CN117946914A - High-yield rhamnolipid strain, microbial inoculum, method for producing rhamnolipid and application - Google Patents

Abstract

The invention belongs to the technical field of microbial strain fermentation, and particularly relates to a high-yield rhamnolipid strain, a microbial inoculum, a method for producing rhamnolipid and application thereof, wherein the high-yield rhamnolipid strain is pseudomonas Pseudomonas aeruginosa, and is preserved in China Center for Type Culture Collection (CCTCC) No. M20232111.

Description

High-yield rhamnolipid strain, microbial inoculum, method for producing rhamnolipid and application

Technical Field

The invention belongs to the technical field of microbial strain fermentation, and particularly relates to a high-yield rhamnolipid strain, a microbial agent, a method for producing rhamnolipid and application of the rhamnolipid strain.

Background

Compared with the traditional chemical surfactant, the rhamnolipid has the advantages of more excellent surface activity and stability, no toxicity, no pollution, biodegradability, good biocompatibility and the like, thereby showing unique application prospect in various fields of medicine, food, agriculture, petroleum exploitation, environmental pollution restoration and the like.

Rhamnolipid synthase systems are naturally occurring in a variety of organisms, among which one is more involved in the synthesis of rhamnolipids in the genera Pseudomonas and bordetella (Burkholderia). The pseudomonas has higher yield of the synthesized rhamnolipid under the aerobic condition, and is a mainstream strain in the current industrial production. The biosynthetic pathway of rhamnolipids in the genus Pseudomonas has been studied to date well, dTDP-L-rhamnose and beta-hydroxy fatty acids are 2 precursor substances for rhamnolipid synthesis, dTDP-L-rhamnose is a glycosyl donor, and beta-hydroxy fatty acids are glycosyl acceptors. All of these 2 precursor species are derived from the central metabolic pathway of Pseudomonas aeruginosa, the synthesis of dTDP-L-rhamnose is derived from the ED pathway (2-keto-3-deoxy-6-phosphogluconate pathway) and the gluconeogenic pathway, and the synthesis of beta-hydroxy fatty acids is derived from the de novo fatty acid synthesis pathway and the beta oxidation pathway of fatty acids.

The rhamnolipid has wide application prospect in the fields of petroleum industry, environmental remediation, agriculture, food, medicine and the like, however, compared with a chemical surfactant, the higher raw material cost and the relatively lower synthetic yield lead to higher production cost of the rhamnolipid, and limit popularization and application of the rhamnolipid. The screening and genetic engineering of the current high-yield strain are main strategies for improving the yield of rhamnolipid and reducing the production cost.

Disclosure of Invention

The invention aims to solve the technical problem of providing a high-yield rhamnolipid strain, a microbial inoculum, a method for producing rhamnolipid and application thereof, so that the production efficiency is improved, and the production cost is reduced.

The embodiment of the invention provides a high-yield rhamnolipid strain which is pseudomonas Pseudomonas aeruginosa and is preserved in China Center for Type Culture Collection (CCTCC) No. M20232111.

The embodiment of the invention provides a microbial inoculum, which comprises the high-yield rhamnolipid strain.

The embodiment of the invention provides a method for producing rhamnolipid, which comprises the following steps of inoculating a microbial inoculum to a fermentation medium for fermentation, and separating and purifying to obtain rhamnolipid. The inoculation amount is 1-10% (v/v).

In one embodiment, the fermentation medium comprises a substrate that is rapeseed oil, coconut oil, corn oil, soybean oil, yeast extract, and/or peptone.

In one embodiment, the fermentation medium comprises 1-5g/L sodium nitrate, 0.1-0.3g/L magnesium sulfate heptahydrate, 6-8g/L disodium hydrogen phosphate, 1-3g/L potassium dihydrogen phosphate, 30-50g/L rapeseed oil and 0.1-0.3g/L potassium chloride.

Preferably, the fermentation medium comprises 3g/L of sodium nitrate, 0.2g/L of magnesium sulfate heptahydrate, 7.13g/L of disodium hydrogen phosphate, 1.76g/L of monopotassium phosphate, 40g/L of rapeseed oil and 0.2g/L of potassium chloride.

In one embodiment, the fermentation medium comprises trace elements with the addition amount of 1ml/L, wherein the trace elements contain 0.1g/L ferric chloride hexahydrate, 0.75g/L zinc sulfate heptahydrate, 0.12g/L cobalt chloride hexahydrate, 0.075g/L copper sulfate pentahydrate, 0.75g/L manganese sulfate monohydrate, 0.15g/L boric acid and 0.05g/L sodium molybdate dihydrate. The solvent of the trace element is water.

In one embodiment, the fermentation temperature is 25-37℃and the fermentation speed is 150-220 rpm.

In one embodiment, the incubation time is 72-168 hours.

The microbial inoculum is obtained by inoculating a high-yield rhamnolipid strain into an LB culture medium and culturing for 12-36 hours under the conditions of 25-37 ℃ and 150-220 rpm.

The embodiment of the invention provides application of a high-yield rhamnolipid strain, which is used for preparing and producing rhamnolipid.

The invention has the beneficial effects that the invention provides the high-yield rhamnolipid strain which is obtained through the screening method of the high-yield rhamnolipid strain, and the steps of normal pressure room temperature plasma mutagenesis, gene editing, CTAB plate screening and the like.

Compared with the related report, the shake flask fermentation unit yield is improved by 61%, and the method is at a higher production level; the rhamnolipid is prepared by fermenting the high-yield rhamnolipid strain obtained by the screening breeding method and the gene mutation method, so that the production efficiency can be greatly improved, and the production cost can be reduced.

Drawings

FIG. 1 is a graph showing the results of 72-hour fermentation of different strains.

FIG. 2 is a graph showing fermentation results of PX-S20 (PAX-1) strain.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.

Thus, all other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are intended to be within the scope of the invention.

The genetic engineering strain PAX-1 is preserved in China Center for Type Culture Collection (CCTCC), address: wuhan city Wuchang's Lojia mountain, post code: 430072, date of deposit: 2023, 11, 02, deposit number: cctccc NO: m20232111.

In the following examples, the final concentration of carbenicillin in the medium was 100ug/mL.

The primer sequence information used in examples 1 to 11 is shown in Table 1.

TABLE 1 primer sequences

The culture medium used in the examples of the present invention is as follows:

CTAB solid medium: 0.7g of potassium dihydrogen phosphate, 0.9g of disodium hydrogen phosphate, 2g of sodium nitrate, 0.4g of magnesium sulfate, 0.1g of calcium chloride, 20g of glycerin, 1mL of microelement solution and 15g of agar powder, carrying out constant volume sterilization with deionized water to 900mL, carrying out steam sterilization at 121 ℃ for 15min, cooling the temperature of the culture medium to 65 ℃, weighing 0.2g of cetyltrimethylammonium bromide and 0.005g of methylene blue, dissolving in 100mL of deionized water, adding into the culture medium through a 0.22 mu m filter membrane, uniformly mixing, and pouring into a flat plate.

LB solid medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 25g of agar and 1000mL of deionized water, and sterilizing at 121 ℃ for 15min.

LB liquid medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 1000mL of deionized water and sterilizing at 121 ℃ for 15min.

Fermentation medium: 3g of sodium nitrate, 0.2g of magnesium sulfate heptahydrate, 7.13g of disodium hydrogen phosphate, 1.76g of potassium dihydrogen phosphate, 40g of rapeseed oil, 1mL of trace elements (0.1 g/L ferric chloride hexahydrate, 0.75g/L zinc sulfate heptahydrate, 0.12g/L cobalt chloride hexahydrate, 0.075g/L copper sulfate pentahydrate, 0.75g/L manganese sulfate monohydrate, 0.15g/L boric acid, 0.05g/L sodium molybdate dihydrate, and the balance water), 0.2g of potassium chloride, 1000mL of deionized water, and sterilizing at 121 ℃ for 15min.

EXAMPLE 1 high yield rhamnolipid Pseudomonas aeruginosa screening

1) And (3) strain primary screening: sampling in a sewage disposal treatment chamber of a certain factory in Changde City of Hunan province and residual oil waste of dining halls, preliminarily filtering and separating to obtain a sample, adding 100mL of sterile normal saline into the environmental sample for dilution, shaking and uniformly mixing, standing for 1 hour, taking supernatant fluid for gradient dilution and coating on an LB plate, placing in a constant temperature incubator at 37 ℃ for culturing for 48 hours, and then picking colony clusters carrying blue-green pigment; further gradient dilution was applied to CTAB plates, and after repeated incubation at 37℃for 72 hours, colonies with dark blue halos around were picked up until 181 strains were obtained.

2) Mutagenesis screening: culturing each strain obtained by primary screening to logarithmic phase, mixing at equal ratio, centrifuging to collect thallus, washing with sterile physiological saline for 2 times, and re-suspending thallus with appropriate amount of sterile physiological saline to concentration of 10 ⁶～10⁸; roasting a metal slide glass in an ultra-clean workbench for 30s, cooling, and uniformly coating 10 mu l of bacterial liquid on the slide glass; loading the slide on an operation bin plate of an atmospheric pressure room temperature plasma (ARTP) system, and moving to a position, at which the slide is positioned at an air flow port by 2 mm; setting the power to be 100W, the air flow to be 10SLM, after 45s treatment, putting the slide into an EP tube filled with 1mL of sterile physiological saline, and vibrating for 1min; the suspension is coated on a CTAB plate for culture after being diluted in a gradient way, and colonies with large deep blue halos are picked out after the culture is carried out for 72 hours at 37 ℃; repeating the steps, carrying out mutagenesis and screening for 4 rounds, obtaining a strain of bacteria PX-F4, and streaking, purifying and storing.

3) And (3) strain identification: inoculating the strain into LB culture medium, shake culturing at 37 ℃ for 16 hours, collecting the strain, extracting bacterial genome DNA by using genome DNA extraction kit; performing PCR amplification reaction by using a 16S rDNA universal primer pair 27F/1492R, wherein the total volume of a PCR reaction system is 25 mu L, and the PCR amplification condition is 94 ℃ for 5min;94 ℃ 45s,55 ℃ 45s,72 ℃ 45s,30 cycles; 72 ℃ for 10min; the size of the PCR product is identified by using 1% agarose gel electrophoresis, a purified PCR amplified fragment is obtained by using a gel recovery kit, and sequence information is obtained by sequencing; the obtained sequence information is compared with NCBI database, and BLAST search comparison is performed to identify the strain PX-F4 as pseudomonas aeruginosa.

EXAMPLE 2 construction of genetically engineered plasmids

1) Amplifying the target fragment: performing high-fidelity PCR amplification reaction of estA gene by using genome DNA of the obtained strain as a template and using primers estA-F and estA-R, detecting the obtained PCR fragment by 1% agarose gel electrophoresis, and recovering and purifying the PCR product by using a gel recovery kit to obtain an amplified estA gene fragment (see SEQ ID NO. 1);

2) Linearization of plasmid vector: using plasmid pBBR1MCS4 as a template, amplifying a plasmid sequence by using primers pMCS4-F and pMCS4-R through high-fidelity polymerase, detecting and identifying an obtained PCR product through 1% agarose gel electrophoresis, then digesting an original plasmid template by using DpnI enzyme, reacting at 37 ℃ for 1 hour, and then carrying out glue recovery and purification of the PCR product to obtain a linearized pBBR1MCS4 plasmid vector fragment (see SEQ ID NO. 2);

3) Construction of genetic engineering plasmids: determining the concentration of estA gene fragments and pBBR1MCS4 plasmid vector fragments by using a micro nucleic acid quantitative instrument, calculating the volume ratio according to the molecular weight of the fragments and the concentration of nucleic acid, fusing the fragments by adopting an In-fusion method, and reacting at 50 ℃ for 15min to obtain recombinant plasmids;

4) Verification and amplification of recombinant plasmids: and (3) transforming the recombinant plasmid into competent cells E.coli DH5 alpha, coating and screening on a resistance plate carrying carbenicillin, selecting a monoclonal to perform colony PCR verification, selecting a colony with correct band under 1% agarose gel electrophoresis to perform plasmid extraction and sequencing identification, and finally obtaining the constructed pBBR1MCS4-estA plasmid.

Example 3 screening of high yield rhamnolipid strains based on genetic engineering methods

EstA gene codes esterase, is positioned on the outer membrane of pseudomonas aeruginosa cells, is an automatic transport protein, belongs to a V-type secretion system, and is closely related to cell movement, biofilm formation and rhamnolipid production. The gene is over-expressed, so that the generation and the excretion of the rhamnolipid of the pseudomonas aeruginosa can be promoted, and detection signals are amplified, so that the pseudomonas aeruginosa has higher and more accurate detection efficiency, and the pseudomonas aeruginosa with high rhamnolipid yield can be obtained by combining with an ARTP system mutagenesis and genetic engineering method.

1) Preparation of shock competent bacteria: inoculating PX-F4 to 5mL LB medium, and culturing at 37 ℃ and 220rpm for 16 hours; inoculating 1mL of bacterial liquid into 50mL of LB culture medium in the next day, and continuing to culture at 37 ℃ and 220rpm until the OD600 is 0.4-0.6; centrifuging at 4 ℃ and 4000rpm for 3min to collect thalli; re-suspending the thalli by using 30mL of 0.3M precooled sterile sucrose solution in ice bath, and centrifugally collecting the thalli after washing for 2 times; then 30mL of precooled 10% glycerol ice bath is used for resuspension of the thalli, after washing for 1 time, 1mL of 10% glycerol is added for resuspension, and 100 mu L of the thalli are respectively packaged in 1.5mL of EP pipes for standby;

2) Electrotransformation of recombinant plasmids: adding 2 mu L of plasmid (200 ng) into 100 mu L of electrotransformation competence, uniformly mixing, carrying out ice bath for 5min, transferring into a precooled 0.2cm electrotransformation cup, and carrying out electrotransformation under the conditions of 1.6kV, 25 mu F and 200ohms by using an electrotransformation instrument; after electric shock, 1mL of LB culture medium is added into an electric shock cup, bacterial liquid is transferred into a 1.5mL EP tube after being gently blown and evenly beaten, and the bacterial liquid is cultured for 1 hour at 37 ℃ and 220 rpm; after resuscitating and culturing, 200 mu L of bacterial liquid is coated on an LB solid plate containing 100 mu g/mL of carbenicillin, and after culturing for 16 hours at 37 ℃, monoclonal is selected, and PX-F4/pBBR1MCS4-estA bacterial strain is obtained;

3) Mutagenesis screening: PX-F4/pBBR1MCS4-estA is inoculated in a carbenicillin-resistant LB medium, cultured for 16 hours at 37 ℃ and 220rpm, and then inoculated with 2% v/v for continuous culture to the logarithmic phase; collecting thalli at 4 ℃, washing for 2 times by using sterile physiological saline, re-suspending and coating on an ARTP system metal slide, and carrying out mutagenesis treatment for 45s; resuspension metal slide glass with sterile physiological saline and shake for 1min, coating the diluted suspension on CTAB plate, culturing at 37 deg.C for 72 hr, picking colony with larger halo diameter/colony diameter ratio, performing next round of mutagenesis and screening, repeating for 20 rounds to obtain strain PXP-S20;

4) Purifying and preserving seeds: strains PXP-S1, PXP-S5, PXP-S10 and PXP-S20 of the 1 st round, the 5 th round, the 10 th round and the 20 th round in the mutagenesis process are selected, inoculated in an antibiotic-free LB culture medium, and continuously subjected to subculture for 10 generations at 37 ℃ and 220 rpm; then taking a bacterial solution three-area line to culture on an antibiotic-free LB plate for 16 hours at 37 ℃, picking a monoclonal spot to seed on the LB plate carrying the resistance of carbenicillin, culturing for 24 hours at 37 ℃ to obtain pBBR1MCS4-estA plasmid lost strains which are PX-S1, PX-S5, PX-S10 and PX-S20 (PAX-1) respectively, wherein the PAX-1 is the Pseudomonas pseudomonad PAX-1Pseudomonas aeruginosa PAX-1 of the preserved strain, and the bacterial forms of the PAX-1 strain are as follows: on an LB solid culture medium plate, a monoclonal colony is round and light yellowish in color; after gram staining, it is in the shape of a short bar under a microscope, rose red, about 0.5-1.0 x 1.2-2.0 μm.

Example 4 quantitative detection method of rhamnolipid

Sulfuric acid-anthrone reagent: 87mL of analytically pure sulfuric acid (98%) was slowly added to 13mL of water at a sulfuric acid concentration of 85%, 0.2g of anthrone was added, and stirred until the anthrone was dissolved, and the mixture was ready for use.

The rhamnolipid content was determined using the sulfuric acid-anthrone method. Diluting the fermentation liquor to a certain volume, taking 0.5mL of diluent (or rhamnose standard samples with different concentrations), adding 1mL of sulfuric acid-anthranone solution, mixing while adding, carrying out ice water bath reaction for 10min, then carrying out boiling water bath reaction for 8min, cooling to room temperature, taking deionized water as a blank control, and measuring the absorbance value at 625nm by using an enzyme-labeling instrument. Meanwhile, standard curves are established by using standard samples of rhamnose with different concentrations, and the rhamnolipid content in the fermentation broth is expressed by the measured content of the rhamnose. Multiplying the content of the rhamnolipid by a coefficient of 3.4 according to the relation between the rhamnolipid structure and the molecular weight to obtain the concentration of the rhamnolipid.

Example 5 fermentation production and yield detection

Strains PX-F4, PX-S1, PX-S5, PX-S10 and PX-S20 (PAX-1) were inoculated into LB medium, cultured at 37℃and 220rpm for 12 hours, and then inoculated into fermentation medium at 3% v/v for 100 hours at 37℃and 220 rpm. Taking bacterial liquid samples at various time points in the fermentation process; 1mL of the sample was used to determine pH and diluted 100-fold to determine OD600 absorbance; another 1mL sample was centrifuged at 4000rpm for 5min, the supernatant was diluted 50-fold, and the yield of rhamnolipid was determined according to the method of example 4; OD600 and rhamnolipid titers at 72 hours are shown in FIG. 1, pH values of PX-S20 (PAX-1) change with time, OD600 and rhamnolipid titers are shown in FIG. 2, the OD600 at 72 hours reaches 8.6, the rhamnolipid titer is 10.9g/L, and the unit yield is improved by about 61% compared with the corresponding report yield of the model strain PAO1 of 6.77 g/L.

Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of protection of the application is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the application, the steps may be implemented in any order and there are many other variations of the different aspects of one or more embodiments of the application as described above, which are not provided in detail for the sake of brevity.

One or more embodiments of the present application are intended to embrace all such alternatives, modifications and variations as fall within the broad scope of the present application. Accordingly, any omissions, modifications, equivalents, improvements and others which are within the spirit and principles of the one or more embodiments of the application are intended to be included within the scope of the application.

Claims (10)

1. A high-yield rhamnolipid strain is characterized by being pseudomonas Pseudomonas aeruginosa, and is preserved in China Center for Type Culture Collection (CCTCC) No. M20232111.

2. A microbial agent comprising the high yielding rhamnolipid strain of claim 1.

3. The method for producing rhamnolipid is characterized by comprising the following steps of inoculating the microbial inoculum of claim 2 into a fermentation medium for fermentation, and separating and purifying to obtain rhamnolipid.

4. A method of producing rhamnolipids according to claim 3, in which the fermentation medium comprises a substrate, which is rapeseed oil, coconut oil, corn oil, soybean oil, yeast extract and/or peptone.

5. The method for producing rhamnolipid according to claim 4, wherein the fermentation medium comprises 1 to 5g/L of sodium nitrate, 0.1 to 0.3g/L of magnesium sulfate heptahydrate, 6 to 8g/L of disodium hydrogen phosphate, 1 to 3g/L of potassium dihydrogen phosphate, 30 to 50g/L of rapeseed oil, and 0.1 to 0.3g/L of potassium chloride.

6. The method of producing rhamnolipid of claim 5 wherein the fermentation medium comprises 3g/L sodium nitrate, 0.2g/L magnesium sulfate heptahydrate, 7.13g/L disodium hydrogen phosphate, 1.76g/L potassium dihydrogen phosphate, 40g/L rapeseed oil, 0.2g/L potassium chloride.

7. The method for producing rhamnolipid according to any one of claims 4 to 6, wherein the fermentation medium comprises trace elements, the trace elements are added in an amount of 1ml/L, and the trace elements comprise 0.1g/L ferric chloride hexahydrate, 0.75g/L zinc sulfate heptahydrate, 0.12g/L cobalt chloride hexahydrate, 0.075g/L copper sulfate pentahydrate, 0.75g/L manganese sulfate monohydrate, 0.15g/L boric acid, and 0.05g/L sodium molybdate dihydrate.

8. The method for producing rhamnolipid according to any one of claims 4 to 6, wherein the fermentation temperature is 25 to 37 ℃ and the fermentation speed is 150to 220rpm.

9. The method for producing rhamnolipid of claim 8 wherein the incubation time is 72 to 168 hours.

10. Use of the high rhamnolipid producing strain of claim 1 for the preparation of a rhamnolipid producing strain.