Method for rapidly preparing archaea cell membrane lipid intermediate product 2, 3-di-O-geranyl-geranyl-glycerol (DGGGOH)

Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for rapidly preparing an archaea cell membrane lipid intermediate product 2, 3-di-O-geranyl geranyl-glycerol (DGGGOH). The method comprises the following steps: constructing engineering escherichia coli for expressing archaea membrane lipid synthesis genes (crtE, GGPPS, G1PDH, GGGPS and DGGGPS) and mevalonic acid (MVA), and carrying out induced fermentation, cell disruption, organic solvent extraction and semi-preparative C18 reversed-phase chromatographic column purification to realize efficient preparation of DGGGOH. The method provided by the invention solves the technical bottlenecks of tedious operation steps, low yield, low recovery rate and the like of traditional chemical synthesis and other microbial fermentation preparation of DGGGOH, and provides reliable technical support for archaea membrane lipid research and large-scale production of DGGGOH.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and to a method for rapidly preparing the archaea cell membrane lipid intermediate 2,3-di-O-geranylgeranyl-glycerol (DGGGOH). Specifically, it relates to a method for rapidly preparing the key archaea cell membrane lipid intermediate DGGGOH using engineered Escherichia coli strains combined with high performance liquid chromatography (HPLC) technology. Background Technology

[0002] Archaea cell membrane lipids (such as GDGTs) are composed of unique isoprene-chain glycerol diethers. DGGGOH is a key precursor for the synthesis of dialkylglycerol tetraethers (GDGTs), containing two geraniol-geraniol (GG) side chains. It serves as a model molecule for studying the carbon-carbon bond formation mechanism and membrane lipid cyclization modification of GDGTs. Its efficient preparation is crucial for investigating archaea membrane structure and environmental adaptation mechanisms. Rapidly obtaining high-purity DGGGOH can provide a core tool for elucidating the dynamic regulatory network of archaea membrane lipids and developing resources from microorganisms operating in extreme environments.

[0003] Existing preparation methods mostly rely on chemical synthesis or complex biological extraction, which have problems such as cumbersome steps, low yield, and insufficient purity.

[0004] Chemical synthesis: Using 3,4-dimethoxybenzyl alcohol, epichlorohydrin, and trans-farnesyl bromide as starting materials, the target product DGGGOH can be synthesized through at least 11 reaction steps. After multiple reaction steps and purification, the final overall yield of DGGGOH is only 5.27%, indicating a lengthy and inefficient process.

[0005] Archaea heterologous expression: Although archaea heterologous expression systems can utilize model species such as methanogenic archaea to catalyze GGPP condensation to generate target products, the product accumulation efficiency is difficult to improve due to the long archaea culture cycle (several weeks to several months), the lack of genetic manipulation tools, and the harsh growth conditions.

[0006] In recent years, metabolic engineering of model microorganisms such as Saccharomyces cerevisiae has provided new strategies for the synthesis of terpenoids. For example, by overexpressing rate-limiting enzymes of the MVA pathway (such as tHMG1), introducing heterologous GGPP synthases, and optimizing acetyl-CoA supply, efficient GGPP supply can be achieved. However, after introducing archaeal membrane lipid synthesis genes into yeast, the endogenous diacylglycerol O-acyltransferase (DGAT) recognizes and utilizes DGGGOH to catalyze the production of triglyceride analogs DGGGO-FAs, resulting in the loss of the target product. In contrast, Escherichia coli, lacking DGAT homologs, although exhibiting trace amounts of byproduct synthesis, demonstrates a significantly better retention capacity of the main product DGGGOH compared to the yeast system.

[0007] This invention provides a universal and efficient method for preparing DGGGOH based on fermentation of engineered Escherichia coli strains and HPLC semi-preparation technology. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing DGGGOH that is simple to operate, has a short cycle (<60 hours), and high purity (>80%), and achieves large-scale production by combining engineered Escherichia coli fermentation with universal HPLC semi-preparation technology.

[0009] The technical solution adopted in this invention is as follows:

[0010] A method for rapidly preparing the archaea cell membrane lipid intermediate 2,3-di-O-geranylgeranyl-glycerol, comprising the following steps:

[0011] (1) Fermentation by engineered bacteria: Escherichia coli carrying two plasmids containing archaeal membrane lipid synthesis genes were inoculated into a culture medium containing ampicillin and chloramphenicol, and cultured at 30-37℃ and 150-200 rpm until OD. 600 =0.6-0.8, then add 0.5-1.0mM IPTG to induce expression for 36-48 hours;

[0012] (2) Membrane lipid extraction: high pressure homogenization or ultrasonic disruption;

[0013] (3) HPLC purification: A semi-preparative C18 reversed-phase column was used. The mobile phase A was acetonitrile-water (6-7:2-4, v / v) containing 0.05-0.01% ammonia, and the mobile phase B was acetonitrile-isopropanol (8-10:0.5-1, v / v). The gradient elution program was: 0 min 58% B → 10 min 80% B → 30 min 92% B → 36 min 100% B → 46 min 100% B. The flow rate was 1.7 mL / min, and the detection was performed using 210 nm UV. The fraction with a retention time of 23-30 min was collected.

[0014] (4) Purity and Quantitative Detection: Using DGGGOH standard, the purity and quantification of the collected and concentrated DGGGOH were determined by LC-MS or HPLC.

[0015] Preferably, the fermentation process uses shake flasks, fermenters, or bioreactors of different volumes.

[0016] Preferably, the culture medium used for the above fermentation is one or more of TB (Terrific Broth), LB (Luria-Bertani), SOB (Super Optimal Broth), SOC (Super Optimal Broth with Catabolite Repression), SB (Super Broth), M9, or MBL.

[0017] Preferably, the Escherichia coli is BL21(DE3), TOP10, or K12, wherein plasmid 1 is ptrc-Crte_Ma, which includes five genes: crtE, GGPPS, G1PDH, GGGPS, and DGGGPS, and plasmid 2 is pJBEI 2997, which includes the MVA pathway.

[0018] Preferably, the method for extracting membrane lipids includes: collecting cell cells by centrifuging the fermentation broth at 4000-8000 rpm for 5-10 minutes; disrupting the cells 3-5 times using a high-pressure homogenizer or ultrasonic disruptor; centrifuging at 4-16℃ and 8000-12000 rpm for 10-20 minutes to obtain a supernatant; adding an equal volume of dichloromethane-methanol-water (1-3:0.5-2:0.5-2, v / v) for extraction 3-5 times; combining the organic phases and concentrating under reduced pressure to dryness to obtain the membrane lipid extract;

[0019] Preferably, the solvent used for extraction is chloroform or dichloromethane.

[0020] Preferably, the semi-preparative C18 reversed-phase chromatographic column has an inner diameter of 10 mm, a packing particle size of 5-10 μm, and a pore size of [missing information]. The filler material with a length (150-250 mm) and pore size is octadecylsilane (C18).

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. High efficiency: The yield of engineered bacteria induced fermentation for 48 hours was 35 mg / L (shaking flask);

[0023] 2. Rapid separation: A single HPLC purification process takes only 50 minutes;

[0024] 3. Universality: Uses a conventional semi-preparative C18 packed column;

[0025] 4. High purity: The purity of the final product is >80%, meeting the requirements of DGGGOH standard. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the pTrc-Crte_Ma plasmid.

[0027] Figure 2 The chemical structure is DGGGOH;

[0028] Figure 3 The HPLC chromatogram of DGGGOH in the extract;

[0029] Figure 4 This is the HPLC chromatogram of purified DGGGOH. Detailed Implementation

[0030] To enable a better understanding of the objectives, technical solutions, and advantages of this invention, the invention will be described in detail below with reference to examples. The examples described below are only some implementation cases of this invention. Other similar techniques that use archaeal membrane lipid synthesis genes and add crtE genes to purify DGGGOH using HPLC combined with C18 semi-preparative columns are all within the scope of protection of this invention.

[0031] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0032] The genes encoding the DGGGOH synthesis pathway include geraniol geraniol pyrophosphate synthase (GGPPS), glycerol-1-phosphate dehydrogenase (G1PDH), geraniol geraniol glycerol phosphate synthase (GGGPS), and digeraniol geraniol glycerol phosphate synthase (DGGGPS). These genes are derived from Methanosarcina acetivorans, have undergone codon optimization, and are integrated into the same E. coli expression plasmid.

[0033] The codon optimization upstream of the GGPPS gene in the expression plasmid was derived from the diterpene synthesis gene crtE of Phaffia rhodozyma.

[0034] The amino acid sequences of the genes involved are as follows:

[0035] The amino acid sequence of crtE is: Accession:CDZ97186.1;

[0036] The amino acid sequence of GGPPS: Accession: AAM04050.1;

[0037] The amino acid sequence of G1PDH: Accession:WP_011023593.1;

[0038] The amino acid sequence of GGGPS: Accession:WP_011023864.1;

[0039] The amino acid sequence of DGGGPS: Accession:WP_011020999.1.

[0040] Example 1: Preparation of DGGGOH by shake-flask fermentation (250 mL scale)

[0041] The specific implementation steps and procedures are as follows:

[0042] Strains: The top 10 strains containing the expression plasmid ptrc-Crte_Ma(crtE+GGPPS+G1PDH+GGGPS+DGGGPS) and pJBEI2997 are numbered DC-DGGGOH_1;

[0043] Culture conditions: LB medium (containing ampicillin and chloramphenicol), cultured at 37°C and 200 rpm until OD. 600 =0.6, add 0.5mM IPTG for 48 hours of induction.

[0044] Extraction: Collect bacterial cells by centrifugation (6000 rpm, 10 min); homogenize under high pressure (3 times); extract 3 times with dichloromethane-methanol-water (2:1:1), and concentrate the combined organic phases under reduced pressure.

[0045] HPLC purification: Column: YMC-Pack ODS-A (250×10.0mm, 5μm), Mobile phase: Phase A (acetonitrile-water = 7:4 + 0.01% ammonia), Phase B (acetonitrile-isopropanol = 10:1); Gradient: 0 min → 58% B, 10 min → 80% B, 30 min → 92% B, 36 min → 100% B, 46 min → 100% B; Flow rate: 1.7 mL / min, Detection wavelength: 210 nm, DGGGOH retention time: 28.2 min.

[0046] Purity and Quantitative Detection: Using DGGGOH standard, the purity and quantification of the collected and concentrated DGGGOH were determined by LC-MS or HPLC.

[0047] Example 2: Effects of different gene combinations on DGGGOH yield

[0048] The specific implementation steps and procedures are as follows:

[0049] The fermentation preparation steps for DGGGOH in this embodiment are the same as those in Example 1.

[0050] Differences: The following gene combinations were constructed in the same plasmid backbone pTrc (plasmid 1 backbone), and were combined with pJBEI 2997 plasmid or without the plasmid. The results were then transformed into E. coli TOP10 for fermentation to verify the yield of DGGGOH. This example mainly investigates the effect of different gene combinations in plasmid 1 on the yield of DGGGOH.

[0051] Table 1. Effects of different gene combinations in plasmid 1 on DGGGOH yield.

[0052] Group Gene / plasmid combination DGGGOH yield (mg / L) control group crtE+GGPPS+G1PDH+GGGPS+DGGGPS 35.2±2.1 Experimental group 1 Knock out G1PDH 22.5±1.8 Experimental group 2 Knock out GGGPS <0.5 Experimental group 3 Knock out DGGGPS <0.5 Experimental group 4 Knock out GGPPS <0.5 Experimental group 5 pJBEI2997 plasmid not added 8.7±1.5 Experimental group 6 No crtE gene added 20.3±1.2

[0053] Table 1 shows that the DGGGOH production was highest in the control group. Because the *E. coli* genome contains genes with functions similar to G1PDH, DGGGOH synthesis was still detectable in experimental group 1 after the G1PDH gene was knocked out, but its production was significantly lower than the control group. Since *E. coli* has a terpene-synthesizing metabolic pathway, even with co-expression using plasmid 2, the strain could still maintain DGGGOH synthesis through its own endogenous terpene synthesis pathway, but the production was low. When individual genes GGGPS, DGGGPS, and GGPPS in plasmid 1 were knocked out individually, the DGGGOH production plummeted below the detection limit, confirming that *E. coli* lacks endogenous isoenzymes with corresponding catalytic activities. Furthermore, in experimental group 6, where the crtE gene was not added, the DGGGOH production was lower than the control group, indicating that the addition of crtE in plasmid 1 significantly promoted DGGGOH accumulation. In summary, the combination of five genes in plasmid 1 resulted in the highest DGGGOH production.

[0054] Example 3: Scale-up production in a 5L fermenter

[0055] The strain is the same as in Example 1.

[0056] Culture conditions: 3.0 L LB medium (containing ampicillin and chloramphenicol), inoculation amount 1% (v / v), cultured at 37℃ and 200 rpm until OD. 600 =0.8-1.2, add 0.5mM IPTG and induce fermentation at 30℃ for 48 hours. After adding IPTG, replenish 100mL of solution every 6-8 hours, containing 2g tryptone and 1g yeast extract. During fermentation, control dissolved oxygen above 30% and pH between 6.5 and 7.2. Extraction, HPLC purification and other steps are the same as in Example 1. Quantitative analysis showed that the yield of DGGGOH was 170.2mg / L.

[0057] Example 4: Optimization of Semi-Preparative Chromatography Column Parameters

[0058] The fermentation preparation of DGGGOH in this embodiment is the same as that in Example 1. The difference is that semi-preparative chromatographic columns of different lengths and particle sizes were used to investigate the separation effect of different semi-preparative chromatographic columns on DGGGOH. Specific results are shown in Table 2 below.

[0059] Table 2 Comparison of separation effects of different chromatographic columns on DGGGOH

[0060] Column parameters DGGGOH purity (%) DGGGOH retention time (min) Column length 150mm, particle size 5μm 70.2 20.3 Column length 250mm, particle size 5μm 87.6 25.6 Column length 250mm, particle size 10μm 75.1 26.2

[0061] As shown in Table 2, the optimal separation effect was achieved when the column length was 250 mm and the particle size was 5 μm, yielding a DGGGOH fraction with a purity of 87.6% in a single collection. Under the same particle size (5 μm), shortening the column length to 150 mm significantly reduced the separation efficiency, with a purity of only 70.2%. This data indicates that increasing the column length improves separation efficiency more effectively than decreasing the particle size, and there is a synergistic effect between column length and particle size; the 250 mm × 5 μm parameter combination can achieve highly efficient separation.

[0062] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for rapidly preparing the archaea cell membrane lipid intermediate 2,3-di-O-geranylgeranyl-glycerol (DGGGOH), characterized in that, Includes the following steps: (1) Fermentation by engineered bacteria: Escherichia coli carrying two plasmids containing archaeal membrane lipid synthesis genes were inoculated into a culture medium containing ampicillin and chloramphenicol, and cultured at 30-37℃ and 150-200 rpm until OD. 600 =0.6-0.8, add 0.5-1.0mM IPTG to induce expression for 36-48 hours; (2) Membrane lipid extraction: After the fermentation broth is centrifuged to collect the cells, the cells are homogenized by high pressure or ultrasonically broken, and membrane lipids are extracted by organic solvents. (3) HPLC purification: The target product was separated using a semi-preparative C18 reversed-phase column with a gradient elution program at a flow rate of 1.7 mL / min and 210 nm UV detection. The fraction with a retention time of 23-30 min was collected. (4) Purity and quantification: The purity and quantification of the collected fraction were analyzed by LC-MS or HPLC using DGGGOH standard.

2. The method according to claim 1, characterized in that, The fermentation device used for the engineered bacteria fermentation is one or more of the following: shake flask, fermenter, or bioreactor.

3. The method according to claim 1, characterized in that, The fermentation medium is one or more of TB, LB, SOB, SOC, SB, M9 or MBL media.

4. The method according to claim 1, characterized in that, The *E. coli* strain is BL21(DE3), TOP10, or K12; the two plasmids are: Plasmid 1: ptrc-Crte_Ma, containing the crtE, GGPPS, G1PDH, GGGPS and DGGGPS genes; Plasmid 2: pJBEI 2997, contains genes related to the MVA pathway.

5. The method according to claim 1, characterized in that, The membrane lipid extraction step includes: (1) Collect the cells by centrifuging the fermentation broth at 4000-8000 rpm for 5-10 minutes; (2) High-pressure homogenization or ultrasonic disruption of cells 3-5 times; (3) Centrifuge at 4-16℃ and 8000-12000rpm for 10-20 minutes to obtain the supernatant; (4) Add an equal volume of dichloromethane-methanol-water (1-3:0.5-2:0.5-2, v / v) and extract 3-5 times. Combine the organic phases and concentrate under reduced pressure to dryness to obtain the membrane lipid extract.

6. The method according to claim 5, characterized in that, The organic solvent is chloroform or dichloromethane.

7. The method according to claim 1, characterized in that, In the HPLC purification step, the parameters of the semi-preparative C18 reversed-phase column are: inner diameter 10 mm, packing particle size 5-10 μm, pore size... Length 150-250mm, filler is octadecylsilane (C18).

8. The method according to claim 1, characterized in that, In the HPLC purification step, mobile phase A is acetonitrile-water (6-7:2-4, v / v) containing 0.05-0.01% ammonia, and mobile phase B is acetonitrile-isopropanol (8-10:0.5-1, v / v); the gradient elution program is: 0 min 58% B → 10 min 80% B → 30 min 92% B → 36 min 100% B → 46 min 100% B.

9. The method according to claim 1, characterized in that, In the purity and quantitative detection steps, qualitative analysis is performed by comparing the retention time and mass spectrometry characteristic peaks of the DGGGOH standard with those of LC-MS or HPLC, and quantitative calculation is performed by peak area or mass spectrometry intensity.

10. The method according to any one of claims 1-9, characterized in that, The method is suitable for large-scale preparation of DGGGOH, with a total cycle time of no more than 72 hours and a product purity of ≥95%.

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