu, Zhao Zongbao. Journal of Bioengineering 2012, 28(1) , 57-65].

On the basis of Example 1, the bacteria samples were commissioned by BGI to extract RNA and then conduct digital gene expression profiling analysis of R. toruloides under nitrogen-limited and non-nitrogen-limited culture conditions. Samples were analyzed in triplicate, and data analysis was based on the accumulation of differentially expressed genes identified in the triplicate analyses.

The results showed that when R.toruloides np11 was cultured with MM and MM-N, there was a significant difference in the oil content. %, which is 22.8% higher than that under the condition of non-limiting nitrogen culture (MM culture). By detecting the concentration of nitrogen in the supernatant of the culture solution, it shows that the nitrogen concentration is 46.97mM under the condition of non-limiting nitrogen, while under the condition of nitrogen limitation (MM-N), the concentration of nitrogen has been lower than the detection limit, which indicates that the bacterium was cultivated using the A stable nitrogen-limited and non-nitrogen-limited environment.

After removing N bases and low-quality data, 119,192 (MM-N) and 92,644 (MM) differential clean Tags were obtained by digital gene expression profiling analysis. On the basis of these clean Tags, the following analysis is carried out, and the Tag positioning is matched to the reference gene to obtain the expression level of the gene. If the Tag that cannot be located and matched to the gene is located, its positioning is matched to the genome, and finally it cannot be A matching Tag is considered an unknown Tag. After filtering, 56,846 MM Tags (3,454,492 in total) and 60,010 MM-N Tags (3,600,362 in total) were obtained. After normalization processing and statistical testing, select the expression abundance ratio ≥ 2, control the false positive FDR ≤ 0.001, and finally get 1176 up-regulated genes, including three Tags with an expression abundance ratio ≥ 10: CATG CCGCACTCGCCGGCCCC, CATG CAGGACATCGAGGTCGT, and ATCT TCATCCAGCCCTTCA, derived from three transcripts with nucleotide sequence lengths of 3.8kb, 8.8kb and 3.4kb, respectively (numbers RHTO_02032.t1, RHTO_02139.t1, and RHTO_03915.t1 in the R. toruloides local transcriptome database) , respectively encoding fatty acid synthase beta subunit, alpha subunit and ATP citrate lyase protein. Among them, the expression level of RHTO_03915.t1 increases with the increase of lipid content in the lipid droplet, and its encoded amino acid sequence is shown in SEQ ID NO: 3, named rtaclp. The coding gene was named RT-ACL cDNA.

Example 3: Acquisition of Rhodosporidium toruloides AS2.1389RT-ACL genomic DNA

Design gene-specific primers according to the nucleotide sequence of Rt-ACL as follows:

ACL-orf-P1: 5'-ATGTCGGCAAAGCCTATCCGCGA-3'

ACL-orf-P2: 5'-CTACTGGCGCTGCTGGACGAGGAT-3'

Genomic DNA of Rhodosporidium toruloides AS2.1389 was extracted according to the method described in "Guide to Molecular Biology Experiments (Fourth Edition) (Osper et al., translated by Yan Ziying, et al., published by Science Press), UV/visible light spectrum Analysis showed that OD ₂₆₀ /OD ₂₈₀ of the sample was 1.88, indicating that the genomic DNA was of good quality. Freeze at -20°C for later use.

Using the genomic DNA of Rhodosporidium toruloides AS2.1389 as a template, using two primers ACL-orf-P1 and ACL-orf-P2, PCR amplification was performed according to conventional methods, and a PCR of about 4.3 kb was obtained (Fig. 3). The PCR amplification product was recovered according to the operation steps in Example 2, cloned into the pMD18-T vector, and sequenced. The sequencing results showed that the amplified nucleotide sequence was SEQ ID NO: 2, and the length was 4428bp. The nucleotide shown in SEQ ID NO: 2 was named RT-ACLgDNA, and the obtained positive recombinant plasmid was named pMD18T-RTACLg.

Example 4: Functional verification of Rhodosporidium toruloides RT-ACL gene

Design the following primers according to the nucleotide sequence of RT-ACL, be used to construct the vector that RT-ACL expresses in Saccharomyces cerevisiae (the underlined part of primer ACL-PYX-BamH I is the BamH I restriction site, primer ACL-PYX- The underlined part of HindIII is the HindIII restriction site):

ACL-PYX-BamH I: 5'-TCAG GGATCC AAAAATGTCGGCAAAGCCTATCCGCGA-3'

ACL-PYX-HindIII: 5'-ACTG AAGCTT CTAATGGTGATGGTGATGATGCTGGCGCTGCTGGACGAGG-3'

Step 1 Construction of RT-ACL yeast recombinant vector pYX212-Rt-ACL

ACL-PYX-BamH I and ACL-PYX-HindIII primers were used to amplify the fragment, and after double digestion with BamHI/HindIII, it was ligated into the same digested pYX212 (purchased from Biovector). The recombinant plasmid that released about 3.5kb insert fragment identified by BamHI/HindIII digestion was sent to TaKaRa for sequencing, and the correctly sequenced recombinant plasmid was named pYX212-ACL (Figure 4).

Step 2 Construction of RT-ACL recombinant yeast expression strain

pYX212-ACL was transformed into Saccharomyces cerevisiae INVSc1 (purchased from Invitrogen, genotype: MATa/α, his3Δ1/his3Δ1, leu2/leu2, trp1-289/trp1-289, ura3-52/ura3-52). The transformation method is as follows: a single yeast colony is inoculated in 5ml YPD medium (20g/L peptone, 10g/L yeast extract and 20g/L glucose, pH 6.0), cultured overnight; inoculated at 1:50 in 100ml fresh YPD medium At this time, the OD value is about 1.0-1.2, and the ice bath is 15 minutes; 4°C, 2000×g centrifugation for 10 minutes to collect the bacteria, suspend in 50ml ice-cold ddH ₂ O, and centrifuge at 2000×g for 10 minutes to collect the bacteria , then suspended in 20ml of ice-cold 1M sorbitol, centrifuged at 2000×g for 10min to collect the cells, poured out the solution, and suspended the cells in 0.5-1.0ml of ice-cold sorbitol, at this time the OD value was about 100-200. 50μl electroporation Add no more than 5 μl of plasmid DNA (0.5-1 μg) to the competent cells, place on ice for 10 minutes, transfer to an ice-cold electroporation cup, and transform with a voltage of 1500V. Under this condition, the electric shock time is about 5ms. After electric shock, add 1ml of ice-cold sorrel Alcohol, incubating on a shaker at 30°C for 2h, spread on SC-Uracil plate (20g/L glucose, 6.7g/L YNB W/O amino acids but with ammonium sulfate (basic nitrogen source of yeast without amino acid, containing ammonium sulfate, purchased from BD Dific, product number: 291920), 1.9g/L Yeast Synthetic Drop-out Medium Supplements (purchased from sigma, product number Y1501)), cultured at 30°C for 3-4 days until transformants grew out. Transformants were inoculated in SC-Ura liquid medium (0.67% YNB W/O amino acids but with ammonium sulfate (same as above), 2% glucose, 0.01% leucine, 0.01% tryptophan, 0.005% histidine) overnight , 3-4ml bacterial liquid was collected by centrifugation, the cells were broken with glass beads, the plasmid DNA was extracted and transformed into E.coli DH5a, the transformants were extracted with plasmids, and the recombinant bacteria were named INVSc1-pYX212-ACL after digestion with BamHI/HindIII to verify the correct source of the plasmid. .

Step 3 Preparation of Enzyme Solution

The correct recombinant strain INVSc1-pYX212-ACL was inoculated in 5ml SC-Ura liquid medium as a seed night and cultivated overnight. Then inoculated into 100ml SC-Ura liquid medium with an inoculation amount of 1:100 (v/v) until the OD value reached 4.2.

The supernatant was discarded from the bacterial solution and washed once with the same volume of distilled water. Suspend in 5 ml of extraction buffer (50 Mm Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM DTT, 1 mM PMSF), and crush the bacterial cells at 12 kPa using a Fischer crusher. Centrifuge at 20,000g for 15 minutes at 4°C and collect the supernatant. On a PD-10 chromatographic column filled with Shehadex G-25 (GEHEALTHCAREBIO-SCIENCES Company), use elution buffer (10mM sodium phosphate (pH7.4), 1mM MgCl ₂ , 0.1mM EDTA, 1mM DTT) to elute, elute Remove the liquid as the enzyme solution.

Step 4 ACL Activity Determination

The activity of ACL is determined by the amount of oxaloacetate it catalyzes the generation of citric acid, and the amount of NADH reduction in the reaction process of oxaloacetate and NADH catalyzed by malate dehydrogenase. The amount of change in NADH can be calculated from the change at _A340 .

The reaction formula is as follows:

(1) Citric acid + CoA + ATP → oxaloacetic acid + ADP + Pi + acetyl CoA

(2) Oxaloacetic acid + NADH → malic acid + NAD ⁺

The composition of the reaction solution is 10mM Tris-HCl (pH8.4), 10mM MgCl ₂ , 1mM DTT, 10mM ATP, 10mM citric acid, 0.2mM CoA, 6 units of malate dehydrogenase, 0.1mMNADH, 50μl enzyme solution, the total amount is 1ml. Addition of CoA started the reaction, which was carried out at 28°C.

The results are shown in Figure 5. It can be seen that only the strain expressing the empty vector pYX212 in Saccharomyces cerevisiae could not detect the activity of ACL, on the contrary, the strain expressing pYX212-ACL showed the activity of ACL, and it was significant. Therefore, we can determine that the expressed Rt-ACL protein can decompose citric acid into oxaloacetate and acetyl CoA in Saccharomyces cerevisiae, that is, it has the activity of ATP citrate lyase.

Embodiment 5: Construction of Rt-ACL expression recombinant Rhodosporidium toruloides strain

Design the following primers according to the nucleotide sequence of Rt-ACL (the underlined part of the primer FBA-F-SpeI is the SpeI restriction site, and the underlined part of the primer ACL-R-EcoRV is the EcoRV restriction site):

FBA-F-SpeI: 5'-ACTG ACTAGTCTCTGCTCTCGCTCGCTGTGGCTTG -3'

FBA-R: 5’-GATTCCACGCACCTTTGCCGACATTGTAGCTAGTTAGTGTTAGAAGTG-3’

ACL-F: 5'-CACTTCTAACACTAACTAGCTACAATGTCGGCAAAGGTGCGTGGAATC-3'

ACL-R-EcoRV: 5'-CCGT GATATC CTACTGGCGCTGCTGGACGAGG-3'

The primers were all in the 5' to 3' direction.

Using the cloning vector pMD18T-RTACLg constructed in Example 3 as a template, use the primers ACL-F and ACL-R-EcoRV to amplify the RT-ACL sequence, and use the Rhodosporidium toruloides AS2.1389 genome as a template and use the primer FBA -F-SpeI and FBA-R amplify the FBA promoter region. The two PCR fragments were fused using the method in the literature [Zhou YJ, Gao W, et al. J. Am. Chem. Soc2012, 134: 3234-3241.]. The obtained fusion fragment was digested by EcoRV/SpeI and ligated into pRH203 [Liu Y, Koh CM, Sun L, Hlaing MM, Du M, Peng N, Ji L. Appl Microbiol Biotechnol.2013Jan; 97( 2): 719-729] in vector. The ligation product was transformed into E.coli DH5a chemically competent cells, and the streptomycin-resistant transformants were selected for enrichment culture and plasmid extraction (Biyuntian Plasmid Small Extraction Kit, Cat. No.: D0003). The recombinant plasmid samples were sent to TaKaRa Company for sequencing, and the correctly constructed recombinant plasmid was named pRH203-RTACLg after sequencing verification.

pRH203-RTACLg was transformed into Agrobacterium AGL1 [Lazo GR, Stein PA, Ludwig RA. Biotechnology (NY). 1991, 9(10), 963-967.]. Spectinomycin-resistant transformants were picked and identified by PCR using primers ACL-F and ACL-R-EcoRV, and the positive recombinant was named AGL1/pRH203-RTACLg.

The recombinant Agrobacterium AGL1/pRH203-RTACLg was transformed into Rhodosporidium toruloides ATCC10788 according to the method of Yanbin Liu et al., and hygromycin-resistant transformants were picked for PCR identification. The positive recombinant was named R.toruloides ATCC10788RTACLg[ Liu Y, Koh CM, Sun L, Hlaing MM, Du M, Peng N, Ji L. Appl Microbiol Biotechnol. 2013 Jan;97(2):719-729].

The starting strain R. toruloides ATCC10788 and the recombinant R. toruloides ATCC10788RTACLg were inoculated into YEPD medium respectively, cultured at 30°C and 200 rpm for 3 days, and samples were taken every 24 hours for oil content analysis and fluorescence observation [Wu SG, Zhao X, Shen HW, Wang Q, Zhao ZK. Bioresour. Technol. 2011, 102(2), 1803-1807.]. It was found that under the YEPD culture conditions that are not conducive to oil accumulation, compared with the control strain ATCC10788, the intracellular oil content of recombinant Rhodosporidium toruloides ATCC10788RTACLg increased from 14% to 35%, an increase of 2.5 times. This proves that the RL-ACL gene can promote a significant increase in the oil content of R. toruloides.

Embodiment 6: Construction of Rt-ACL recombinant Rhodococcus opacus

Design the following primers according to the coding nucleotide sequence of Rt-ACL (the underlined part of the primer ACL-F-BamHI is the BamHI restriction site, and the underlined part of the primer ACL-R-XbaI is the XbaI restriction site):

ACL-F-BamHI: 5'-ATGC GGATCC ATGTCGGCAAAGGTGCGTGGAATC-3'

ACL-R-XbaI: 5'-TGAC TCTAGA CTACTGGCGCTGCTGGACGAGGAT-3'

The primers were all in the 5' to 3' direction.

Using the cloning vector pMD19T-RTACLc constructed in Example 2 as a template, the primers ACL-F-BamHI and ACL-R-XbaI were used to amplify the sequence of the RT-ACL coding region, and the PCR fragment was double digested with BamHI/XbaI, and the DNA was recovered Kit (purchased from Beijing Zhoudingguo, article number: NEP013-2), according to the supplier's recommended steps to purify the large fragment; pJAM2 plasmid [Triccas, J.A., T.Parish, W.J.Britton, and B.Giquel. FEMS Microbiol. Lett.1998, 167, 151-156] using BamHI/XbaI double digestion to recover large fragments. Mix the RT-ACL cDNA digested large fragment pJAM2 digested large fragment at a molar ratio of 5:1, use T4 DNA ligase to ligate and circularize, transform E.coli DH5a chemically competent cells, and select Amp-resistant transformants for growth. Bacterial culture, plasmid extraction (Biyuntian Plasmid Small Extraction Kit, Cat. No.: D0003). The recombinant plasmid samples were sent to TaKaRa Company for sequencing, and the correctly constructed recombinant plasmid was named pJAM-RL-ACLc after sequencing verification.

Transform the pJAM-RL-ACLc plasmid into competent cells of Rhodococcus opacus (R.opacus) ATCC51881 (purchased from ATCC: American Tissue Culture Colection) by electric shock transformation method, and pick the resistant transformants for PCR identification, and the PCR identification is positive The transformants were named PD630/pJAM-RL-ACLc [Kalscheuer R, M. and A Steinbüchel. Appl Microbiol Biotechnol 1999, 52, 508-515.].

Recombinant Rhodococcus turbide PD630/pJAM-RL-ACLc was inoculated in MSM medium containing 0.01g/l NH ₄ Cl and 10g/l sodium gluconate [Schlegel H G, H.Kaltwasser, and G.Gottschalk.Arch.Mikrobiol. 1961, 38, 209-222.], 28°C, 200rpm, cultivated for 24 hours, took samples every 6 hours, and kept them for oil content analysis [Jan Marc Horst Robenek, Alexander Steinbüchel Appl. Environ. Microbiol. 2006, 72(10), 6743.]. It was found that, compared with the control strain R. opacus PD630, the intracellular lipid content of the recombinant Rhodococcus opacus R. opacus PD630/pJAM-RL-ACLc increased from 30% to 74%. It was proved that the RL-ACL gene can promote a significant increase in the lipid content of Rhodococcus opacus.

Embodiment 7: Construction of Rt-ACL recombinant Chlamydomonas reinhardtii

Design the following primers according to the nucleotide sequence of rldp1 (the underlined part of the primer ACL-F-XbaI is the XbaI restriction site, and the underlined part of the primer ACL-R-NotI is the NotI restriction site):

ACL-F-XbaI: 5'-ATGC TCTAGA ATGTCGGCAAAGGTGCGTGGAATC-3'

ACL-R-NotI: 5'-TGAC GCGGCCGCCTACTGGCGCTGCTGGACGAGGAT -3'

The primers were all in the 5' to 3' direction.

Using the cloning vector pMD19T-RTACLc constructed in Example 2 as a template, primers ACL-F-XbaI and ACL-R-NotI were used to amplify the sequence of the RT-ACL coding region, and the PCR fragment was double digested with XbaI/NotI and ligated into the same Double-digested pChlamy_1 (purchased from Invitrogen, article number: A14258) vector (recovering large fragments for ligation) was transformed into E.coli DH5a chemically competent cells, and Amp-resistant transformants were selected for enrichment culture and plasmid extraction ( Biyuntian plasmid mini-extraction kit, item number: D0003). The recombinant plasmid samples were sent to TaKaRa Company for sequencing, and the correctly constructed recombinant plasmid was named pChlamy_1-RLACLc after sequencing verification. Use ScaI to linearize the recombinant plasmid pChlamy_1-RLDP1, and then use the DNA recovery kit (purchased from Beijing Zhoudingguo, article number: NEP013-2) to purify the PCR product according to the supplier's recommended steps (NEP013-2 instructions), -20 °C Save for later.

Chlamydomonas reinhardtii (C. reinhardtii) ATCC-50049 (purchased from ATCC: American Tissue Culture Colection) was cultured with GibcoR TAP medium (purchased from Invitrogen, product number: A1379801) until the OD750 value was 0.5, according to the kit instructions (product number: A14258) Prepare Chlamydomonas reinhardtii CC-849 electric shock competent cells, take 250 μl competent cells, add 2 μg of the aforementioned linearized pChlamy_1-RLACLc recombinant plasmid, incubate at room temperature for 5 min, according to the following parameters (voltage 600V, capacitance 50 μF, resistance Infinity), using Bio-RadGene Pulser II for electroporation transformation. Immediately after transformation, add 5ml TAP-40mM sucrose solution (TAP is purchased from Invitrogen, product number: A1379801, add 40mM sucrose), recover in 6-well culture plate at 28°C, 50μE m-2s-1 for 24h, then centrifuge at 2000rpm for 5min to collect Algae, spread on TAP-agar-Hygromycin plate (TAP purchased from Invitrogen, product number: A1379801, supplemented with 2% agar powder, 100 μg/ml hygromycin), cultured at 28°C, 50 μE m-2s-1 for 5 days , Pick hygromycin-resistant transformants, use ACL-F-XbaI and ACL-R-Not for PCR identification, and the positive clone is named CC-849/pChlamy_1-RLACLc.

Recombinant Chlamydomonas reinhardtii CC-849/pChlamy_1-RLACLc was inoculated with TAP nitrogen-limited medium (abbreviated as TAP-N: Tris base 20mM, MgSO4·7H2O0.83mM, CaCl2·2H2O0.45mM, K2HPO41.65mM, KH2PO41.05mM, glacial acetic acid 0.1% (V/V), trace element solution 1ml/l medium, trace element solution formula: Na2EDTA·2H2O5g/100ml, ZnSO4·7H2O2.2g/100ml, H3BO31.14g/100ml, MnCl2·4H2O0.5g/ 100ml, FeSO4 7H2O 0.5g/100ml, CoCl2 6H2O 0.16g/100ml, CuSO4 5H2O 0.16g/100ml, (NH4) 6Mo7O24 4H2O 0.11g/100ml) 28°C, continuous light 70μE m-2s-1 After culturing for 4 days, samples were taken every 24 hours for oil content analysis and fluorescence observation [Moellering EricR., Benning1Christoph, Eukaryotic Cell2010, 9, 97-106]. It was found that, compared with the control strain Chlamydomonas reinhardtii CC-849, the intracellular lipid content of the recombinant Chlamydomonas reinhardtii CC-849/pChlamy_1-RLACLc increased from 18% to 39%. It is proved that RL-ACL gene can promote the significant increase of oil content in Chlamydomonas reinhardtii.

It should be understood that while the invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that, without departing from the spirit and scope of the invention as defined by the appended claims, Various changes in form and details can be made therein, and any combination of various embodiments can be made.

Claims (10)

1. an ATP citrate-lyase, described ATP citrate-lyase is by following (a) or protein (b):

(a) protein being formed by the aminoacid sequence shown in SEQ ID NO:3;

(b) by the amino acid of SEQ ID NO:3 through one or several amino acid whose replacement and/or disappearance and/or interpolation and with have grease storing and metabolic regulation related activity by the derivative protein of SEQ ID NO:3.

Coding ATP citrate-lyase claimed in claim 1 nucleotide sequence.

3. nucleotide sequence according to claim 2, it is SEQ ID NO:1.

4. the recombinant vectors that comprises the nucleotide sequence of claim 2 or 3.

5. a reconstitution cell, described cell imports recombinant vectors claimed in claim 4.

6. build a method for grease production reconstitution cell, described method comprises: the nucleotide sequence described in claim 2 or 3 is imported in host cell, obtain grease production reconstitution cell.

7. method according to claim 6, wherein by importing the nucleotide sequence described in claim 2 or 3 with recombinant vectors transformed host cell claimed in claim 4.

8. method according to claim 6, wherein said host cell is selected from yeast, micro-algae, rhodococcus or intestinal bacteria.

9. method according to claim 8, wherein said yeast is selected from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or circle red winter spore yeast (R.toruloides), described micro-algae is selected from chlamydomonas or synechococcus, and described rhodococcus is muddy rhodococcus (R.opacus).

10. method according to claim 9, wherein said chlamydomonas is Chlamydomonas reinhardtii (C.reinhardtii).