A method for preparing eucommia gum by fermenting eucommia leaves with recombinant yeast

Abstract

The present invention discloses a method for producing eucommia gum by fermenting eucommia leaves with recombinant yeast. The main steps of the method include: constructing an expression vector containing the cellulase genes Cel5A / Cel6A of Trichoderma reesei, introducing Saccharomyces cerevisiae to construct an engineered yeast, and using the engineered yeast to further enzymatically hydrolyze the eucommia leaves after ultrasonic pretreatment to achieve complete release of eucommia gum, and using Soxhlet extraction to purify the eucommia gum from the fermentation product to achieve full extraction of the eucommia gum. The method of the present invention does not require high-concentration alkaline and acid solution cracking and repeated rinsing, thus avoiding the destruction of the rubber morphology during the extraction process and reducing the generation of harmful waste liquid during the extraction process. It has guiding significance for achieving green extraction of eucommia gum.

Description

Technical Field

[0001] The invention belongs to the fields of agricultural and forestry biotechnology and microbial genetic engineering, and specifically is a method for extracting eucommia gum by cracking eucommia leaves through recombinant saccharomyces cerevisiae mediation. Background Art

[0002] Eucommia ulmoides Oliver is a gum-producing plant endemic to my country, and its gum products hold great potential for development in the fields of medicine, industry, and agriculture. Eucommia gum (Eucommia ulmoides gum, EuG) is a natural polymer material that is an isomer of natural rubber (NR). EuG is a secondary metabolite of Eucommia cells, primarily synthesized and stored in the gum-containing cells. Studies have found that the content of Eucommia gum varies among different Eucommia tissues. For example, Eucommia samaras contain the highest EuG content, reaching 12-18%, while the leaves contain the lowest, at only 2-3.5%. Despite their low gum content, Eucommia leaves possess the largest biomass, making them an ideal raw material for extracting Eucommia gum. Currently, Eucommia gum extraction primarily relies on physical, chemical, and biological methods. However, the mechanical impact and shearing produced by physical methods can easily damage the gum fibers, making it difficult to separate non-gum impurities. Chemical methods, on the other hand, consume high amounts of reagents, create difficult waste disposal processes, and can easily pollute the environment. Although biological methods are mild and largely preserve the native structure of EuG, enzymatic degradation of the cell wall is inefficient, significantly impacting EuG extraction yield. Recently, researchers have discovered that some fungi (such as Trichoderma reesei and Aspergillus niger) can secrete highly active cellulases and hemicellulases. However, these fungi have complex genetic backgrounds and produce numerous secondary metabolites, making enzymatic fermentation of these enzymes unsuitable for the isolation and purification of target products. Therefore, genetically engineering yeast to enhance the functionality of these engineered bacteria has become a hot topic for many researchers. Summary of the Invention

[0003] To address the needs in the aforementioned fields, the present invention provides a method for producing eucommia gum by fermenting eucommia leaves with recombinant yeast. By constructing an expression vector containing the exogenous Trichoderma reesei cellulose-degrading genes Cel5A / Cel6A and introducing it into the recipient yeast Saccharomyces cerevisiae (S. cerevisiae), the engineered yeast is fermented with the recombinant yeast. Compared with traditional eucommia gum extraction methods, this method reduces pollution, achieves a higher extraction yield, and does not affect the properties of the gum. This method can lay the foundation for the future large-scale extraction and promotion of eucommia gum.

[0004] A recombinant expression vector is characterized in that: using pAUR101 as a base vector, the alcohol dehydrogenase (ADH) gene promoter sequence (PADH1) and the nitric oxide synthase (NOS) gene terminator sequence are ligated to the expression vector pAUR101 to obtain an intermediate expression vector pAUR101-PADH1, and then the Trichoderma reesei (T. reesei) endoglucanase Cel5A gene or the Trichoderma reesei (T. reesei) exoglucanase Cel6A gene are respectively ligated to the intermediate vector to obtain a recombinant expression vector, the nucleotide sequence of the Cel5A gene is shown in SEQ ID No. 1, and the nucleotide sequence of the Cel6A gene is shown in SEQ ID No. 2.

[0005] The recombinant expression vector is named pAUR101-PADH1::Cel5A or pAUR101-PADH1::Cel6A, and its structure is as follows Figure 2 or Figure 3 shown.

[0006] The recombinant Saccharomyces cerevisiae engineered bacteria contains the above-mentioned recombinant expression vector pAUR101-PADH1::Cel5A or pAUR101-PADH1::Cel6A.

[0007] The Saccharomyces cerevisiae strain is INVSc1 # , resistant to ampicillin and cannabinoids.

[0008] A method for producing eucommia gum by fermenting eucommia leaves with recombinant saccharomyces cerevisiae comprises the following steps:

[0009] 1) Eucommia leaf fermentation pretreatment: Naturally air-dried Eucommia leaves were crushed into 2-4 mm tissue fragments, added with 0.1 g / L SDS and 50% ethanol, and sonicated; then boiled to remove chlorophyll, filtered, and set aside;

[0010] 2) Eucommia ulmoides leaf fermentation process: activating the two recombinant Saccharomyces cerevisiae engineered bacteria containing the Cel5A gene and the Cel6A gene of claim 3 or 4, adding pretreated Eucommia ulmoides leaves, and fermenting under the following fermentation process conditions: a solid-liquid ratio of 1:10, fermentation at 30°C, pH 7, and fermentation for 120 hours;

[0011] 3) Extracting Eucommia gum: taking out the fermented solids, filtering, air-drying, and then heating and refluxing with petroleum ether as solvent, and then cooling the solution to precipitate, and air-drying the precipitate to obtain Eucommia gum.

[0012] The volume ratio of the two recombinant Saccharomyces cerevisiae engineered strains is 1:1.

[0013] The activation is to inoculate the recombinant Saccharomyces cerevisiae engineered bacteria into 5L potato dextrose liquid culture medium (Potato Dextrose, PD) at an inoculation ratio of 1:100 and ferment. The preparation method of the recombinant Saccharomyces cerevisiae engineered bacteria is to introduce the expression vector pAUR101-PADH1::Cel5A or pAUR101-PADH1::Cel6A in claim 2 into Escherichia coli (E. coli DH5α) after enzyme digestion verification; then linearize it using Stu I enzyme digestion, and transform the recipient Saccharomyces cerevisiae by heat shock method after gel recovery and purification. The heat shock conditions are: 42°C, heat shock for 30 minutes.

[0014] The expression vector pAUR101-PADH1::Cel5A or pAUR101-PADH1::Cel6A is constructed by using pAUR101 as a base vector, connecting the alcohol dehydrogenase (ADH) gene promoter sequence (PADH1) and the nitric oxide synthase (NOS) gene terminator sequence to the expression vector pAUR101 to construct an intermediate expression vector pAUR101-PADH1, and then connecting the Trichoderma reesei (T. reesei) endoglucanase Cel5A gene or the Trichoderma reesei (T. reesei) exoglucanase Cel6A gene to the intermediate vector to obtain the obtained vector. The nucleotide sequence of the Cel5A gene is shown in SEQ ID No. 1, and the nucleotide sequence of the Cel6A gene is shown in SEQ ID No. 2.

[0015] The method for connecting the Cel5A gene or Cel6A gene to the intermediate vector is to use Bam HI and Kpn I to double-digest pAUR101-PADH1, connect the Cel5A and Cel6A genes to the intermediate vector respectively via T4 DNA ligase, and finally construct expression vectors pAUR101-PADH1::Cel5A and pAUR101-PADH1::Cel6A.

[0016] The ultrasonic treatment is: treating at an ultrasonic frequency of 20 kHz, 45° C., and ultrasonicating for 30 minutes, and the boiling temperature is 80° C.; the reflux is hot reflux extraction at 90° C. for 12 hours; and the cooling is cooling the solution at -20° C. for 3 hours.

[0017] The eucommia leaves are all from the middle section of eucommia ulmoides L.var.Huazhong No.6, a 3-year-old tree.

[0018] The method of the present invention for preparing eucommia gum by fermenting eucommia leaves with recombinant yeast is as follows:

[0019] 1) Construction of recombinant yeast

[0020] 2) Collecting Eucommia ulmoides leaves and air-drying them;

[0021] 3) Eucommia leaf fermentation pretreatment: First, crush the Eucommia leaves into approximately 3mm tissue fragments, ultrasonicate them, add 0.1g / L SDS and 50% ethanol, and sonicate for 30 minutes at a frequency of 20kHz and 45°C. Then, boil at 80°C to remove chlorophyll, filter, and set aside.

[0022] 4) Using recombinant Saccharomyces cerevisiae to activate the culture, inoculate 5 L of potato dextrose liquid medium at a ratio of 1:100 and ferment. After 48 hours of fermentation, add Eucommia ulmoides leaves;

[0023] 5) The main fermentation conditions were a solid-liquid ratio of 1:10, 30°C, pH 7, and fermentation for 120 h;

[0024] 6) The fermented solids were removed, filtered, air-dried, and then extracted with petroleum ether at 90°C for 12 hours under reflux.

[0025] 7) Cool the extract at -20°C for 3 h to separate out the precipitate, discard the petroleum ether (petroleum ether can be recycled), and allow the precipitate to air-dry naturally to obtain the Eucommia ulmoides fine gum.

[0026] Note:

[0027] ① After the above pretreatment, the Eucommia leaves need to be sterilized at high temperature before fermentation;

[0028] ② The above recombinant Saccharomyces cerevisiae is divided into two types, namely recombinant Cel5A genotype and recombinant Cel6A genotype. When the strains are activated and inoculated, the volume ratio of the two strains is 1:1.

[0029] ③The recombinant yeast expression vector is pAUR101, which is a chromosome-integrated shuttle vector of Saccharomyces cerevisiae. It cannot replicate autonomously in yeast cells and can only exist stably by integrating it into the yeast chromosome through a recombinase. pAUR101 contains the mutant AUR1-C gene as a screening marker. During yeast transformation, Aureobasidin A (AbA) can be used for resistance screening. The vector can be linearized by a single-cutting restriction enzyme (BstP I, EcoO65 I, BsiW I or Stu I) in the AUR1-C gene, and then effectively integrated into the host chromosome by homologous recombination;

[0030] ④ The Saccharomyces cerevisiae strain is INVSc1 # This strain is a fast-growing diploid yeast cell that is auxotrophic for His, Leu, Trp, and Ura and cannot grow in SC minimal media lacking histidine, leucine, tryptophan, and uracil. Its genotype is as follows: MATa his3Δ1 leu2 trp1-289 ura3-52 / MATα his3Δ1 leu2 trp1-289 ura3-52;

[0031] ⑤ The Eucommia leaves are all from the middle sections of 3-year-old Eucommia trees in the Eucommia resource garden of the Agricultural Bioengineering Research Institute of Guizhou University. The Eucommia variety is: Huazhong No. 6 (Eucommia ulmoides L.var. Huazhong No. 6).

[0032] The invention establishes an expression vector containing the cellulase gene Cel5A / Cel6A of Trichoderma reesei, introduces Saccharomyces cerevisiae into the vector to construct an engineered yeast, and uses the engineered yeast to further enzymatically hydrolyze eucommia leaves after ultrasonic pretreatment to achieve complete release of eucommia gum. The fermentation product is subjected to Soxhlet extraction to purify the eucommia gum, thereby achieving full extraction of the eucommia gum.

[0033] The present invention does not require acid and alkali treatment and large amounts of water washing, and has the following advantages:

[0034] First, this method better preserves the morphological structure of Eucommia rubber. The exogenous cellulase enzymes from engineered Saccharomyces cerevisiae effectively enzymatically degrade the cell walls of Eucommia leaves, enabling efficient extraction of Eucommia rubber. Furthermore, compared to traditional Eucommia rubber extraction methods, this method offers less pollution, a higher extraction yield, and no compromise of Eucommia rubber's properties.

[0035] Second: The Eucommia rubber extraction method obtained in this study can improve the extraction rate of Eucommia leaves to a certain extent, with an extraction rate of 2.35%

[0036] Third: High repeatability and universality. The processing method of materials is unified and easy to repeat.

[0037] Fourth: The eucommia rubber obtained by the eucommia rubber extraction method in this study is mainly filamentous eucommia rubber with the highest average molecular weight is 29.283×10 4 The obtained Eucommia gum is a high molecular weight gum. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Intermediate vector construction and enzyme digestion diagram (lane 1 in the figure is the undigested control group, and lane 2 is the enzyme digestion group)

[0039] Figure 2 Cel5A vector construction and enzyme digestion diagram (lane 1 in the figure is the undigested control group, and lane 2 is the enzyme digestion group)

[0040] Figure 3 Cel6A vector construction and enzyme digestion diagram (lane 1 in the figure is the undigested control group, and lane 2 is the enzyme digestion group)

[0041] Figure 4 Yeast transformation colony screening

[0042] A, Transformed Cel5A-positive colony. B, Transformed Cel6A-positive colony

[0043] Figure 5 PCR electrophoresis diagram of yeast transformation screening colonies,

[0044] A, PCR of positive colonies transformed with Cel5A. B, PCR of positive colonies transformed with Cel6A.

[0045] (Lanes 1, 2, 3, 4, and 5 are strain-positive colony PCR)

[0046] Figure 6 Eucommia gum preparation process flow chart,

[0047] Figure 7 Picture of Eucommia gum after fermentation,

[0048] Figure 8 Molecular weight distribution of Eucommia gum after fermentation and extraction by alkali leaching method, DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to the embodiments.

[0050] Terms and culture medium of the present invention:

[0051] The yeast culture in the present invention is generally cultured using PDA medium (yeast extract 10 g / L + peptone 10 g / L + glucose 10 g / L, pH 7.2).

[0052] 1. Obtaining recombinant yeast

[0053] 1.1 Target gene selection and optimization

[0054] Based on literature reports, the coding sequences of the T. reesei endoglucanase Cel5A (Gene ID: 18482842) and exoglucanase Cel6A (Gene ID: 18488147) genes were retrieved using NCBI accession numbers. Codons with a coding frequency of less than 20% were codon-optimized using the online software ExpOptimizer (https: / / www.novopro.cn / tools / codon-optimization.html) (Akcapinar et al. 2011). Furthermore, to achieve targeted expression of the target genes, an α-factor signal peptide was added to the front of both genes, and the GC content was adjusted to 40%-50%. The optimized gene sequences were fully synthesized by Jin Kairui (Wuhan). See SEQ ID No. 1 and SEQ ID No. 2.

[0055] 1.2 Expression vector construction

[0056] The artificially synthesized S.cerevisiae alcohol dehydrogenase (ADH) gene promoter sequence (PADH1) and nitric oxide synthase (NOS) gene terminator sequence (see SEQ ID No. 3) were connected to the expression vector pAUR101 to construct the intermediate expression vector pAUR101-PADH1 ( Figure 1 , intermediate vector construction and enzyme digestion map). pAUR101-PADH1 was then double-digested with Bam HI and Kpn I, and the Cel5A and Cel6A genes were ligated to the intermediate vector using T4 DNA ligase, and finally the expression vector pAUR101-PADH1::Cel5A ( Figure 2 Cel5A vector construction and enzyme digestion map) and pAUR101-PADH1::Cel6A ( Figure 3 Cel6A vector construction and enzyme digestion map). At the same time, the two constructed expression vectors were verified by enzyme digestion and then introduced into Escherichia coli (E. coli DH5α) for storage.

[0057] 1.3 Yeast transformation and PCR verification

[0058] The expression vector was linearized using Stu I digestion and purified by gel extraction. The recipient Saccharomyces cerevisiae was transformed using the LiAC / PEG heat shock method. Heat shock conditions were: 42°C for 30 minutes. 200 μL of the resuspended bacteria were then plated on PDA medium containing AbA (0.3 μml / L) for screening. Figure 4 ), the obtained positive colonies were verified by PCR amplification ( Figure 5), colony amplification system see Table 1, primers see Table 2, and positive strains Cel5A-S were obtained respectively. # , and Cel6A-S # .

[0059] The above colony PCR amplification system is as follows:

[0060] Table 1 Cel5A / Cel6A gene colony PCR amplification system

[0061]

[0062] Table 2 PCR verification primer sequences

[0063]

[0064] 2. Recombinant Saccharomyces cerevisiae fermentation

[0065] 2.1 Pre-fermentation treatment

[0066] Eucommia leaves were collected and air-dried. For fermentation pretreatment, 10 g of dry Eucommia leaves were crushed into approximately 3 mm fragments. Ultrasonic treatment was then performed. 0.1 g / L SDS and 50% ethanol were added and ultrasonicated for 30 minutes at a frequency of 20 kHz and 45°C. The mixture was then boiled at 80°C to remove chlorophyll. The mixture was filtered and set aside.

[0067] 2.2 Activation of fermentation bacteria

[0068] The cells were activated using recombinant Saccharomyces cerevisiae engineering bacteria at an inoculation ratio of 1:100 (Cel5A recombinant Saccharomyces cerevisiae engineering bacteria: Cel56 recombinant Saccharomyces cerevisiae engineering bacteria volume ratio of 1:1), inoculated into 5 L of yeast glucose liquid medium (YPD) and fermented. Eucommia ulmoides leaves were added after 48 h of fermentation.

[0069] 2.3 Fermentation conditions

[0070] The main fermentation conditions were a material-liquid ratio of 1:10, 30°C, pH 7, and fermentation for 120 h ( Figure 6 ).

[0071] 3. Soxhlet extraction and molecular weight determination

[0072] 3.1 Soxhlet extraction conditions

[0073] The fermentation solids were taken out, filtered, and naturally air-dried, and then extracted with petroleum ether as the liquid phase at 90 ° C for 12 hours under hot reflux; the extract was placed at -20 ° C for 3 hours to precipitate, and the petroleum ether was discarded (petroleum ether can be recycled). The precipitate was naturally air-dried to obtain Eucommia ulmoides fine gum ( Figure 7 );

[0074] 3.2 Calculation of Eucommia Gum Yield

[0075] After extracting the crude gum from Eucommia ulmoides, the gum extraction rate was calculated using the following formula:

[0076]

[0077] Where: P1 is the yield of Eucommia ulmoides fine rubber, that is, the proportion of Eucommia ulmoides fine rubber in the dry weight of leaves;

[0078] M1 is the dry mass of Eucommia ulmoides leaves;

[0079] M2 is the dry mass of the refined rubber obtained;

[0080] The extraction rates of the three control groups were 2.67%, 2.10%, and 2.28%, respectively. After statistical analysis, the extraction rate was 2.35±0.016%. The rubber yields of the alkaline hydrolysis control group were 0.65%, 0.61%, and 0.57%, with an average of 0.61%. (See control group experiment)

[0081] 3.2 Molecular weight determination

[0082] In order to verify the difference in the characteristic properties of the molecular weight of Eucommia gum in this experiment and that of the alkali extraction method, the Eucommia gum extracted above was placed at 37°C and dried to constant weight. 10-20 mg of the sample was accurately weighed and dissolved in 10 mL of THF solution at 37°C and filtered with a 0.22 μL organic phase filter. The molecular weight of Eucommia gum was determined by size exclusion chromatography (SEC). Chromatographic conditions: the mobile phase was tetrahydrofuran (THF), the liquid phase flow rate was 0.35 mL / min, the sample was diluted to 1-2 mg / mL, the column temperature was set to 40°C, 6 replicates were set for each group, and a standard curve was established based on the peak elution times (5.972, 7.273, 9.198, 10.73, 10.833, 11.165) corresponding to the mixed standard (Mw = 706000, 96400, 5970, 474, 370, 266). The weight-average molecular weight of Eucommia gum can be directly output by GPC dedicated software (https: / / www.agilent.com.cn / ) Figure 8 ).

[0083] Comparative experiment: Extraction of Eucommia gum by alkali leaching (control group)

[0084] Accurately weigh 8.00 g of Eucommia leaf powder, add 100 mL of 10% NaOH solution, pretreat the Eucommia peel at 90°C for 3 hours, filter, rinse with distilled water to a pH of 7.0, and then dry in a 50°C oven to constant mass to obtain crude Eucommia gum. Extract with petroleum ether at reflux at 90°C for 26 hours. The extract is frozen at -20°C for 3 hours. After the Eucommia gum precipitates, remove (natural evaporation) or recover (centrifugal collection) the petroleum ether. The extracted Eucommia gum is then dried in a 37°C oven.

[0085] The results showed that the molecular weight of the Eucommia gum extracted from different groups showed five distinct peaks, which was similar to the results reported. In the high molecular weight distribution peak, the average molecular weight of the Eucommia gum extracted from each group ( - Mw) were significantly higher than those of the control group (without pretreatment). Among them, the average molecular weight of the fermentation group was the highest at 29.283×10 4 , which is nearly 20 times higher than the control group (the molecular weight of the control group was 1.701×10 4 ).

Claims

1. A method for producing eucommia gum by fermenting eucommia leaves with recombinant Saccharomyces cerevisiae, comprising the following steps: 1) Eucommia leaf fermentation pretreatment: Naturally air-dried Eucommia leaves were crushed into 2-4 mm tissue fragments, added with 0.1 g / L LSDS and 50% ethanol, and sonicated. The leaves were then boiled to remove chlorophyll and filtered for later use. 2) Eucommia ulmoides leaf fermentation process: Activation of exogenous Cel5A Gene or Cel6A Two recombinant Saccharomyces cerevisiae engineered strains with the same gene were added with pretreated Eucommia ulmoides leaves for fermentation. The fermentation process conditions were a solid-liquid ratio of 1:10, fermentation at 30°C, pH 7, and fermentation for 120 h. 3) Extraction of Eucommia gum: take out the fermented solids, filter, air dry and then heat with petroleum ether as solvent, reflux, then cool the solution to precipitate, and air dry the precipitate to obtain Eucommia gum. Cel5A Gene or Cel6A The gene is derived from Trichoderma reesei.

2. The method according to claim 1, wherein the volume ratio of the two recombinant Saccharomyces cerevisiae engineered bacteria is 1:

1.

3. The method according to claim 2, wherein the activation is performed by inoculating the recombinant Saccharomyces cerevisiae engineered bacteria into 5 L of potato dextrose liquid culture medium at an inoculation ratio of 1:100 and fermenting.

4. The method according to claim 3, wherein the preparation method of the recombinant Saccharomyces cerevisiae engineered bacteria comprises the following steps: PADH1::Cel5A or pAUR101- PADH1::Cel6A After enzyme digestion verification, the cells were introduced into Escherichia coli DH5α. The cells were then linearized using Stu I enzyme digestion, purified by gel recovery, and transformed into the recipient Saccharomyces cerevisiae using the heat shock method. The heat shock conditions were: 42°C, heat shock for 30 min. The expression vector pAUR101- PADH1::Cel5A or pAUR101- PADH1::Cel6A The construction method is as follows: using pAUR101 as the base vector, the promoter sequence of the alcohol dehydrogenase (ADH) gene and the terminator sequence of the nitric oxide synthase (NOS) gene are connected to the expression vector pAUR101 to construct the intermediate expression vector pAUR101-PADH1, and then the Trichoderma reesei endoglucanase is inserted into the expression vector pAUR101-PADH1. Cel5A Trichoderma reesei exoglucanase Cel6A The gene is connected to the intermediate vector and obtained. Cel5A The nucleotide sequence of the gene is shown in SEQ ID No.

1. Cel6A The nucleotide sequence of the gene is shown in SEQ ID No.

2.

5. The method according to claim 4, Cel5A Gene or Cel6A The method of connecting genes to intermediate vectors is to use Bam HI and Kpn I double enzyme digestion of pAUR101- PADH1 , ligated by T4 DNA ligase Cel5A or Cel6A The genes were connected to the intermediate vectors and finally constructed into the expression vector pAUR101- PADH1::Cel5A or pAUR101- PADH1:: Cel6A .

6. The method according to claim 1, wherein the ultrasonic treatment is: ultrasonic treatment at an ultrasonic frequency of 20 kHz, 45°C, and ultrasonic treatment for 30 minutes, the boiling temperature is 80°C; the reflux is hot reflux extraction at 90°C for 12 hours; the cooling is cooling the solution at -20°C for 3 hours; the Eucommia leaves are all from the middle section of Eucommia ulmoides variety Huazhong No. 6 and are 3-year-old trees.

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