Construction and application of engineering Rhodococcus etherivorans for synthesizing isocasponge alkene by using inferior biomass lignin

Abstract

The invention discloses construction and application of engineering Rhodococcus etherivorans for synthesizing isocasponge alkene by using inferior biomass lignin, Rhodococcus etherivorans capable of degrading various aromatic phenolic compounds is used as an original strain, and a strain with enhanced utilization capability of lignin-derived aromatic phenolic compounds is obtained through metabolic engineering modification. The strain can take inferior biomass lignin as a carbon source to realize synthesis of isospongiene. The yield and content of isosponge alkene synthesized by the strain reach 18.22 mg / L and 12.24 mg / g, and compared with a strain N1, the yield is increased by 116%, and the content is increased by 265%. The invention provides a method for efficiently synthesizing a high-value chemical isospongiene from lignin.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the construction and application of an engineered Rhodococcus etherivorans strain which utilizes low-quality biomass lignin to synthesize isospongene. Background Art

[0002] Isosponge is a rare carotenoid with a benzene ring end group. The aromatic rings at both ends can form conjugated large π bonds with the symmetrical isoprene carbon chain. This structure gives it stronger stability. In addition to having high antioxidant capacity, isosponge can also effectively absorb ultraviolet radiation, so it has a cell photoprotective effect. As a carotenoid with a more stable structure and better functions, isosponge has shown great application potential in functional foods and medicines.

[0003] Lignocellulose, such as straw, is the most abundant renewable resource on earth. The second-generation biorefining technology using lignocellulose as raw material has attracted much attention and has now been applied to the synthesis of biomass fuels and high-value chemicals. However, lignin is complex in structure and heterogeneous. Therefore, it is often used as an obstacle and waste for the high-value conversion of lignocellulose in the biorefining process. In the papermaking industry, lignocellulose also needs to be delignified. Therefore, a large amount of waste lignin is generated during the biorefining and utilization of lignocellulose, resulting in waste of resources and environmental pollution. Therefore, using low-quality carbon resource lignin as raw material to synthesize high-value chemicals can not only realize the full component utilization of lignocellulose, but also benefit environmental protection and the efficient utilization of renewable resources.

[0004] The present invention intends to utilize low-quality carbon resource lignin as a carbon source and develop a technology that can utilize lignin to synthesize high-value chemical isospongene, thereby realizing the conversion of waste carbon resources into high-value chemicals and providing a new method for the utilization of all components of lignocellulose in the biorefining process of lignocellulose. Summary of the invention

[0005] The purpose of the invention is to provide a method for constructing an engineered Rhodococcus etheriferus capable of efficiently degrading lignin.

[0006] Another object of the present invention is to provide a method for synthesizing isospongene using low-quality biomass lignin.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] An engineered Rhodococcus etherivorans strain for synthesizing isospongene using low-quality biomass lignin. The engineered Rhodococcus etherivorans strain is constructed by introducing syringate demethylase and 3-o-methylgallate 3,4-dioxygenase into Rhodococcus etherivorans strain. The coding gene sequence of the syringate demethylase is shown in SEQ ID No. 1; the coding gene sequence of the 3-o-methylgallate 3,4-dioxygenase is shown in SEQ ID No. 2.

[0009] It also includes introducing a urea-inducible promoter nucleic acid sequence pami, wherein the urea-inducible promoter nucleic acid sequence pami is shown in SEQ ID No.3.

[0010] The Rhodococcus aetherivorans is Rhodococcus aetherivorans N1, and its preservation number is CCTCC NO: M 20221270.

[0011] The gene is introduced into the Rhodococcus aetherovorus N1 by constructing the Rhodococcus shuttle plasmid pNV-dmm-mdo through the expression plasmid pNV18.1.

[0012] The reverse amplified fragment of plasmid pNV18.1, the complete fragments of genes dmm, mdo and promoter pami were obtained respectively, and the plasmid pNV-dmm-mdo was constructed by multi-fragment homologous recombination.

[0013] Homologous recombination of multiple fragments connects dmm, pami and dmo in sequence.

[0014] The plasmid contains a kanamycin marker gene.

[0015] The construction method of the engineered Rhodococcus etheriferus comprises using the expression plasmid pNV18.1 to construct a Rhodococcus shuttle plasmid pNV-dmm-dmo containing a syringate demethylase encoding gene dmm, a 3-o-methylgallate 3,4-dioxygenase encoding gene dmo, and a urea-inducible promoter nucleic acid sequence pami; the syringate demethylase encoding gene dmm sequence is SEQ ID No. 1. The 3-o-methylgallate 3,4-dioxygenase encoding gene dmo sequence is SEQ ID No. 2, and the urea-inducible promoter nucleic acid sequence pami sequence is SEQ ID No. 3;

[0016] The plasmid pNV-dmm-dmo is introduced into Rhodococcus aetherovorans N1 by electrotransformation to obtain engineered Rhodococcus aetherovorans N1-dmm-dmo.

[0017] The invention discloses an application of the syringate demethylase (DMM) and 3-o-methylgallate 3,4-dioxygenase (MDO) in synthesizing isospongene by utilizing lignin.

[0018] The two genes DMM and MDO synergistically improve the efficiency of Rhodococcus etheriferus in utilizing lignin, thereby increasing the yield of isospongene synthesized by Rhodococcus etheriferus using lignin.

[0019] The application of the engineered Rhodococcus etheriferus in the production of isospongene.

[0020] Using lignin as carbon source, engineered Rhodococcus etherivorans was fermented and cultured under aerobic conditions to produce isospongene.

[0021] The fermentation culture conditions are:

[0022] 1) Plate culture: Streak the engineered Rhodococcus etherivorans on LB solid medium containing 50 mg / L kanamycin, culture temperature 25-35°C, culture time 45-50h;

[0023] 2) Seed culture: inoculate the colonies on the solid culture medium into LB seed culture medium containing 50 mg / L kanamycin, culture temperature 25-35°C, 180 rpm, culture time 28-32 h;

[0024] 3) Fermentation culture: The seed culture solution is inoculated into the fermentation medium, the inoculation amount is 3-5% v / v, the fermentation temperature is 25-35° C., and the fermentation culture time is 120-144 h.

[0025] The formula of the fermentation medium is 0.5-10 g / L of lignin, 1-2.5 g / L of corn syrup dry powder, 1-3 g / L of urea, and 0.5-2 mol / L of MgCl2.

[0026] More preferably, the fermentation pH is 6, the temperature is 30° C., and the fermentation culture time is 144 h.

[0027] Lignin was used as the carbon source, a fermentation medium containing different concentrations of lignin was prepared, and the engineered Rhodococcus etheriferus N1-dmm-dmo was used as the fermentation strain to ferment and produce isospongene.

[0028] The lignin concentration in the fermentation medium is 0.5 g / L to 10 g / L, preferably 5 g / L to 10 g / L, and more preferably 8 g / L.

[0029] The remaining ingredients in the culture medium are 1-2.5 g / L corn steep liquor powder, 1-3 g / L urea, and 0.5-2 mol / L MgCl2. Preferably, the corn steep liquor powder is 2.5 g / L, the urea is 1 g / L, and the MgCl2 is 1 mol / L.

[0030] The Rhodococcus etherivorans is activated by streaking culture on a solid culture medium, the activated strain is transferred to a seed culture medium, and the seed liquid is inoculated into a fermentation culture medium for fermentation. The preferred seed activation condition is to culture the streaked plate in an incubator at 30°C for 50 hours, and then pick the bacteria into a liquid culture medium at 30°C and 180rpm for 30 hours. The preferred fermentation culture condition is to inoculate the seed liquid into a fermentation culture medium at 30°C and 180rpm for 144 hours at an inoculation rate of 5%.

[0031] The fermentation broth was subjected to liquid chromatography-mass spectrometry to detect isospongene.

[0032] The invention discloses a method for synthesizing isospongene by using low-quality biomass lignin as a carbon source. The method uses lignin as a carbon source and ferments and cultivates engineered Rhodococcus etheriferus under aerobic conditions to produce isospongene.

[0033] The Rhodococcus aetherivorans is Rhodococcus aetherivorans N1 or engineered Rhodococcus aetherivorans N1-dmm-dmo.

[0034] Beneficial effects:

[0035] 1. The present invention is based on a strain of Rhodococcus etheriferus with excellent performance. By introducing exogenous genes syringate demethylase (DMM) and 3-o-methylgallate 3,4-dioxygenase (DMO), the Rhodococcus etheriferus constructed using urea-inducible promoter pami can efficiently utilize lignin as a carbon source for growth metabolism.

[0036] 2. The engineered Rhodococcus etheriferus constructed by the present invention can utilize lignin as a carbon source to synthesize isospongene, and the content of synthesized isospongene is 12.35 mg / g, which is the highest content of isospongene synthesized by microorganisms reported so far. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the plasmid map of pNV-dmm-dmo;

[0038] Figure 2 The growth OD of Rhodococcus etherivorans N1 using different concentrations of lignin for fermentation 600 Detection chart;

[0039] Figure 3 This is a graph showing the production of isospongene produced by fermentation of lignin with different concentrations by Rhodococcus etherivorans N1;

[0040] Figure 4 The growth OD of Rhodococcus etherivorans N1-dmm-dmo fermented with different concentrations of lignin 600 Detection chart;

[0041] Figure 5This is a graph showing the production of isospongene produced by fermentation of lignin with different concentrations by Rhodococcus etheriferus N1-dmm-dmo;

[0042] Figure 6 This is a liquid phase detection diagram of the fermentation product of Rhodococcus etherivorans N1-dmm-dmo;

[0043] Figure 7 Mass spectrometry detection results of fermentation products of Rhodococcus etherovorans N1-dmm-dmo. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with specific embodiments.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] The strain used in the present invention is Rhodococcus aetherivorans N1, with a deposit number of CCTCC NO: M 20221270. The strain has been disclosed in patent CN116769864A.

[0048] The lignin used in the culture medium in the following examples comes from Sigma-Aldrich Company, CAS No.: 8068-05-1.

[0049] Example 1

[0050] Construction of engineered Rhodococcus etherivorans N1-dmm-dmo containing genes dmm and dmo

[0051] (1) Obtaining target genes: According to the coding genes of syringate demethylase (DMM) and 3-o-methylgallate 3,4-dioxygenase (DMO) from Sphingobium sp.SYK-6 provided by NCBI, the dmm gene sequence is shown in SEQ ID No.1; the dmo gene is shown in SEQ ID No.2.

[0052] (2) GenScript Biotech Co., Ltd. was commissioned to synthesize the gene sequences of dmm, dmo and the complete sequence of the urea promoter pami, and PNV-U and PNV-D were used as primers to obtain a plasmid reverse amplification template, dmm-U and dmm-D were used as primers to obtain a dmm gene fragment containing homology arms, pami-U and pami-D were used as primers to obtain a promoter pami complete fragment containing homology arms, and dmo-U and dmo-D were used as primers to obtain a dmo gene fragment containing homology arms. All primer sequences are shown in Table 1.

[0053] Table 1 Primer sequences required for construction

[0054]

[0055] The PCR enzyme used for amplification was Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozyme Biotechnology Co., Ltd. The system is shown in Table 2

[0056] Table 2 PCR system configuration table

[0057] system 50μL Phanta Max Super-Fidelity DNA Polymerase 1μL buffer 25μL Distilled water 20μL DNTP 1μL Quote 1μL quoted below 1μL template 1μL

[0058] Amplification Procedure:

[0059]

[0060] The corresponding fragment lengths, primers, reaction temperatures and times for different plasmid templates are shown in Table 3

[0061] Plasmid template fragment length Upstream primer Downstream primer Temperature a Time b

[0062] Table 3 Conditions required for fragment amplification

[0063]

[0064]

[0065] ClonExpress MultiS One Step Cloning Kit was used for multi-fragment ligation, and the ratio of vector to fragment in the reaction system was 1:2. The ligated reaction solution was introduced into the competent E. coli, ice-water bath for 30 minutes, 42°C water bath for 90 seconds, ice-water bath for 2 minutes, and then 900 μL LB was added, incubated at 37°C, 180 rpm, and incubated for 45-60 minutes, and screened with kanamycin screening plates.

[0066] The constructed and verified correct plasmid was introduced into the original Rhodococcus etherivorans N1 by point transformation, with a voltage of 1500V, a resistance of 400Ω, a capacitor of 25μF, and an electric shock time of 5-6ms. 400μL LB medium was added immediately after electric shock, and the culture was shaken at 30°C for 3h. The strain was screened on a kanamycin screening plate and sent for strain verification to obtain the engineered Rhodococcus etherivorans N1-dmm-dmo.

[0067] Example 2

[0068] Growth and fermentation of R.aetherivorans N1-dmm-dmo using different concentrations of lignin:

[0069] (1) Activation of bacterial strains: The engineered Rhodococcus etheriferus N1-dmm-dmo was streaked and cultured on LB solid medium containing 50 mg / L kanamycin at 30°C for 50 h;

[0070] (2) Seed culture: Pick a single colony of strain N1-dmm-dmo from the solid plate culture medium and inoculate it into 5 mL LB liquid test tube culture medium containing 50 mg / L kanamycin, and culture it at 30°C and 180 rpm for 30 h.

[0071] (3) Fermentation culture: The seed liquid was inoculated at 5% v / v into a fermentation medium containing 50 mg / L kanamycin and different concentrations of lignin, and fermented at 30° C. and 180 rpm for 144 h.

[0072] The fermentation medium formula is: 0.5-10 g / L lignin, 2.5 g / L corn syrup dry powder, 1 g / L urea. The pH is adjusted to 6.

[0073] Example 3

[0074] Growth and fermentation of R.aetherivorans N1 using different concentrations of lignin:

[0075] (1) Activation of bacterial strains: The engineered Rhodococcus etherivorans N1 was streaked on LB solid medium and cultured at 30°C for 50 h;

[0076] (2) Seed culture: Pick a single colony of strain N1 from the solid plate culture medium and inoculate it into 5 mL of LB liquid test tube culture medium, and culture it at 30°C and 180 rpm for 30 h.

[0077] (3) Fermentation culture: The seed liquid was inoculated into fermentation medium containing different concentrations of lignin at 5% v / v and fermented at 30°C and 180 rpm for 144 h.

[0078] The fermentation culture conditions were consistent with those in Example 2, and the strain N1 control of Example 2 was used. Under the condition of 5 g / L lignin as the carbon source, the strain N1 grew best, and the production and content of isosponge were also the highest at 8.44 mg / L, and the content was 3.35 mg / g. Under the condition of 8 g / L lignin as the carbon source, the strain N1-dmm-dmo grew best, and the production and content of isosponge were also the highest at 18.22 mg / L and 12.24 mg / g, wherein the production of isosponge was increased by 116% compared with the production of strain N1, and the content was increased by 265%.

[0079] Example 4

[0080] Take 2 ml of fermentation liquid, centrifuge at 12000 rpm for 3 min, wash twice with ultrapure water, discard the supernatant, add 2 ml of dimethyl sulfoxide, shake well, incubate at 55°C in a water bath for 15 min, add 2 ml of acetone, incubate at 55°C in a water bath away from light for 15 min, centrifuge at 12000 rpm for 3 min, take the supernatant, and wait for the next step of detection.

[0081] The fermentation product is extracted with dimethyl sulfoxide and acetone to obtain isospongene, and the product isospongene is qualitatively detected by LC-MS analysis and its yield is detected by high performance liquid chromatography.

[0082] The liquid phase conditions are:

[0083] Chromatographic column: C30 chromatographic column; UV absorption wavelength of 450nm; injection volume of 10uL; mobile phase A is methanol and B is methyl tert-butyl ether, flow rate: 1mL / min; elution procedure: first stage: use a mixed solution of mobile phase A and mobile phase B for elution for 30 minutes, wherein the volume percentage of mobile phase A changes from 95% to 70%, and the volume percentage of mobile phase B changes from 5% to 30%; second stage: use a mixed solution of mobile phase A and mobile phase B for elution for 20 minutes, wherein the volume percentage of mobile phase A changes from 70% to 50%, and the volume percentage of mobile phase B changes from 30% to 50%; third stage: use a mixed solution of mobile phase A and mobile phase B for elution for 10 minutes, wherein the volume percentage of mobile phase A changes from 50% to 95%, and the volume percentage of mobile phase B changes from 50% to 5%. Figure 4 The results are from liquid phase testing.

[0084] The mass spectrometry conditions were:

[0085] Acquisition mode: MSE (low energy / high energy switching scan); electrospray positive ion / negative ion scanning respectively; capillary voltage 2KV; cone voltage 40V; nebulizer gas temperature: 450℃; nebulizer gas flow rate 900L / h; cone backflush gas 50L / h; ion source temperature 115℃; scanning range 50~1000m / z; scanning speed 0.2s; collision energy 6eV / 20~45eV; online lock mass; 250pg / uL leucine enkephalin continuous injection, flow rate 10ul / min; acquisition interval 0.5s; acquisition time 0.5s. Figure 5 The results of mass spectrometry.

[0086] SEQ ID No.1

[0087]

[0088] SEQ ID No.2

[0089]

[0090] SEQ ID No.3

[0091]

Claims

1. An engineered Rhodococcus etheriferus strain that synthesizes isospongene using low-quality biomass lignin, characterized in that: The engineered Rhodococcus aetherobacterium is constructed by introducing syringate demethylase and 3-o-methylgallate 3,4-dioxygenase into Rhodococcus aetherobacterium, the coding gene sequence of the syringate demethylase is shown in SEQ ID No.1; the coding gene sequence of the 3-o-methylgallate 3,4-dioxygenase is shown in SEQ ID No.

2.

2. The engineered Rhodococcus etherivorans strain for synthesizing isospongene using low-quality biomass lignin according to claim 1, characterized in that: The invention also includes introducing a urea-inducible promoter nucleic acid sequence pami, wherein the urea-inducible promoter nucleic acid sequence pami The sequence is shown as SEQ ID No.

3.

3. The engineered Rhodococcus etherivorans strain for synthesizing isospongene using low-quality biomass lignin according to claim 1, characterized in that: The ether-loving Rhodococcus is ether-loving Rhodococcus ( Rhodococcus aetherivorans )N1, the deposit number is CCTCC NO: M 20221270.

4. The engineered Rhodococcus etherivorans strain for synthesizing isospongene using low-quality biomass lignin according to claim 1, characterized in that: The gene was expressed in plasmid pNV18.1 to construct the Rhodococcus shuttle plasmid pNV- dmm - mdo Introduced into the Rhodococcus aetherovorans N1.

5. The method for constructing the engineered Rhodococcus etherivorans according to any one of claims 1 to 4, characterized in that: Construction of expression plasmid pNV18.1 containing the gene encoding syringate demethylase dmm , 3-o-methylgallate 3,4-dioxygenase encoding gene dmo and the urea-inducible promoter nucleic acid sequence pami, a shuttle plasmid of Rhodococcus sp. dmm - dmo ; The plasmid pNV- dmm - dmo Introduced into Rhodococcus etheriferus N1 to obtain engineered Rhodococcus etheriferus N1- dmm - dmo .

6. Use of the engineered Rhodococcus etherivorans according to any one of claims 1 to 4 in the production of isospongene.

7. The use according to claim 6, characterized in that: Using lignin as carbon source, engineered Rhodococcus etherivorans was fermented and cultured under aerobic conditions to produce isospongene.

8. The use according to claim 7, characterized in that: The fermentation culture conditions are: 1) Plate culture: Streak the engineered Rhodococcus etherivorans on LB solid medium containing kanamycin at a temperature of 25-35°C for 45-50 h; 2) Seed culture: inoculate the colonies on the solid culture medium into LB seed culture medium containing kanamycin, culture temperature 25-35°C, 180rpm, culture time 28-32 h; 3) Fermentation culture: inoculate the seed culture solution into the fermentation medium, the fermentation temperature is 25-35℃, and the fermentation culture time is 120-144 h.

9. The use according to claim 7, characterized in that: The formula of the fermentation medium is 0.5-10 g / L of lignin, 1-2.5 g / L of corn syrup dry powder, 1-3 g / L of urea, and 0.5-2 mol / L of MgCl2.

10. The use according to claim 7, characterized in that pH 6, temperature 30 ℃, fermentation time is 144h.

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