CN102586129B - Produce oil rhodococcus Rhodococcus sp.RHA-MLDS knocking out MLDS gene and uses thereof - Google Patents

Abstract

The present invention relates to the oleaginous Rhodococcus Rhodococcus? which knocks out the MLDS gene? sp. RHA-MLDS and its uses. Specifically, the present invention relates to a Rhodococcus oleaginous bacterium Rhodococcus? sp.RHA-MLDS and its use, and related to the application of inhibitors of MLDS gene and/or MLDS protein in the production of biodiesel, MLDS gene and/or MLDS protein and protein expression products containing recombinant MLDS gene, MLDS gene or containing MLDS Use of the gene recombinant expression carrier composition in the preparation of medicines for preventing and/or treating metabolic diseases related to lipid metabolism and storage disorders. The invention provides a high-efficiency microbial strain for the production of biodiesel. At the same time, the findings of the present invention provide a drug candidate for the treatment of metabolic diseases related to lipid metabolism and storage disorders.

Description

Rhodococcus sp.RHA-MLDS with knockout MLDS gene and application thereof

technical field

The present invention relates to Rhodococcus sp. RHA-MLDS of MLDS gene knockout and application thereof, and relates to the application of inhibitors of MLDS gene and/or MLDS protein in the production of biodiesel, MLDS gene and/or MLDS protein and comprising Use of recombinant MLDS gene protein expression product, MLDS gene or the composition of recombinant expression vector containing MLDS gene in the preparation of medicines for preventing and/or treating metabolic diseases related to lipid metabolism and storage disorders.

Background technique

The shortage of energy and the pollution of the environment have seriously restricted the development of the world economy and the improvement of living standards. Therefore, it is imperative to develop new energy sources, especially sustainable and renewable clean bioenergy. From the fact that my country's investment in green and clean energy (34.6 billion U.S. dollars) in 2009 was higher than a quarter of the world's total investment and twice that of the United States, it can be seen that my country's demand and attention to green and clean energy . The energy conversion efficiency of microorganisms is much higher than that of other biological species. According to the research report of the US Department of Energy, the oil production of microalgae per unit area of land is 100 times that of ground plants [1]. Therefore, the use of microorganisms to produce bioenergy has become an important direction for human beings to develop sustainable and renewable energy. Among them, the use of microorganisms to produce biodiesel is the top priority [2].

In bacteria, triglycerides, the precursor to biodiesel, are stored in organelles called lipid droplets. Therefore, elucidating the relevant molecular mechanisms regulating the formation and dynamic changes of lipid droplets will greatly help to improve the yield of biodiesel and reduce the production cost. At the same time, a variety of metabolic diseases in the human body are closely related to the metabolism and storage of lipids [3]. So far, the formation mechanism of lipid droplets is still under hypothesis, and its molecular mechanism remains to be studied. Obviously, the determination of lipid droplet binding protein will provide powerful clues and basis for revealing the mechanism of lipid droplet formation. Although lipid droplet binding proteins in eukaryotic cells have been mostly identified [4-7], bacterial lipid droplet binding proteins are still blank. At the same time, the PAT family proteins that have a great effect on lipid droplets in animal cells have no homologous proteins in bacteria. Therefore, we selected Rhodococcus sp.RHA1[8] as the research material to analyze and study the related proteins of bacterial lipid droplets, especially the proteins that regulate the formation and size changes of lipid droplets. Rodococcussp.RHA1 was originally a strain isolated from pesticide-contaminated soil, which can utilize a variety of organic compounds as carbon sources, such as carbohydrates, steroids, aromatic compounds, nitrile compounds, and so on. Recent studies mainly focus on its biodegradation function, including nitrile, eugenol, sterolone, benzoate, etc. [9-11]. This bacterium has a strong ability to accumulate triglycerides in the body, a potentially renewable resource for the production of fatty acid methyl esters. Therefore, we choose this bacterium as the research material, which will not only help the development of biodiesel, but also provide a theoretical basis for the treatment of human metabolic diseases.

Contents of the invention

Therefore, the technical purpose of the present invention is to explore the related proteins of bacterial lipid droplets and explore the application of the related proteins in the production of biodiesel and the treatment of human metabolic diseases.

Therefore, the first aspect of the present invention relates to Rhodococcus oleaginousus Rhodococcussp.RHA-MLDS with MLDS gene knockout, which was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms on November 30, 2010, and its address is the People's Republic of China No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, its classification name is Rhodococcus sp., its preservation number is CGMCC No. 4379, and the NCBI accession number of the MLDS gene is 4218150.

The second aspect of the present invention relates to the application of Rhodococcus sp. RHA-MLDS with MLDS gene knockout in the production of biodiesel.

The third aspect of the present invention relates to a method for producing biodiesel using Rhodococcus oleaginum Rhodococcus sp. RHA-MLDS with MLDS gene knocked out.

The fourth aspect of the present invention relates to the application of inhibitors of MLDS gene and/or MLDS protein in the production of biodiesel, wherein the NCBI accession number of the MLDS gene is 4218150. Preferably, the inhibitor is siRNA and/or a specific antibody against MLDS protein, and the siRNA and/or specific antibody against MLDS protein can silence and/or reduce the expression of MLDS gene and neutralize MLDS protein.

The fifth aspect of the present invention relates to the use of MLDS gene and/or MLDS protein in the preparation of drugs for the prevention and/or treatment of metabolic diseases related to lipid metabolism and storage disorders, wherein the NCBI accession number of the MLDS gene is 4218150. Preferably, the disease is selected from obesity, fatty liver or atherosclerosis. Preferably, the drug is selected from recombinant MLDS gene protein expression products, MLDS genes, recombinant expression vectors comprising MLDS genes or host cells comprising MLDS gene recombinant expression vectors, preferably, said recombinant MLDS gene protein expression products are produced by prokaryotic expression system or eukaryotic expression system, preferably, the recombinant expression vector is a recombinant expression vector suitable for gene therapy.

The sixth aspect of the present invention relates to a composition comprising recombinant MLDS gene protein expression product, MLDS gene or a recombinant expression vector comprising MLDS gene in the preparation of a medicament for preventing and/or treating metabolic diseases related to lipid metabolism and storage disorders , wherein the NCBI accession number of the MLDS gene is 4218150. Preferably, the disease is selected from obesity, fatty liver or atherosclerosis.

In other words, the formation and dynamic changes of lipid droplets, the organelles that store triglycerides, are not only directly related to human metabolic diseases, but also directly determine the ability of organisms to synthesize biodiesel. Therefore, revealing the molecular mechanism and related genes that regulate the formation and dynamic changes of lipid droplets is of great significance for the prevention and treatment of metabolic diseases and the development of biodiesel. The present invention selects related genes by analyzing the genome and proteome of Rhodococcus oleaginous bacteria Rhodococcussp. Analyze and observe, and finally use other biochemical and molecular biology methods to analyze and study related strains, focusing on the determination of triglyceride content. The present invention finds that knocking out the gene ro2104 (NCBI accession number: 4218150) can significantly increase the size of the lipid droplet of Rhodococcus oleaginum, and at the same time significantly increase the triglyceride content of the bacteria. In addition, by using the expression vector JAM-egfp to form a fusion protein between green fluorescent protein and ro2104 and express it in Rhodococcus oleaginous, the localization of the protein in bacteria was also observed. The present invention found that the PD2104 protein is completely localized on lipid droplets. Therefore, ro2104 may be a protein that exists on lipid droplets and protects small lipid droplets from being fused into large lipid droplets. Therefore, the present invention names it as "MicroorganismLipidDropletSmall", "MLDS" for short. Since MLDS prevents the fusion of small lipid droplets into large lipid droplets, the lipid droplets in the Rhodococcus oleaginous bacteria Rhodococcussp. The bacterium has been preserved in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on November 30, 2010. Its address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, People's Republic of China, Institute of Microbiology, Chinese Academy of Sciences, The preservation number is CGMCCNo.4379, and its classification name is Rhodococcus (the Latin name is Rhodococcus sp.). Because the bacterium can make the fat droplet in the bacterium obviously bigger and the triglyceride content obviously increase, so it can be used for producing biodiesel. Since MLDS prevents the fusion of small lipid droplets into large lipid droplets, MLDS is a negative factor for the production of biodiesel, therefore, inhibitors of MLDS genes and/or MLDS proteins can promote related organisms (such as Rhodococcus oleaginous) cells The lipid droplets inside become larger and significantly increase the triglyceride content, that is, inhibitors of MLDS gene and/or MLDS protein can be used to produce biodiesel. The inhibitor of MLDS gene can be small interfering RNA (siRNA), microRNA (miRNA) that can specifically knock out or knock down the expression of MLDS gene, or other chemical substances that can reduce the expression of MLDS gene. Those skilled in the art are well aware of how to design siRNA specifically knocking out or knocking down the expression of a certain gene and how to screen other chemical substances that can reduce the expression of MLDS genes. At the same time, since the specific binding of antigen and antibody can neutralize the biological activity of the antigen, the antibody specifically binding to MLDS protein can also be used to produce biodiesel. Since MLDS can protect small lipid droplets from being fused into large lipid droplets, MLDS genes and/or MLDS proteins and compositions comprising MLDS genes and/or MLDS proteins can be used to prevent and/or treat lipid metabolism and Metabolic diseases associated with storage impairment, such diseases can be obesity, fatty liver or atherosclerosis. MLDS can be applied in the form of MLDS protein, MLDS gene or recombinant expression vector comprising MLDS gene.

The oleaginous Rhodococcus sp.RHA-MLDS with the MLDS gene knocked out has been preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on November 30, 2010, and its address is Beichen West Road, Chaoyang District, Beijing, People's Republic of China No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCCNo.4379, and its taxonomic name is Rhodococcus sp.

Rhodococcus oleaginum Rhodococcussp.RHA1 was preserved in the General Microorganism Center (CGMCC) of China Committee for the Collection of Microbial Cultures (CGMCC) on December 8, 2010. Its address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, People's Republic of China, Chinese Academy of Sciences Institute of Microbiology, its classification name is Rhodococcus sp., and its preservation number is CGMCCNo.4423.

The Escherichia coli pJAM2-egfp/S17-1 containing the pJAM2-eGFP expression plasmid was deposited in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on December 8, 2010, and its address is Beichen West, Chaoyang District, Beijing, People's Republic of China Road No. 1, Yard No. 3, Institute of Microbiology, Chinese Academy of Sciences, classified as Escherichia coli, and the preservation number is CGMCCNo.4422.

Description of drawings

Figure 1: The mutation strategy for MLDS gene knockout and the positions of PCR primers.

Figure 2: Electropherogram of the MLDS gene knockout construct, in which the PCR amplified fragment (lane 2) of the knockout strain is about 640 bp, which is the same as the constructed gene knockout plasmid (lane 1). However, the wild-type fragment was PCR-amplified with the same primers to about 1470bp (lane 3). In order to further confirm the accuracy of the deletion, we also detected the target gene and the sequences on both sides by PCR. The results showed that the sequences (271bp and 372bp) on both sides of the target gene were consistent with the wild-type strain (lane 4-7), while the target gene (831bp) was only present in the wild-type strain, and could not be detected by PCR in the deletion strain (lane 8 -9), indicating that the deletion strain has been constructed successfully. 1. For the positive control, the gene knockout plasmid is used as a template, and Primera and Primerd are used as primers. 2. Use the ro02104 deletion bacterial genome as a template, and Primera and Primerd as PCR primers. 3. For the negative control, use the wild-type strain genome as a template, and Primera and Primerd as PCR primers. 4-5, PCR results using wild-type and gene knockout strains as templates, and Primera and Primerb as primers. 6-7, PCR results using wild-type and gene knockout strains as templates, and Primerc and Primerd as primers. 8-9, PCR results using wild-type and gene knockout strains as templates, and Primerf and Primerr as primers.

Figure 3: Electron micrographs, in which negative staining electron microscopy (a and c) and transmission ultrathin section electron microscopy (b and d) were used to observe the size of lipid droplets in wild-type bacteria and MLDS gene knockout bacteria, and it was found that in gene knockout bacteria Medium lipid droplets are much larger than those of wild type.

Figure 4: TLC experiment results, wherein TLC showed that the triglyceride content in the MLDS gene deletion bacteria increased.

Figure 5: Localization of MLDS fusion protein detected by Western blot.

Figure 6: Fluorescence micrographs of localizing MLDS with bacteria expressing MLDS-GFP fusion protein, in which, a, bacteria transfected with empty vector JAM-egfp under bright field; b, bacteria transfected with empty vector JAM-egfp under fluorescent field Bacteria; c, bacteria transfected with the target gene JAM-MLDS-egfp under bright field; d, bacteria transfected with the target gene JAM-MLDS-egfp under fluorescent field (the bar in the figure is 5 μm).

Detailed ways

The present invention will be further illustrated by the following non-limiting examples below. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

Example

Example 1

Experimental materials and equipment

1. Strains and plasmids

Rhodococcus sp.RHA1 was donated by Professor Lindsay D.Eltis of the University of British Columbia (University of British Columbia) (it was preserved in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on December 8, 2010, and its address is People's Republic of China No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as Rhodococcus sp., and the preservation number is CGMCCNo.4423; Escherichia coli S17-1 (ATCC47055) and pK18mobsacB (ATCC87097) integrated plasmid Purchased from ATCC, U.S.; pJAM2-eGFP expression plasmid was Daniel from the University of Munster (University of Munster), Germany Gifted by the professor, the Escherichia coli pJAM2-egfp/S17-1 containing the pJAM2-eGFP expression plasmid was deposited in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on December 8, 2010, and its address is Chaoyang, Beijing, People's Republic of China No. 3, No. 1 Yard, Beichen West Road, Chinese Academy of Sciences, Institute of Microbiology, Chinese Academy of Sciences, the classification name is Escherichia coli, and the preservation number is CGMCCNo.4422; Escherichia coli DH5α was purchased from TIANGEN Company.

2. Restriction enzymes and other modifying enzymes

Restriction enzymes and ExTaq DNA polymerase for PCR were purchased from TaKaRa Company; T vectors for cloning were purchased from Genstar Company.

3. Other main reagents

Kanamycin sulfate (Kanamycin) and nalidixic acid (NalidixicAcid) were purchased from Sigma Company; yeast powder (YeastExtrant) and tryptone (Tryptone) were purchased from Oxide Company; prestained protein Marker was a product of Fermentas Company; plasmid mini-extraction reagents The kit and gel recovery kit were purchased from Tiangen Company; other reagents were of analytical grade.

4. Main instruments and equipment

The electron microscope (FEITecnaiG2F20) is a product of FEI Company in the United States; the model of the fluorescence microscope is Zeiss-AXIO-Imager; the electrotransformer is BIO-RADMicopulser165-2100; the electric constant temperature incubator (DPX-9002B-1) is Shanghai Fuma experimental equipment Co., Ltd. products; double-layer small-capacity full-temperature constant temperature shaker (ZHWY-2102C) is a product of Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; clean bench (SW-CJ-1FD) is a product of Suzhou Antai Air Technology Co., Ltd.; The instrument is a product of BIO-RAD Company of the United States; the ultrapure water preparation device is a product of PALL Company of the United States; the PCR thermal cycler (Dongsheng Long EDC-810) is a product of Beijing Dongsheng Innovation Biotechnology Co., Ltd.; the ultraviolet spectrophotometer (D -22331Hamburg) is a product of Eppendorf Company; centrifuges (microfuge16, centrifuge5417R and 3K30) are products of Sigma, Eppendorf and Beckman Company respectively; gel imaging system (AlphalmagerEP) is a product of AlphaInnotech Company of the United States; ultraviolet transmission reflectance analyzer (ZF-4) It is a product of Shanghai Changming Optical Electronic Instrument Factory; the electric heating constant temperature water tank (DK-8D) is a product of Shanghai Yiheng Technology Co., Ltd.; the high-pressure steam sterilizer (YXQ-LS-50SII) is a product of Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory ; Electrophoresis apparatus and electrophoresis tank are products of Beijing Liuyi Factory; microwave ovens are products of LG and Galanz; various refrigerators for experiments are products of Haier Company; miniature desktop vacuum pump (CL-802B) is Haimen Qilinbeier Instrument Manufacturing Co., Ltd. Product; 0.22 μm filter is a product of Sartorius, Germany.

experimental method

1 is used for the construction of the carrier of deletion ro02104 (MLDS) gene

1.1 Design of primers for ro02104 (MLDS) gene

Find the target gene RHA1MLDS and the sequences on both sides thereof (respectively ro02103, NCBI accession number is 4218149, and ro02105, NCBI accession number is 4218151) from the NCBI database, amplify its upstream 271bp segment and downstream 372bp segment by PCR method The sequence and primers were designed by primer5.0 software. Among them, the 271bp sequence is amplified by Primera (SEQ ID NO: 1) and Primerb (SEQ ID NO: 2), and the 5' end of Primera is an EcoRI restriction site. The 372bp sequence was amplified with Primerc (SEQ ID NO: 3) and Primerd (SEQ ID NO: 4) primers, and the 5' end of Primerd was a HindIII restriction site. There is a 19bp cohesive complementary region between the Primerb and Primerc primers, which is used to connect the AB segment and the CD segment together during PCR amplification. The primers used for detection are Primerf (SEQ ID NO:5) and Primerr (SEQ ID NO:6), which are used to amplify the target gene ro02104.

Primera5′-CG GAATTC GGGAACAGCAGGAGGACGAGGAAT-3′

Primerb5′- CCATCCACTAAACTTAAAC AGGCTTGATCCTTCACGC

TTTTCG-3'

Primerc5′ -GTTTAAGTTTAGTGGATGG TCTTGACGCTGTCGATGG

TCTTCT-3'

Primerd 5′-CAC AAGCTT TCTGAGTCAGATCGAGCGTGGGTT-3′

Primerf5′-ATGACTGACCAGAAGACCATCGACAG-3′

Primerr 5′-TCAAGCCTTCTTGGCCGGAGCAGC-3′

1.2 Amplification of MLDS gene

The AB segment was amplified with primera and primerb, the CD segment was amplified with primerc and primerd, and the template was the genome of wild-type RHA1.

The PCR system is:

The PCR conditions are:

Recycle AB segment and CD segment respectively

Use the recovered AB and CD segments as templates, use primera and primerd as primers to amplify AD segments, and the PCR conditions are the same as AB and CD segments.

1.3 Agarose gel electrophoresis of PCR products

Amplified products and ligated products were subjected to agarose gel electrophoresis, the concentration of agarose was 0.8%, and Goldview nucleic acid dye (purchased from Saibaisheng, catalog number HGV-1); The gels were analyzed and photographed.

1.4 Recovery of PCR products

The AD segment was recovered by gel, and the operation steps were carried out according to the instructions in the gel recovery kit (purchased from TIANGEN Company). The recovered AD segment was the DNA sequence in which the target gene MLDS was deleted in vitro.

2 Construction of plasmid integration vector

2.1 Connection of MLDS gene and T carrier

The recovered AD segment sequence was used as an exogenous fragment and connected to the T vector. The connection system was: 4 μL of the exogenous fragment, 1 μL of the T vector, and 5 μL of 2× premixed buffer. Ligation conditions were 16°C overnight.

2.2 Preparation of DH5α Competent Cells by CaCl ₂ Method

Pick a single colony of newly activated DH5α from the LB plate, inoculate it in 5 mL LB liquid medium, and cultivate it with shaking at 37°C for about 12 hours. Then the bacterial solution was inoculated in 100 mL LB liquid medium at a ratio of 1:100, and cultured at 37° C. with shaking at 220 rpm until the OD ₆₀₀ was 0.6. Place on ice for 20min. Centrifuge at 5000 rpm for 5 min at 4°C, discard the supernatant, and suck up the remaining medium with a suction tip. Add appropriate amount of MgCl ₂ -CaCl ₂ solution to wash twice. Add an appropriate amount of ice-cold 0.1mol/LCaCl ₂ to resuspend. Competent cells at this time can be transformed or stored at -80°C after adding glycerol.

2. Transformation of 3T vector ligation products

The ligation product was added to 50 μL DH5α competent cells, mixed gently, placed on ice for 20 hr, and heat-shocked (42° C., 90 sec). Place on ice for 2-3min, add 0.8mL LB medium, incubate at 37°C for 45min, resuspend with 200μl LB medium after centrifugation, take 100μL ampicillin-coated IPTG and X-gal plates, and culture overnight at 37°C.

2.4 Extraction of transformed colony plasmid

The plasmids extracted from the grown colonies were verified, and the plasmids were extracted using the Tiangen Plasmid Small Amount Rapid Extraction Kit. The extracted plasmids were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing results showed that the obtained recombinant gene sequence was completely correct.

2.5 Construction of PK18 plasmid integration vector containing MLDS gene

1) Perform EcoRI and HindIII double enzyme digestion on the positive clone with correct sequencing and the vector pK18mobsacB. The enzyme digestion system is as shown in the table below. The enzyme digestion temperature is 37°C and the enzyme digestion time is 4hr.

2) Run electrophoresis on the digested product and perform gel recovery according to the instructions of the gel recovery kit.

3) Ligate the recovered small fragments as exogenous fragments with the vector pK18mobsacB. The connection system is: 4 μL of exogenous fragment, 1 μL of T carrier, and 5 μL of 2× premixed buffer. Ligation conditions were 16°C overnight.

4) Transform the ligation product into competent cells S17-1 prepared by the CaCl ₂ method, and the transformation method is as in 2.3.

5) Apply kanamycin to a plate and culture overnight at 37°C. The grown colonies are positive colonies containing recombinant plasmids.

Knockout of MLDS Gene in 3RHA1 Strain Genome

3.1 Culture of S17-1 and RHA1 bacteria

Pick a single colony of Escherichia coli S17-1 into 10 mL of LB medium containing 50 μg/mL kanamycin, and culture it at 37°C. Pick a single colony of wild-type RHA1 to 10 mL of LB medium containing 30 μg/mL nalidixic acid, and culture at 30°C.

3.2 Combination of S17-1 strain and RHA1 strain

The donor bacterium S17-1 and the recipient bacterium wild-type RHA1 were cultured to the end of the bacterial growth index. Take 100 μL of each solid LB plate coated with no resistance and incubate overnight at 30°C. At this time, the plasmid will be transferred from the donor bacterium to the recipient bacterium RHA1.

3.3 Integration screening of MLDS mutant strains

Add 2 mL of liquid LB medium to the plate cultured overnight, scrape off the cells with a coating stick, dilute it to 10 ^-1 , 10 ^-2 , 10 ^-3 , and coat the NA+Kan plate (nalidixic acid+Kana Mycin plate), cultured at 30°C for 2-3 days. The bacteria that grow out are the first integration. Identify the first integration of the grown colonies: pick a single colony and put it on the NA+Kan plate and the NA+Kan+10% sucrose plate respectively, and pick the growth on the NA+Kan plate and the NA+Kan+10% sucrose plate. Colonies that cannot grow on the plate. Such a colony is the first successfully integrated bacterium. Streak the successfully integrated colony for the first time on a 10% sucrose plate and culture at 30°C for 2-3 days. The bacteria that grow out are the second integration. The grown colony is identified for the second integration: pick a single colony and place it on the NA+Kan plate and the NA plate respectively, and pick the colony that grows on the NA plate but cannot grow on the NA+Kan plate. Such colonies are bacteria that successfully integrated for the second time.

Molecular Biological Level Verification of 4MLDS Mutants

4.1 Genome extraction

The extraction method was carried out according to the Bacterial Genomic DNA Extraction Kit of TIANGEN Company

4.2 PCR identification of deletion mutants

The extracted genome was used as a template for PCR verification, the positive control was a recombinant plasmid constructed in vitro as a template, and the negative control was a wild-type RHA1 genome as a template. At the same time, the MLDS gene was amplified with primerf and primerr, and the deletion of the target gene was confirmed again.

5 Electron microscope to observe the morphology of bacteria

5.1 Preparation of electron microscope sample slices

Bacterial collection: 2 mL of bacterial cells were collected by centrifugation at 3800 g for 2 min, and the bacterial cells were washed twice with 1 mL of 0.1 MPBS; embedding: adding 5% gelatin (w/v), standing at room temperature for 1 hr, and standing at 4°C for 30 min, and then Cut the embedded block into small pieces ^of about 1mm3; pre-fix: fix the above small pieces with 2.5% (w/v) glutaraldehyde (pH7.4, 0.1MPBS), overnight at 4°C; wash: use 1mL0.1MPBS Wash at 4°C to remove glutaraldehyde, wash 3 times, each time for 10min; postfix: fix the sample with 200μl 1% (w/v) osmic acid at 4°C for 24hr; wash: wash with 1mL0.1MPBS at 4°C to remove osmic acid, Wash 3 times, each 10min; Gradient ethanol dehydration: dehydrate with 1mL 30%, 50%, 70%, 90% and 95% ethanol for 10min at room temperature, and finally dehydrate with 100% ethanol for 3 times, each 10min; alternative: room temperature Add 1 mL of propylene oxide to replace ethanol at low temperature, replace 3 times, 10 min each time; permeate: at room temperature, infiltrate with propylene oxide: Quetol812 at a ratio of 3:1, 1:1, 1:3 for 8hr, 10hr, 12hr, and finally Infiltrate with pure Quetol812 resin for 12hr; embedding and polymerization: Embed the sample with Quetol812 full resin added with accelerator, and polymerize at 35°C for 12hr, at 45°C for 12hr, and at 60°C for 24hr; slice: use ultrathin slices Machine LeicaEMUC6 (Leica company) cut the embedded sample into 90nm ultrathin sections; staining: the ultrathin sections were stained with 2% (w/v) uranyl acetate at room temperature for 20min, washed 3 times with water, and then washed with 2 % Lead citrate was stained at room temperature for 5 min, washed 3 times with water, and the ultrathin section was observed with an electron microscope FEITecnai20 (FEIcompany).

Overexpression of the 6ro02104 gene

6.1 Construction of expression plasmid

6.1.1 Amplification of the target gene

The sequence of the target gene RHA1MLDS was found from the NCBI database, and its sequence was amplified by PCR, and the primers were designed by primer5.0 software. A BamHI restriction site was added to both the 5' and 3' ends of the gene. The primers used were PrimerF (SEQ ID NO: 7) and Primer R (SEQ ID NO: 8):

PrimerF5'-CA GGATCC ACTGACCAGAAGACCATCGACAGCGT-3'

PrimerR5'-CA GGATCC AGCCTTCTTGGCCGGAGCAGCCTT-3'

The PCR system is:

The PCR conditions are:

6.1.2

For gel electrophoresis and recovery of PCR products, please refer to Sections 1.3 and 1.4 respectively.

For ligation and transformation of the target gene into T vector, please refer to Section 2.1 and 2.3 respectively.

For the extraction of transformant plasmids, please refer to Section 2.4.

6.1.3 Construction of JAM-egfp plasmid integration vector containing ro02104 gene

1) Perform BamHI digestion on the positive clones with correct sequencing and the vector pJAM-egfp. The digestion system is as shown in the table below. The digestion temperature is 30°C and the digestion time is 4hr.

2) Gel recovery after electrophoresis of the digested product, and gel recovery according to the kit instructions.

3) Ligate the recovered small fragments as exogenous fragments with the vector pJAM-egf. The connection system is: 4 μL of exogenous fragment, 1 μL of carrier, and 5 μL of 2× premixed buffer. Ligation conditions were 16°C overnight.

4) Transform the ligation product into DH5α competent cells, the method is the same as 2.3, and culture overnight at 37°C.

5) Extract the plasmid from the transformant and digest it to identify whether there is an insertion of a foreign fragment, the method is the same as 2.4.

6.2 Preparation of Competent Cells for Electrotransformation

Pick a single colony of newly activated RHA1 from the LB plate, inoculate it in 10 mL LB liquid medium, and cultivate it with shaking at 30°C for about 48 hours. Then the bacterial solution was inoculated in 100 mL LB liquid medium at a ratio of 1:100, and cultured at 30° C. with shaking at 220 rpm until the OD ₆₀₀ was 0.6. Take 45mL of each bacterial solution and add it to two sterile 50mL centrifuge tubes. Immediately placed on ice for 20 min, the culture was cooled to 0 ° C to stop the growth of bacteria. Centrifuge the centrifuge tube at 4°C, 6000 rpm for 10 min, discard the supernatant, and suck up the residual medium with a pipette tip. Add 25 mL of ice-cold sterile water to each centrifuge tube to resuspend. Place on ice for several minutes. Centrifuge at 6000 rpm for 10 min at 4°C to recover the cells. Resuspend with 25 mL of ice-cold sterile water again, discard the supernatant after centrifugation under the same conditions, and suck up the residual medium with a suction tip. Add 2 mL of ice-cold 10% glycerol to each centrifuge tube to resuspend, and distribute to 1.5 mL centrifuge tubes. Store at -80°C.

6.3 Transformation and expression of plasmids

Take 2 μL of the positive clone plasmid and add it to 50 μL of competent cells, mix gently, transfer to a 0.1 cm electric shock cup, and place on ice for 20 min. Select the "Ec3" program (3.0kV, 6.0msec) to shock once. Quickly culture the cells in LB liquid medium prepared by SMM at 30° C., 180 rpm for 4 hours. Apply kanamycin to the plate and incubate at 30°C. (SMM formula: 0.5M sucrose, 20 μM MgCl ₂ , 20 μM maleic acid, adjust the pH to 6.5 with NaOH).

Pick the grown single colonies into 10 mL of LB liquid medium, add kanamycin to a final concentration of 50 μg/mL, and add the inducer acetamide (purchased from sigma-aldrich, catalog number 695122-1G) to a final concentration 0.5% (w/v), cultured at 30°C for about 48 hours.

Centrifuge the bacterial solution (4°C, 8000rpm, 3min), discard the supernatant, add 1 mL of 0.1 MPBS to resuspend, centrifuge again under the same conditions, add 200 μL of PBS to resuspend, and perform fluorescence microscope observation.

7Westernblot identification of fusion proteins

7.1 Preparation of samples

Cultivate the bacteria containing the inducer acetamide to the end of exponential growth, take 0.5mL of the bacterial liquid and centrifuge (4°C, 8000rpm, 3min), discard the supernatant, add 1mL of 0.1MPBS to resuspend, centrifuge again under the same conditions, add 200μL of 2×samplebuffer (Sampling buffer), 200 watts of power ultrasonic 8 times, ultrasonic cycle condition is ultrasonic 3s, pause 3s. Then place it on a metal heater and heat it to 95°C for 5 minutes, and put the sample in a refrigerator at 4°C.

7.2 Electrophoresis

Preparation of 10% polyacrylamide gel: the formula is shown in the table below

Preparation of separation gel: first install the special device for glue preparation, the items used must be fully washed and dried, and can only be carried out after testing for no water leakage. After preparing the separating gel according to the recipe on ice, mix well. Then suck 7.5mL of separation gel and slowly add it into the glue making tank, then slowly add 2mL of deionized water with a pipette to press the glue. The stacking gel can be prepared after at least 45 minutes of polymerization. Preparation of stacking gel: first blot the water of pressing the gel, and insert a suitable sample comb. Then prepare 5mL stacking gel according to the recipe on ice and mix well. Then use a pipette to carefully add the stacking gel to the tank until it is full. Wait for the gel to polymerize. During this process, there may be shrinkage phenomenon, and the concentrated gel needs to be supplemented. It takes about 30 minutes to polymerize.

Spotting: Pull out the comb from the gel, install the electrophoresis tank and fill it with electrophoresis buffer, then use a pipette to spot the sample, 10 microliters of sample per well, and 10 microliters of pre-stained marker (molecular weight standard). Empty lanes were filled with loading buffer.

Electrophoresis: constant current electrophoresis, 25mA per gel, electrophoresis stops when the front of bromophenol blue is 0.5cm away from the bottom of the gel, this process takes about 100min.

7.3 Transfer film

Preparation before transfer: Remove the gel, remove the stacking gel, and equilibrate in the transfer buffer for 15 minutes. Cut the PVDF membrane into a size of 6.5cm*8.3cm, activate it in methanol for 30sec, and then equilibrate it in transfer buffer for 15min. At the same time, cut 2 sheets of Bio-rad special electro-transfer filter paper, the size is 8cm*1cm, and put them in the electro-transfer solution for 5 minutes to balance. Transfer: Take a clean 25cm diameter large round plate and fill it with transfer buffer. Then put the black side of the transfer clip down, and spread the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad sequentially. During this process, use a thick comb to remove the air bubbles in the gel and PVDF membrane, and finally clamp the transfer clip. Place the printing clip in the transfer tank correctly in the order of positive and negative electrodes, fill with transfer buffer, and cover the electrode cover. Place the whole on ice, set constant current mode, 200mA, and transfer for 2hr.

7.4 Hybridization

Remove the PVDF membrane, place it in a clean small square box, and rinse it twice with TBS, 1 min each time. The PVDF membrane was placed in blocking solution and incubated for 2 hr at room temperature with shaking. The blocked membrane was rinsed twice with TBS, 1 min each time. Add 10 mL of GFP mouse monoclonal antibody prepared with antibody diluent, the dilution ratio is 1:2000, and shake and incubate overnight on a shaker at 4°C. Wash the primary antibody 3 times with membrane washing buffer. Add 20 mL of goat anti-mouse secondary antibody at a dilution ratio of 1:10000, and incubate at room temperature for 1 hr with shaking. Wash 3 times with membrane washing buffer, 3 min each time.

7.5 Detection of chemiluminescent reagents

Substrate incubation: Take 0.5 mL each of substrate A and B, mix well on the Parafilm membrane and set aside. Remove the PVDF and place it on filter paper to absorb excess liquid. Then spread it face down on the uniformly mixed substrate, incubate for 30 sec, drain the excess substrate, then wrap the film with plastic wrap and place it in an X-ray film box, taking care to prevent air bubbles from appearing. Exposure: Operate under the red light in the darkroom, quickly attach the film to the PVDF film, close the lid of the X-ray film box tightly, and control the exposure time between 10sec-2min according to the fluorescence intensity. Develop under red light to a suitable strip depth, rinse with clean water to stop developing, then fix for about 45 seconds until the film is completely transparent, and finally rinse with clean water twice. Put the film on the shelf to dry, mark the position of the pre-stained marker (molecular weight standard) on the film, and finally scan the film to obtain the data.

7.6 Formulation of related solutions

Tris-glycine electrophoresis buffer: 25mM Tris, 250mM glycine, 0.1% (m/v) SDS; electrophoresis transfer buffer: 25mM Tris, 192mM glycine, 15% methanol (v/v), cooled to 4°C for later use; 2×sample buffer Liquid: 100mMTris-HCl (pH6.8), 8% SDS (m/v), 20% glycerol (v/v), 0.2% bromophenol blue (m/v), 200mMDTT; TBS buffer: 20mMTris, 150mM NaCl, pH7.5; washing buffer: TBS buffer + 0.2% milk powder + 0.2% Tween20; blocking solution: TBS buffer + 5% skimmed milk + 0.2% Tween20; antibody diluent: TBS buffer + 1% skimmed milk + 0.2% Tween20.

Experimental results:

1. MLDS gene knockout

Rhodococcus oleaginousus Rodococcussp.RHA1 was originally isolated from pesticide-contaminated soil, and it can use a variety of organic compounds as carbon sources, such as carbohydrates, steroids, aromatic compounds, nitrile compounds and so on. In experiments, cell growth was maintained with nutrient broth (NB) medium, with mineral salt medium (MSM), including a small amount of nitrogen, and with sufficient sodium gluconate as a carbon source for triglyceride TAG accumulation. In the results of comparative proteomics, it was observed that the number of peptides of the MLDS (GI111019097) protein was significantly reduced in the lipid droplets in the MSM environment, indicating a decrease in protein expression. Interestingly, through homologous sequence comparison ( http://pfam.janelia.org ), the homologous protein of this gene is apolipoprotein. In order to study the function of the protein, we used pK18mobsacB to knock out the gene through homologous recombination [12-13]. Figure 1 shows the design scheme of the PCR primers for the deletion site and the sequences on both sides. And the knockout of the gene was confirmed by PCR method ( FIG. 2 ). The results showed that compared with the positive plasmid control (swimming lane 1), the MLDS (831bp) gene was completely knocked out (swimming lane 2), while the bacterial genome was used as a negative control (swimming lane 3), and the knockout site and the sequences on both sides were also carried out PCR detection was performed to further confirm the success of gene knockout. In order to further analyze the phenotype of the MLDS gene knockout bacteria, the wild bacteria and the MLDS gene knockout bacteria were observed by negative staining electron microscopy and transmission ultrathin section electron microscope (TLC), and it was found that the lipid droplets in the MLDS gene knockout bacteria changed significantly. Big (Figure 3). TLC results showed that the triglyceride content of the MLDS gene knockout bacteria increased ( FIG. 4 ).

2. Localization results of MLDS protein

JAM-egfp is a vector that can express foreign proteins in microorganisms such as actinomycetes. In order to study the localization of MLDS protein that affects the lipid droplet size, we respectively used JAM-egfp empty vector and JAM-MLDS-egfp in wild Overexpression in the type strain [14-16]. At the same time, the expression of the fusion protein was identified by Western blot. The results of Western blot showed that the content of the fusion protein MLDS-GFP was very high, but there was almost no single GFP (Fig. 5). This ensures that the green we see under the fluorescence microscope is the color emitted by the fusion protein and accurately reflects the localization of MLDS. Through the observation of fluorescence microscope, we can see that when only the JAM-egfp carrier is transferred into the cells, the whole bacterium is evenly distributed green, and when JAM-MLDS-egfp is transferred into the cells, it can be seen that there are The distribution of spherical particles (Fig. 6), which indicates that MLDS protein is localized on the surface of lipid droplets.

There are two possibilities for lipid droplets to grow from small to large. One is that lipid droplets grow up by themselves, and the other is that small lipid droplets fuse into large lipid droplets. Our results support the second possibility, since the number of lipid droplets is significantly reduced in MMLDS knockout mutants. In the process of lipid droplet fusion, there are two possible mechanisms for regulation. One is the positive regulation of certain proteins, such as SNARE and ADRP, which can promote the fusion of small lipid droplets into large lipid droplets; the other is the positive regulation of certain proteins. The negative regulation of some proteins, such as MMLDS, which protects the lipid droplets and prevents the fusion of lipid droplets, these two aspects reach a dynamic balance in the cell, and when we knock out the negative regulatory MMLDS, the Will make lipid droplets tend to fuse.

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Claims (3)

1. Rhodococcus oleaginousus Rhodococcussp.RHA-MLDS with knockout MLDS gene, which was preserved in the General Microorganism Center of China Microbiological Culture Collection Committee on November 30, 2010, and the preservation number is CGMCCNo.4379, wherein the MLDS gene NCBI accession number is 4218150. 2. The application of the Rhodococcus oleaginous bacteria Rhodococcussp.RHA-MLDS knocking out the MLDS gene according to claim 1 in the production of biodiesel. 3. A method for producing biodiesel by utilizing the Rhodococcus oleaginous bacteria Rhodococcussp.RHA-MLDS which has knocked out the MLDS gene according to claim 1.