A transformation vector containing a lignin decomposing enzyme gene, an engineering bacterium and its application

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a transformation vector, an engineered bacterium and an application thereof containing a lignin decomposing enzyme gene. The transformation vector provided by the present invention inserts a target gene (lignin decomposing enzyme gene) into the downstream of the α-MF signal peptide gene of a pKLAC1 vector, so that the expressed target protein is secreted outside the cell under the guidance of the α-MF signal peptide; at the same time, the linear expression frame of the pKLAC1 vector cut by SacⅡ does not carry any antibiotic resistance gene, but carries a fungal acetamidase gene (amdS). When constructing an engineered bacterium, 5mM acetamide (acetamide) can be added to a yeast carbon source basal medium (YCB) to screen transformants, and there is no drug resistance or biosafety hazard.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a transformation vector containing a lignin decomposing enzyme gene, an engineering bacterium and applications thereof. Background Art

[0002] Since Glenn et al. (1983) first isolated lignin peroxidase (LIP) from the culture medium of white rot fungi (P. chrysosporium), people have paid extensive attention to lignin decomposing enzymes. Because lignin decomposing enzymes can degrade plant cell wall components with a solid phenylpropane structure, as well as polycyclic aromatic hydrocarbons, chlorinated aromatic hydrocarbons, dyes, pesticides and other difficult-to-degrade benzene ring organic pollutants, they are of great significance in the reuse of highly lignified biomaterials and the control of environmental pollution.

[0003] Since the 1980s, when ligninase was isolated from the culture fluid of white rot fungi, researchers have determined the physical and chemical properties of ligninase through a large number of experiments and studies, and also developed ligninase gene recombinant microorganisms [Tien M, Tu CDP. Cloning and sequencing of a cDNA for a ligninase from phanerochaete chrysosprium. Nature, 1987, 326: 520–523]. In the development and utilization of enzyme-producing bacteria resources, extensive research has been carried out mainly on the screening and cultivation of ligninase and its enzyme-producing bacteria, the optimization of ligninase production process and regulation technology, and some achievements have been made. With the continuous upgrading of molecular biological research methods, there have also been major breakthroughs in the research and development of transgenic engineering bacteria and their application. Through the research of gene cloning and gene recombinant microorganisms, different types of enzyme-producing gene recombinant engineering bacteria have been obtained, including transgenic Escherichia coli, Aspergillus, yeast, etc., and active heterologous expression of ligninase has been successfully obtained, laying the foundation for the production of ligninase using transgenic engineering bacteria.

[0004] However, to realize the industrialized mass production of lignin degrading enzymes, a series of problems still need to be solved. First, the exocytosis performance of heterologous expressed lignin degrading enzymes is poor, and the production of lignin degrading enzymes is generally low; second, in the current preparation process of genetically recombinant enzyme-producing engineered bacteria, most of them use antibiotic resistance screening methods, which intentionally or unintentionally increase the drug-resistant bacteria population, and some engineered bacteria clones are pathogenic (Escherichia coli), which poses a biosafety hazard in practical applications, bringing certain difficulties to the realization of industrialized mass production. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a transformation vector containing a lignin decomposing enzyme gene, an engineered bacterium and its application. The engineered bacterium constructed by the transformation vector provided by the present invention not only has a strong exocrine performance of the expressed protein, but also has no drug resistance or biosafety risks in the screening method.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a transformation vector containing a lignin decomposing enzyme gene, wherein the basic vector of the transformation vector comprises a pKLAC1 vector; the lignin decomposing enzyme gene comprises a lipH8 gene, a lip2 gene, a mnp3 gene or a mnp2 gene;

[0008] When the lignin decomposing enzyme gene is the lipH8 gene, the lipH8 gene is located between the BglⅡ and StuⅠ restriction sites of the basic vector;

[0009] When the lignin decomposing enzyme gene is the lip2 gene, the lip2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector;

[0010] When the lignin decomposing enzyme gene is the mnp2 gene, the mnp2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector;

[0011] When the lignin decomposing enzyme gene is the mnp3 gene, the mnp3 gene is located between the AvrⅡ and StuⅠ restriction sites of the basic vector.

[0012] The present invention also provides an engineering bacterium that secretes lignin decomposing enzymes, and the construction method of the engineering bacterium comprises the following steps:

[0013] The above transformation vector is cut with SacⅡ enzyme to obtain a linear expression frame containing the lignin decomposing enzyme gene;

[0014] The linear expression frame was inserted into the LAC4 promoter homology frame of the lactic acid Kluyveromyces genome, and engineered bacteria that secrete lignin decomposing enzymes were screened and named GG799-TgN (pKLAC1-lipH8)-LCEL1, GG799-TgN (pKLAC1-lip2)-LCEL1, GG799-TgN (pKLAC1-mnp3)-LCEL1, and GG799-TgN (pKLAC1-mnp2)-LCEL1, respectively.

[0015] Preferably, the screening culture medium comprises: YCB culture medium with acetamide as nitrogen source.

[0016] The present invention also provides the use of the above expression vector or the above engineering bacteria in preparing lignin decomposing enzyme.

[0017] Preferably, the method comprises the following steps:

[0018] The engineered bacteria are cultured in an induction medium containing galactose to obtain a lignin decomposing enzyme.

[0019] Preferably, the induction medium comprises a carbon source basal medium, acetamide 5 mmol / L and galactose 20 g / L, or a YP medium and galactose 20 g / L;

[0020] The carbon source basic culture medium includes YCB culture medium, YCB Gal Culture medium;

[0021] Preferably, the YCB Gal The culture medium is prepared by replacing the glucose in the YCB medium with an equal mass of galactose;

[0022] The YP culture medium includes: 10 g / L yeast extract and 20 g / L peptone.

[0023] Preferably, the induction medium further comprises 33-100 mg / L of hemin and 0-100 μL / L of composite protease inhibitor.

[0024] Preferably, the culture temperature is 20-30° C. and the culture time is 24-60 h.

[0025] Preferably, before culturing the engineered bacteria in the induction medium, the process further comprises: pre-culturing the engineered bacteria in a YCB medium with acetamide as a nitrogen source for 12 to 48 hours.

[0026] Preferably, after the culturing, the method further comprises: centrifuging the culture solution after the culturing to obtain a supernatant and a precipitate; the supernatant contains a soluble lignin decomposing enzyme;

[0027] The precipitate is mixed with an extract to obtain a lignin decomposing enzyme and a bacterial precipitate dissolved in the extract;

[0028] The extract comprises glycerol and a buffer; the buffer comprises a sodium tartrate buffer or a sodium succinate buffer.

[0029] Beneficial effects:

[0030] The invention provides a transformation vector containing a lignin decomposing enzyme gene, wherein the basic vector of the transformation vector comprises a pKLAC1 vector; the lignin decomposing enzyme gene comprises a lipH8 gene, a lip2 gene, an mnp3 gene or an mnp2 gene; when the lignin decomposing enzyme gene is a lipH8 gene, the lipH8 gene is located between BglⅡ and StuⅠ restriction sites of the basic vector; when the lignin decomposing enzyme gene is a lip2 gene, the lip2 gene is located between BglⅡ and KpnⅠ restriction sites of the basic vector; when the lignin decomposing enzyme gene is a mnp2 gene, the mnp2 gene is located between BglⅡ and KpnⅠ restriction sites of the basic vector; when the lignin decomposing enzyme gene is a mnp3 gene, the mnp3 gene is located between AvrⅡ and StuⅠ restriction sites of the basic vector. The transformation vector provided by the present invention inserts the target gene (lignin decomposing enzyme gene) into the downstream of the α-MF signal peptide gene of the pKLAC1 vector, so that the expressed target protein is secreted outside the cell under the guidance of the α-MF signal peptide; at the same time, the linear expression frame of the SacⅡ-cut pKLAC1 vector does not carry any antibiotic resistance gene, but carries the fungal acetamidase gene (amdS). When constructing the engineered bacteria, 5mM acetamide can be added to the yeast carbon source basal medium (YCB) to screen the transformants, and there is no drug resistance or biosafety hazard. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0032] Figure 1 The cDNA fragments of target enzymes (Lip, Mnp) were cloned from the hyphae of white rot fungi cultured under low nitrogen conditions by RT-PCR.

[0033] Figure 2 The target gene and vector DNA fragments are cut out using specific restriction endonucleases after amplifying the target gene using the pGEMT Easy vector;

[0034] Figure 3 It is the target gene DNA band purified by gel purification system;

[0035] Figure 4 To construct the target enzyme expression vector, i.e., PKLAC1-lip, schematic diagram of the mnp gene structure;

[0036] Figure 5 PCR identification of clones for constructing pKLAC1-target enzyme expression vector;

[0037] Figure 6The amplified expression vector is cut with SacⅡ for linear expression frame DNA and vector DNA; a is the linear expression frame DNA and vector DNA, and b is the linear expression frame DNA after gel purification;

[0038] Figure 7 Schematic diagram of the linear expression cassette gene structure cut with SacⅡ;

[0039] Figure 8 This is the result of PCR identification of clones after transformation of K. lactis with linear expression cassette DNA;

[0040] Fig. 9 For gene-transformed K. lactis strains screened with acetamide selection medium;

[0041] Fig.10 It is the SDS-PAGE result of expressing the target enzyme protein under specific nitrogen source (acetamide) conditions; wherein C is the control group, L8-48 is the protein expression result of the engineered bacteria prepared in Example 5 after 48h of induced culture, L2-48 is the protein expression result of the engineered bacteria prepared in Example 6 after 48h of induced culture, M3-48 is the protein expression result of the engineered bacteria prepared in Example 7 after 48h of induced culture, M2-48 is the protein expression result of the engineered bacteria prepared in Example 8 after 48h of induced culture, L8-72 is the protein expression result of the engineered bacteria prepared in Example 5 after 72h of induced culture, L2-72 is the protein expression result of the engineered bacteria prepared in Example 6 after 72h of induced culture, M3-72 is the protein expression result of the engineered bacteria prepared in Example 7 after 72h of induced culture, and M2-72 is the protein expression result of the engineered bacteria prepared in Example 8 after 72h of induced culture;

[0042] Fig.11 It is the SDS-PAGE result of expressing the target enzyme protein under the segmented culture condition; wherein Con is the control group, L8 is the protein expression result in the enzyme concentrate after segmented culture of the engineering bacteria prepared in Example 5, L2 is the protein expression result in the enzyme concentrate after segmented culture of the engineering bacteria prepared in Example 6, M3 is the protein expression result in the enzyme concentrate after segmented culture of the engineering bacteria prepared in Example 7, M2 is the protein expression result in the enzyme concentrate after segmented culture of the engineering bacteria prepared in Example 8, L8-I is the protein expression result in the glycerol solution extract after segmented culture of the engineering bacteria prepared in Example 5, L2-I is the protein expression result in the glycerol solution extract after segmented culture of the engineering bacteria prepared in Example 6, M3-I is the protein expression result in the glycerol solution extract after segmented culture of the engineering bacteria prepared in Example 7, and M2-I is the protein expression result in the glycerol solution extract after segmented culture of the engineering bacteria prepared in Example 8;

[0043] Fig.12The results of Lip enzyme activity detection in glycerol extracts under segmented culture conditions; wherein L8-3-I-2.7 is the glycerol extract of the engineered bacteria prepared in Example 5 after segmented culture, and the pH value of the enzyme reaction solution is 2.7; L8-3-I-4.5 is the glycerol extract of the engineered bacteria prepared in Example 5 after segmented culture, and the pH value of the enzyme reaction solution is 4.5; L2-5-I-4.5 is the glycerol extract of the engineered bacteria prepared in Example 6 after segmented culture, and the pH value of the enzyme reaction solution is 4.5;

[0044] Fig.13 The results of the decolorization reaction of Azure B dye by Lip enzyme in glycerol extract under segmented culture conditions; C is the control group, 1 to 15 are the amounts of glycerol extract added, in μL;

[0045] Fig.14 The effect of adding different concentrations of Hemin on the Mnp enzyme activity; M3 is the result of Mnp enzyme activity in the enzyme concentrate after segmented culture of the engineered bacteria prepared in Example 7, and M3-I is the result of Mnp enzyme activity in the glycerol extract after segmented culture of the engineered bacteria prepared in Example 7; M2 is the result of Mnp enzyme activity in the enzyme concentrate after segmented culture of the engineered bacteria prepared in Example 8, and M2-I is the result of Mnp enzyme activity in the glycerol extract after segmented culture of the engineered bacteria prepared in Example 8; abc: indicates significant difference in T test, p≤0.05;

[0046] Fig.15 The effect of adding different concentrations of composite protease inhibitor (Pi) on Mnp enzyme activity; M3 is the result of Mnp enzyme activity in the enzyme concentrate after segmented culture of the engineered bacteria prepared in Example 7, and M3-I is the result of Mnp enzyme activity in the glycerol extract after segmented culture of the engineered bacteria prepared in Example 7; M2 is the result of Mnp enzyme activity in the enzyme concentrate after segmented culture of the engineered bacteria prepared in Example 8, and M2-I is the result of Mnp enzyme activity in the glycerol extract after segmented culture of the engineered bacteria prepared in Example 8; abc: indicates significant difference in T test, p≤0.05;

[0047] Fig.16 For YCB Gal SDS-PAGE results of target protein expression under culture conditions; (a) is the SDS-PAGE result of enzyme concentrate under YCB culture conditions; (b) is the SDS-PAGE result of YCB Gal The results of SDS-PAGE of enzyme concentrate under culture conditions; (c) is the results of SDS-PAGE of Kex2 treatment of YCB culture enzyme concentrate; C is the control; L8~M2 is soluble LipH8~Mnp2; -k is Kex2 enzyme treatment;

[0048] Fig.17 For YCB GalComparison of the specific activity of Mnp enzyme produced under different segmented culture conditions; T20:YCB Gal Culture; T13~T19: segmented culture; abcdef: indicates significant difference in T test, p≤0.05;

[0049] Fig.18 is the specific activity of Mnp enzyme in glycerol extract under different segmented culture conditions; T13-I~T19-I: glycerol extracts of different segmented culture conditions; abcd: indicates significant difference in T test, p≤0.05. DETAILED DESCRIPTION

[0050] The present invention provides a transformation vector containing a lignin decomposing enzyme gene, wherein the basic vector of the transformation vector comprises a pKLAC1 vector; the lignin decomposing enzyme gene comprises a lipH8 gene, a lip2 gene, a mnp3 gene or a mnp2 gene;

[0051] When the lignin decomposing enzyme gene is the lipH8 gene, the lipH8 gene is located between the BglⅡ and StuⅠ restriction sites of the basic vector;

[0052] When the lignin decomposing enzyme gene is the lip2 gene, the lip2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector;

[0053] When the lignin decomposing enzyme gene is the mnp3 gene, the mnp3 gene is located between the AvrⅡ and StuⅠ restriction sites of the basic vector;

[0054] When the lignin decomposing enzyme gene is the mnp2 gene, the mnp2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector.

[0055] The present invention also provides an engineering bacterium that secretes lignin decomposing enzymes, and the construction method of the engineering bacterium comprises the following steps:

[0056] The above transformation vector is cut with SacⅡ enzyme to obtain a linear expression frame containing the lignin decomposing enzyme gene;

[0057] The linear expression frame was inserted into the LAC4 promoter homology frame of the lactic acid Kluyveromyces genome, and engineered bacteria that secrete lignin decomposing enzymes were screened and named GG799-TgN (pKLAC1-lipH8)-LCEL1, GG799-TgN (pKLAC1-lip2)-LCEL1, GG799-TgN (pKLAC1-mnp3)-LCEL1, and GG799-TgN (pKLAC1-mnp2)-LCEL1, respectively.

[0058] In the present invention, the screening culture medium preferably includes: YCB culture medium with acetamide as nitrogen source.

[0059] In the screening medium, wild-type yeast cannot utilize acetamide and cannot survive, which can effectively inhibit the transformants from losing the transformation vector and recovering, thereby improving the purity of the transformants. Moreover, the cell density can be increased with the culture time, effectively increasing the effective cell base number involved in expressing the target protein.

[0060] The present invention also provides the use of the above expression vector or the above engineering bacteria in preparing lignin decomposing enzyme.

[0061] The application of the present invention preferably comprises the following steps:

[0062] The engineered bacteria were pre-cultured in a YCB medium with acetamide as a nitrogen source for 12 hours to obtain a pre-culture solution;

[0063] The pre-culture solution is mixed with an induction culture medium containing galactose to obtain a lignin decomposing enzyme.

[0064] In the present invention, the pre-culture conditions preferably include 30° C. and 200 rpm.

[0065] The present invention can effectively increase the effective bacterial base number involved in expressing the target protein through appropriate pre-culture.

[0066] In the present invention, the galactose-containing induction medium is based on YCB medium, and preferably further comprises: acetamide 5mmol / L, galactose 20g / L, hemin 33-100mg / L and composite protease inhibitor 0-100μL / L; more preferably acetamide 5mmol / L, galactose 20g / L, hemin 50mg / L and composite protease inhibitor 50μL / L.

[0067] In the present invention, the mixed culture conditions preferably include culturing at 20-30° C. and 150-200 rpm for 48 h, and more preferably include culturing at 30° C. and 200 rpm for 48 h.

[0068] After mixed culture, it is preferred to further include: supplementing 10 g / L galactose and 5 mmol / L acetamide, and strengthening the induction culture for 72 hours. The present invention can further increase the expression amount of specific proteins through the strengthened induction culture.

[0069] The composite protease inhibitor (Protease Inhibitor Cocktail) of the present invention is a special mixture composed of AEBSF, aprotinin, benstatin, E64, leupeptin and pepstatin A, which can prevent the target protein from being degraded by endogenous proteases; in order to prevent the exocytotic target protein from being degraded by endogenous proteases produced by bacterial death or lysis during long-term culture, a composite protease inhibitor (Pi) of appropriate concentration is added during the culture process, which not only effectively protects the target protein, but also prevents Pi from inhibiting the normal proliferation of yeast and affecting the expression of the target protein. The composite protease inhibitor of the present invention is preferably purchased from Cell Signaling, TECHNOLOGY.

[0070] The present invention also provides a method for preparing a lignin decomposing enzyme, preferably comprising the following steps:

[0071] The engineered bacteria were pre-cultured in a YCB medium with acetamide as a nitrogen source for 12 hours and expanded for 26 to 36 hours to obtain bacterial precipitates;

[0072] The cells were precipitated in YP Gal The culture was induced in the culture medium to obtain lignin decomposing enzyme.

[0073] In the present invention, the conditions for the pre-culture and the expanded culture preferably include: 30° C., 200 rpm; the time for the expanded culture is preferably 26 to 36 hours, more preferably 36 hours.

[0074] In the present invention, the YP Gal The culture medium preferably comprises the following components: 10 g / L yeast extract, 20 g / L peptone, 5 mmol / L acetamide, 20 g / L galactose, 33-100 mg / L hemin and 0-100 μL / L composite protease inhibitor; more preferably: 10 g / L yeast extract, 20 g / L peptone, 5 mmol / L acetamide, 20 g / L galactose, 50 mg / L hemin and 50 μL / L composite protease inhibitor.

[0075] In the present invention, the induction culture conditions preferably include: induction culture at 20-30°C and 150-200rpm for 24-60h, and more preferably induction culture at 20°C and 150rpm for 48h.

[0076] The present invention adopts the method of using YCB selection medium to cultivate for a certain period of time, centrifuging to remove the YCB medium when the gene transformant reaches a certain abundance, and transferring to YP Gal Continuing to cultivate the culture medium can ensure the purity and abundance of the transformants on the one hand, and enhance the induction effect of lactose on the other hand.

[0077] The induction culture of the present invention preferably further comprises: centrifuging the culture solution after the culture is completed to obtain a supernatant and a precipitate; the supernatant contains a soluble lignin decomposing enzyme;

[0078] The precipitate is mixed with the extract to obtain the lignin decomposing enzyme and bacterial precipitate dissolved in the extract.

[0079] In the present invention, the extract preferably includes glycerol and buffer; the volume concentration of the glycerol in the extract is preferably 20%; the pH value of the buffer is preferably 4.5; the buffer preferably includes sodium tartrate buffer or sodium succinate buffer; the concentration of the buffer is preferably 50mM.

[0080] The present invention is achieved by using YP Gal The culture medium is a basic culture medium for induction culture, and most of the induced lignin decomposing enzymes are insoluble proteins, or adhere to the surface of the bacteria and cannot be detached. When the bacteria are washed with an extract, glycerol and buffer pH partially relieve the hydrophobic pressure of the target protein, or violent mechanical stirring causes the protein to fall out of the solution, or corrects the misfolding of the protein and increases its solubility, thereby extracting the lignin decomposing enzyme.

[0081] The present invention also provides a method for preparing a lignin decomposing enzyme, preferably comprising the following steps:

[0082] The engineered bacteria were pre-cultured in a YCB medium with acetamide as a nitrogen source for 12 hours to obtain bacterial precipitates;

[0083] The bacterial cell precipitate is induced and cultured in an induction medium to obtain a lignin decomposing enzyme.

[0084] In the present invention, the pre-culture conditions preferably include: 30° C., 200 rpm.

[0085] In the present invention, the induction medium is YCB Gal The culture medium is a basic culture medium, preferably further comprising the following components: acetamide 5mmol / L, galactose 20g / L, hemin 33-100mg / L and composite protease inhibitor 0-100μL / L; more preferably acetamide 5mmol / L, galactose 20g / L, hemin 50mg / L and composite protease inhibitor 50μL / L; the YCB Gal The culture medium is preferably a medium prepared by replacing glucose in YCB medium with an equal mass of galactose.

[0086] In the present invention, the induction culture conditions preferably include: induction culture at 30° C. and 200 rpm for 24 to 60 h, and more preferably induction culture at 30° C. and 200 rpm for 48 h.

[0087] The invention adopts a method of using galactose to replace glucose in YCB to culture and transform K. lactis yeast, which can improve the expression of effective target enzyme protein in the culture solution and the activity of the enzyme.

[0088] To further illustrate the present invention, a transformation vector containing a lignin decomposing enzyme gene, an engineering bacterium and applications thereof provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1

[0090] A transformation vector containing the lipH8 gene, wherein the transformation vector is constructed by the following method:

[0091] 1) The lignin peroxidase gene and the manganese peroxidase gene (lip, mnp) were cloned from Phanerochaete chrysosporium (commonly known as white rot fungus); the specific method is as follows:

[0092] A solid plate for culturing spores is prepared, wherein the formula of the culture medium in the solid plate is: 10 g / L glucose, 10 g / L malt extract, 2 g / L peptone, 2 g / L yeast extract, 1 g / L asparagine, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.001 g / L thiamine hydrochloride and 20 g / L agar;

[0093] The spores of white rot fungus were inoculated on a solid plate, and cultured in a 30°C incubator until the mycelium covered the plate. The mycelium was taken and immersed in a low-nitrogen culture solution, which consisted of the following components in volume concentrations: 10% of basic III culture medium, 10% of 10wt.% glucose solution, 10% of 0.1mol / L dimethyl 2,2-succinate (pH 4.2), 10% of 100mg / L thiamine, 2.5% of 8g / L ammonium tartrate, 10% of resveratrol and 6% of trace element solution; the formula of the basic III culture medium was: KH2PO4 20g / L, MgSO4 5g / L and CaCl2 1g / L; the formula of the trace element solution was: MgSO4 3g / L, MnSO4 0.5g / L, NaCl 1g / L, FeSO4·7H2O 0.1g / L, COCl2 0.1g / L, ZnSO4·7H2O ... 0.1 g / L, CuSO4 0.1 g / L, AlK(SO4)2·12H2O 0.01 g / L, H3BO3 0.01 g / L, Na2MoO4·2H2O 0.01 g / L and Nitrilotriacetate 1.5 g / L;

[0094] Filter the mycelium through a sterile filter to prepare a spore solution (OD 650 0.5), inoculated into low nitrogen medium at a volume ratio of 10%, and cultured in a constant temperature shaking incubator at 30°C with shaking (150 rpm). Starting from the third day of culture, mycelium was extracted every day to extract total RNA (Trizol RNA isolation kit), and then the expression of the target gene was detected by RT-PCR (Promega RT kit, M-MLV). The primer pairs used were LipH8F: 5'-AGCTAGATCTATGGCCTTCAAGCAGCTC-3' (SEQ ID NO.1) and LipH8R: 5'-TTCAAGGCCTTTAAGCACCCGGAGGC-3' (SEQ ID NO.2).

[0095] On the 4th day of culture, the expression of lipH8 gene was found in the mycelium ( Figure 1 ), lipH8 cDNA was isolated and purified using a gel purification kit, and sequenced. The sequencing results were compared with the relevant gene sequences in the NCBI gene database for confirmation.

[0096] 2) After the alignment is correct, the isolated and purified lipH8 cDNA is inserted into the pGEMT Easy vector, which is then introduced into E. coli XL-Blue for amplification. The amplified vector DNA is cut out with restriction endonucleases (BglⅡ and StuⅠ) to extract the target gene DNA ( Figure 2 ), and then purify the target gene DNA using a gel purification system ( Figure 3 ).

[0097] 3) Insert the target gene DNA fragment purified in step 2) between the BglⅡ and StuⅠ restriction sites of the pKLAC1 vector to obtain a transformation vector containing the lipH8 gene ( Figure 4 ).

[0098] Example 2

[0099] A transformation vector containing the lip2 gene, wherein the transformation vector is constructed by the following method:

[0100] Step 1) is the same as Example 1) except that: RT-PCR (Promega RT kit, M-MLV) is used to detect the expression of the target gene, and the primer pair used is Lip2F: 5'-AGCTAGATCTATGGCCTTCAAGCAGCTC-3' (SEQ ID NO. 3) and Lip2R: 5'-CGACGGTACCTTAGGCCTTGTGCG-3' (SEQ ID NO. 4).

[0101] On the fourth day of culture, the expression of lip2 gene was found in the mycelium ( Figure 1 ), lip2 cDNA was isolated and purified using a gel purification kit, and sequenced. The sequencing results were compared with the relevant gene sequences in the NCBI gene database for confirmation.

[0102] 2) After the alignment is correct, the isolated and purified lip2 cDNA is inserted into the pGEMT Easy vector, which is then introduced into E. coli XL-Blue for amplification. The amplified vector DNA is cut out with restriction endonucleases (BglⅡ and KpnⅠ) to extract the target gene DNA ( Figure 2 ), and then purify the target gene DNA using a gel purification system ( Figure 3 ).

[0103] 3) Insert the target gene DNA fragment purified in step 2) between the BglⅡ and KpnⅠ restriction sites of the pKLAC1 vector to obtain a transformation vector containing the lip2 gene ( Figure 4 ).

[0104] Example 3

[0105] A transformation vector containing the Mnp3 gene, wherein the transformation vector is constructed by the following method:

[0106] Step 1) is the same as Example 1) except that: RT-PCR (Promega RT kit, M-MLV) is used to detect the expression of the target gene, and the primer pair used is Mnp-3F: 5'-ATCTCCTAGGATGGCCTTTGCATCCTCA-3' (SEQ ID NO. 5) and Mnp-3R: 5'-TTCAAGGCCTTTATGCAGGGCCGTTGAA-3' (SEQ ID NO. 6).

[0107] On the 4th day of culture, the expression of Mnp3 gene was found in the mycelium ( Figure 1 ), Mnp3 cDNA was isolated and purified using a gel purification kit, and sequenced. The sequencing results were compared with the relevant gene sequences in the NCBI gene database for confirmation.

[0108] 2) After the alignment is correct, the isolated and purified Mnp3 cDNA is inserted into the pGEMT Easy vector, which is then introduced into E. coli XL-Blue for amplification. The amplified vector DNA is cut out with restriction endonucleases (AvrⅡ and StuⅠ) to extract the target gene DNA ( Figure 2 ), and then purify the target gene DNA using a gel purification system ( Figure 3 ).

[0109] 3) Insert the target gene DNA fragment purified in step 2) between the AvrⅡ and StuⅠ restriction sites of the pKLAC1 vector to obtain a transformation vector containing the Mnp3 gene ( Figure 4 ).

[0110] Example 4

[0111] A transformation vector containing the Mnp2 gene, wherein the transformation vector is constructed by the following method:

[0112] Step 1) is the same as Example 1) except that: RT-PCR (Promega RT kit, M-MLV) is used to detect the expression of the target gene, and the primer pair used is Mnp-2F: 5'-AGCTAGATCTATGGCCTTCGGTTCTCTC-3' (SEQ ID NO. 7) and Mnp-2R: 5'-TTAAGGTACCTTACGCAGGGCCATCGAA-3' (SEQ ID NO. 8).

[0113] On the 4th day of culture, the expression of Mnp2 gene was found in the mycelium ( Figure 1 ), Mnp2 cDNA was isolated and purified using a gel purification kit, and sequenced. The sequencing results were compared with the relevant gene sequences in the NCBI gene database for confirmation.

[0114] 2) After the alignment is correct, the isolated and purified Mnp2 cDNA is inserted into the pGEMT Easy vector, which is then introduced into E. coli XL-Blue for amplification. The amplified vector DNA is cut out with restriction endonucleases (BglⅡ and KpnⅠ) to extract the target gene DNA ( Figure 2 ), and then purify the target gene DNA using a gel purification system ( Figure 3 ).

[0115] 3) Insert the target gene DNA fragment purified in step 2) between the restriction sites BglⅡ and KpnⅠ of the pKLAC1 vector to obtain a transformation vector containing the Mnp2 gene ( Figure 4 ).

[0116] Example 5

[0117] An engineered bacterium that secretes a lignin decomposing enzyme is constructed by the following steps:

[0118] 1) The expression vector constructed in Example 1 was re-introduced into E. coli XL-Blue to amplify the vector DNA in large quantities. The amplified vector DNA was extracted and the target gene was confirmed by specific cloning PCR method ( Figure 5 ), and then digested with SacⅡ to obtain the linear LipH8 enzyme expression frame ( Figure 6 a) in the figure.

[0119] 2) Purify the linear LipH8 enzyme expression cassette obtained in step 1) using a gel purification system ( Figure 6 b in Figure 7 ); the constructed and purified linear expression cassette DNA was inserted into K. lactis GG799 competent cells using NEB's yeast transformation reagent, and transformants were screened using YCB medium containing acetamide (the concentration of acetamide was 5 mM), and then the colony PCR method was used to confirm whether the transformed gene was inserted.

[0120] The lipH8 gene transformed strain was obtained by specific culture medium screening and PCR confirmation ( Figure 8 , 9 ), named GG799-TgN(pKLAC1-lipH8)-LCEL1.

[0121] Example 6

[0122] An engineered bacterium that secretes a lignin decomposing enzyme is constructed by the following steps:

[0123] 1) The expression vector constructed in Example 2 was re-introduced into E. coli XL-Blue to amplify the vector DNA in large quantities. The amplified vector DNA was extracted and the target gene was confirmed by specific cloning PCR method ( Figure 5), and then digested with SacⅡ to obtain the linear Lip2 enzyme expression cassette ( Figure 6 a) in the figure.

[0124] 2) Purify the linear Lip2 enzyme expression cassette obtained in step 1) using a gel purification system ( Figure 6 b, 7); the constructed and purified linear expression frame DNA was inserted into K. lactis GG799 competent cells using NEB's yeast transformation reagent, and transformants were screened using YCB medium containing acetamide (the concentration of acetamide was 5 mM), and then the colony PCR method was used to confirm whether the transformed gene was inserted.

[0125] Through specific culture medium screening and PCR confirmation, the lip2 gene transformed strain was obtained ( Figure 8 , 9 ), named GG799-TgN(pKLAC1-lip2)-LCEL1.

[0126] Example 7

[0127] An engineered bacterium that secretes a lignin decomposing enzyme is constructed by the following steps:

[0128] 1) The expression vector constructed in Example 3 was re-introduced into E. coli XL-Blue to amplify the vector DNA in large quantities. The amplified vector DNA was extracted and the target gene was confirmed by specific cloning PCR method ( Figure 5 ), and then digested with SacⅡ to obtain the linear mnp3 enzyme expression frame ( Figure 6 a) in the figure.

[0129] 2) Purify the linear mnp3 enzyme expression cassette obtained in step 1) using a gel purification system ( Figure 6 b, 7); the constructed and purified linear expression frame DNA was inserted into K. lactis GG799 competent cells using NEB's yeast transformation reagent, and transformants were screened using YCB medium containing acetamide (the concentration of acetamide was 5 mM), and then the colony PCR method was used to confirm whether the transformed gene was inserted.

[0130] Through specific culture medium screening and PCR confirmation, the mnp3 gene transformed strain was obtained ( Figure 8 , 9 ), named GG799-TgN(pKLAC1-mnp3)-LCEL1.

[0131] Example 8

[0132] An engineered bacterium that secretes a lignin decomposing enzyme is constructed by the following steps:

[0133] 1) The expression vector constructed in Example 4 was re-introduced into E. coli XL-Blue to amplify the vector DNA in large quantities. The amplified vector DNA was extracted and the target gene was confirmed by specific cloning PCR method ( Figure 5 ), and then digested with SacⅡ to obtain the linear mnp2 enzyme expression frame ( Figure 6 a) in the figure.

[0134] 2) Purify the linear mnp2 enzyme expression cassette obtained in step 1) using a gel purification system ( Figure 6 b, 7); the constructed and purified linear expression frame DNA was inserted into K. lactis GG799 competent cells using NEB's yeast transformation reagent, and transformants were screened using YCB medium containing acetamide (the concentration of acetamide was 5 mM), and then the colony PCR method was used to confirm whether the transformed gene was inserted.

[0135] Through specific culture medium screening and PCR confirmation, the mnp2 gene transformed strain was obtained ( Figure 8 , 9 ), named GG799-TgN(pKLAC1-mnp2)-LCEL1.

[0136] Application Example 1

[0137] Galactose induction culture under specific nitrogen source (acetamide) conditions

[0138] In order to prevent the gene transformant from being replaced by wild-type yeast and losing the function of expressing the target protein during the induction culture process, the present invention adopts YCB selection medium (YCB medium also contains 5mM acetamide and 30mM phosphate buffer, pH7.0) to effectively control the growth of the wild type, and then adds galactose to induce the expression of Lip and Mnp proteins.

[0139] The specific method is as follows: the engineered bacteria prepared in Examples 5 to 8 are inoculated (the inoculation amount is a colony of 1 inoculation loop) into 5 ml of YCB selection medium, and after inoculation, they are pre-cultured at 30° C. and 200 rpm for 12 h, and then the pre-cultured liquid is transferred to a 250 ml conical flask, and then 50 ml of YCB selection medium containing galactose is added (the YCB selection medium also contains 20 g / L galactose, 50 mg / L hemin and 50 μL / L composite protease inhibitor), and induced culture is carried out at 30° C. and 200 rpm for 48 h, and then 10 g / L galactose and 5 mmol / L acetamide are supplemented to strengthen the induction culture for 72 h.

[0140] The protein expression in the culture medium after 48h and 72h of induction culture was detected by SDS-PAGE method. The control group in this application example and the following application examples or comparative application examples were all K. lactis GG799 cell culture medium, and the culture medium contained 1% yeast extract, 2% peptone, 20g / L galactose, 50mg / L hemin, and 50μl / L composite protease inhibitor. The culture conditions were the same, and the results are shown in Table 1. Fig.10 .

[0141] Depend on Fig.10 It can be seen that after 48 hours of induction culture, specific protein bands appeared near 45kD and 60kD, and the expression level increased after the enhanced induction culture was tested again after 72 hours. It can be seen that enhanced induction culture can further increase the expression level of specific proteins.

[0142] Application Example 2

[0143] YCB+acetamide and YP Gal Segmented induction culture

[0144] The engineered bacteria prepared in Examples 5 to 8 were inoculated (inoculation amount was one colony of inoculation loop) into 5 ml of YCB selection medium (with the same formulation as in Application Example 1), and after inoculation, they were pre-cultured at 30° C. and 200 rpm for 12 h, and then the pre-cultured liquids were transferred to 250 ml Erlenmeyer flasks, and then 50 ml of YCB selection medium was added, and expanded culture was performed at 30° C. and 200 rpm for 36 h, and then centrifuged at 3000 rpm for 10 min to obtain different bacterial precipitates after expanded culture;

[0145] Add 50 mL of YP to different bacterial precipitates. Gal The culture was induced in a medium (yeast extract 10 g / L, bacterial peptone 20 g / L, galactose 20 g / L, hemin 3.3 mg / L, composite protease inhibitor 70 μL / L, and phosphate buffer 30 mM, pH 7.0) at 20 °C and 150 rpm for 46 h;

[0146] After the induction culture was completed, the culture solutions inoculated with different engineered bacteria were centrifuged at 3000 rpm for 10 min to obtain different bacterial precipitates and supernatants after the induction culture;

[0147] The different supernatants were concentrated using a 10kD centrifugal ultrafiltration membrane to obtain different enzyme concentrates;

[0148] Different bacterial precipitates were washed with 20% (volume percentage) glycerol Tartaric buffer (pH 4.5) to obtain different bacterial glycerol solution extracts.

[0149] Application Example 3

[0150] The protein expression in different enzyme concentrates and different bacterial glycerol solution extracts in Application Example 2 was detected by SDS-PAGE method. The detection results are shown in Fig.11 .

[0151] Depend on Fig.11 It can be seen that the specific protein bands near 60kD and 45kD are not obvious, while the culture fluid of the mnp gene transformed bacteria shows a specific band near 35kD. Compared with the YCB culture system in Application Example 1, the expression effect of the target protein in the culture fluid is not clear. However, after the bacterial part produced during the separation of the culture fluid was extracted with 20% glycerol Tartaric Buffer (pH 4.5), SDS-PAGE analysis showed that very obvious specific protein bands appeared near 60, 45, and 35kD. Therefore, under the condition of segmented culture, most of the target proteins form insoluble proteins, or adhere to the surface of the bacteria and cannot be detached. During the centrifugation process, as the bacteria precipitate, only a small amount of target protein remains in the culture fluid.

[0152] Application Example 4

[0153] The activity of lignin decomposing enzymes in the glycerol solution extracts of different bacteria in Application Example 2 at different enzyme reaction buffer pH (Mnp: 20 mM sodium succinate buffer, pH 4.5; 50 mM sodium tartrate buffer, pH 4.5, pH 2.7) was detected by the method of Vares et al. (APPL. ENVIRON. MICROBIOL [J]., Oct. 1995, p. 3515–3520). The test results are shown in Tables 1 and Fig.12 .

[0154] Table 1 Specific activity of lignin decomposing enzymes in glycerol solution extracts of different bacteria

[0155]

[0156] From the above results, it can be seen that the Lip enzyme specific activity of the bacterial glycerol extract prepared by the present invention can reach 0.72A when the buffer pH is 4.5. 310 / mg / ml / min, Mnp enzyme specific activity up to 3.34A 610 / mg / ml / 5min, all showed high oxidation activity; and when the buffer pH was 2.7, the Lip enzyme specific activity was as high as 1.8A 310 / mg / ml / min.

[0157] Application Example 5

[0158] The different bacterial glycerol solution extracts in Application Example 2 were added to 2 ml test tubes containing 30 μmol Azure B dye. The added volume of each bacterial glycerol solution extract was 1, 5, 7, 10 and 15 μl, respectively. A control group (C) without adding bacterial glycerol solution extract was set up. After 10 minutes of addition, the decolorization effect was observed. The results are shown in Table 1. Fig.13 .

[0159] Depend on Fig.13 It can be seen that the bacterial glycerol solution extract prepared by the present invention has an obvious decolorization effect on Azure B dye. In summary, the precipitated bacterial cells obtained by segmentation experiment and treated with 20% glycerol Tartaric buffer are one of the effective measures to obtain soluble target proteins with higher enzyme activity.

[0160] Comparative application example 1

[0161] Effect of Hemin Addition Amount on Enzyme Activity

[0162] Different bacterial glycerol solution extracts and different enzyme concentrates were prepared by a similar method to Application Example 2, except that the concentration of hemin was 2 mg / 100 mL, to obtain the first group of different bacterial glycerol solution extracts and different enzyme concentrates;

[0163] Different bacterial glycerol solution extracts and different enzyme concentrates were prepared by a similar method to Application Example 2, except that the concentration of hemin was 3.3 mg / 100 mL, to obtain a second group of different bacterial glycerol solution extracts and different enzyme concentrates;

[0164] Different bacterial glycerol solution extracts and different enzyme concentrates were prepared by a method similar to that of Application Example 2, except that the concentration of hemin was 10 mg / 100 mL, to obtain the third group of different bacterial glycerol solution extracts and different enzyme concentrates.

[0165] The activity of lignin decomposing enzymes in the glycerol extracts of different bacterial cells and different enzyme concentrates in Application Example 2, Group 1, Group 2 and Group 3 were tested by the method of Vares et al. (1995). The test results are shown in Tables 2 and Fig.14 .

[0166] Table 2 Ligninolytic enzyme activities in different groups

[0167]

[0168]

[0169] Note: abcd in the table means p≤0.05, and the same applies to the following table.

[0170] From the above results, it can be seen that the Mnp enzyme activity in the culture medium shows a downward trend with the increase of Hemin added concentration, but when the concentration is increased to 10mg / 100ml, the oxidation effect on phenol red suddenly increases. This is related to the oxidation activity of hemin itself. Hemin has the activity of peroxidase and catalase, and Hemin itself participates in the oxidation reaction in the presence of hydrogen peroxide. On the contrary, the Mnp activity in the glycerol tartaric acid buffer extract tends to gradually increase with the increase of Hemin added concentration. This is because during the centrifugal separation of the culture medium, most of the added Hemin remains in the supernatant, while a small amount of insoluble Hemin enters the glycerol tartaric acid extract, and its free heme concentration is not enough to affect the detection of Mnp enzyme activity. However, when it exceeds 3.3mg / ml, the activity of the Mnp enzyme is no longer significantly increased.

[0171] Comparative Application Example 2

[0172] Effects of protease inhibitor cocktail (Pi) on enzyme activity in segmented induction culture

[0173] Different bacterial glycerol solution extracts and different enzyme concentrates were prepared by a method similar to that of Application Example 2, except that the concentration of the composite protease inhibitor was 0 μL / 100 mL, to obtain the first group of different bacterial glycerol solution extracts and different enzyme concentrates;

[0174] A similar method to Application Example 2 was used to prepare different bacterial glycerol solution extracts and different enzyme concentrates, except that the composite protease inhibitor was 10 μL / 100 mL, to obtain a second group of different bacterial glycerol solution extracts and different enzyme concentrates.

[0175] The activity of lignin decomposing enzymes in the glycerol extracts of different bacterial cells and different enzyme concentrates in Application Example 2, Group 1 and Group 2 was detected by the method of Vares et al. (1995), and the total protein production concentration was detected by the Bradford method. The test results are shown in Tables 3, 4 and Fig.15 .

[0176] Table 3 Ligninolytic enzyme activities in different groups

[0177]

[0178] Table 4 Total protein concentration in different groups (mg / L)

[0179]

[0180]

[0181] From the above results, it can be seen that the present invention determines that the Mnp enzyme activity of the 7μl / 100ml Pi addition group is compared with the 0 addition group, although there is no significant difference, it tends to increase, and the total protein amount is significantly higher than the 0 addition group. On the contrary, the protein yield and enzyme activity of the 10μl / 100ml addition group tend to decrease, especially the enzyme activity of the culture solution is significantly inhibited. Therefore, adding too high a concentration of Pi during the culture process affects the yield and activity of the enzyme, while when properly added, it can effectively protect the exocrine protein, increase protein yield, and increase enzyme activity to a certain extent.

[0182] Application Example 6

[0183] Galactose carbon source YCB (YCB Gal )+acetamide induction culture system

[0184] Using YCB basal medium and YP Gal The staged culture method was used to obtain culture fluid and bacterial glycerol washing fluid with peroxidase activity. However, the enzyme activity was mainly present in the bacterial glycerol washing fluid, indicating that most of the peroxidase secreted by the gene-transformed yeast was insoluble protein. During the separation process, as the bacteria sank, only a trace amount was dissolved in the culture fluid. Moreover, through multiple experiments, it was found that the enzyme solution obtained by washing the bacteria with glycerol was inactivated by denaturation, precipitation, and precipitation of the enzyme protein during multiple dissolution and thawing processes. No obvious specific protein bands were found in the culture fluid, and the activities of the Lip and Mnp enzymes in the culture fluid enzyme concentrate were both low. However, when cultured with YCB medium alone, obvious specific protein bands appeared near 45kD and 35kD in the culture fluid. Although they could not be determined as specific bands, obvious thickened protein bands appeared between 55 and 60kD. It was eliminated when treated with Kex2 enzyme, indicating that the protein carried the a-MF signal peptide and was not completely removed during the exocytosis of the target protein. However, the culture fluid showed higher Lip and Mnp enzyme activities, which confirmed that YCB Gal Culture medium promotes the secretion of soluble form of target protein and improves the production of effective enzyme protein. Gal The production of target protein is subject to certain restrictions during culture in the medium, and the overall activity of the enzyme is not as good as that of YP Gal Segmented culture. Because the bacteria are limited by nitrogen sources (can only rely on acetamide), the bacterial growth rate is far less than that of YP GalThe amount of bacteria in the culture medium. The expression of exogenous proteins by yeast is affected by many factors, including the glucose inhibition effect. The carbon source of YCB culture medium is glucose. When yeast is cultured with YCB culture medium, although high concentration (2%) of galactose is added to induce the expression of the target protein, the presence of original glucose in YCB may greatly reduce the induction effect of galactose. In order to avoid the glucose inhibition effect when the engineered bacteria is cultured with YCB culture medium, the present invention adopts the method of replacing glucose in YCB with galactose to improve the expression of the target enzyme protein and its enzyme activity. The specific method is as follows:

[0185] The engineered bacteria prepared in Examples 5 to 8 were inoculated (inoculation amount was 1 colony) into 5 ml of YCB selection medium (the formula was the same as that in Application Example 1), and after inoculation, they were pre-cultured at 30° C. and 200 rpm for 12 h, and then centrifuged at 3000 rpm for 10 min to obtain different bacterial precipitates after expansion culture;

[0186] Add 25 ml of YCB with galactose as the sole carbon source to different bacterial precipitates. Gal The culture medium (YCB selection medium in which glucose is replaced by galactose and contains 50 mg / L hemin and 50 μL / L composite protease inhibitor) was expanded and cultured at 30°C and 200 rpm for 15 h, and then supplemented with 50 ml of YCB medium containing galactose as the carbon source. Gal The culture medium was inducingly cultured at 30°C and 200 rpm for 48 h.

[0187] The supernatant was separated by centrifugation and concentrated 20 times. When SDS-PAGE was performed, a specific protein band at the 45kD position was observed ( Fig.16 b), and a thickened band appeared near 55 kD. However, compared with the YCB culture enzyme concentrate in Application Example 1, the protein bands between 45 and 55 kD were missing, while the protein bands near 35 kD increased significantly ( Fig.16 a and b in the figure). It has been confirmed in previous studies that the 55kD thickened protein band is related to the failure of exocytosis of the exocytosis signal peptide of some target proteases ( Fig.16 c) in.

[0188] Comparative Application Example 3

[0189] The activity of Mnp3 enzyme in different enzyme concentrates in Application Examples 1 and 6 was detected by the method of Vares et al. (1995). The detection results are shown in Table 5.

[0190] Table 5 YCB and YCB Gal Comparison of Mnp enzyme specific activity (ESA) in culture medium systems

[0191]

[0192] From the above results, it can be seen that the Mnp enzyme specific activity of the culture solution obtained by Application Example 6 can reach 3.79±0.16A 610 / mg / ml / 5min, compared with the Mnp enzyme specific activity in the culture medium of the YCB culture system, there was a significant improvement.

[0193] Comparative Application Example 4

[0194] The activity of Mnp3 enzyme in the glycerol solution extracts of different bacteria and different enzyme concentrates in Application Examples 2 and 6 was detected by the method of Vares et al. (1995). The test results are shown in Tables 6 and Fig.17 .

[0195] Table 6 Mnp3 enzyme activity under different culture conditions

[0196]

[0197]

[0198] From the above results, it can be seen that the specific activity of Mnp enzyme in the culture medium of Application Example 6 and Application Example 2, YCB Gal Still presents its advantages ( Fig.17 ). Gal The Mnp enzyme activity of the group was higher than that of any segmented culture group. This indicates that it is a desirable approach to directly use lactose as a carbon source to overcome the glucose inhibition effect when using K. lactis to produce exogenous proteins. This result once again confirms that K. lactis yeast also has a glucose inhibition mechanism. To obtain more effective exogenous enzyme proteins, the yeast basal medium culture method with galactose or lactose as a carbon source can be used to effectively prevent YP Gal Elimination of transformed clones under culture conditions while increasing the production of effective target enzyme protein.

[0199] However, the Mnp enzyme activity in the culture medium was Gal Continuous culture or YCB, YP Gal The culture method was lower than the Mnp enzyme activity in 20% glycerol washing solution (insoluble enzyme protein) of the bacteria ( Fig.18 ), although the protein in this part accounts for about 20% of the total protein in the culture medium (Table 7). This shows that most of the exocrine enzyme protein appears in the culture medium in an insoluble form and precipitates with the bacteria during centrifugation. Gal The insoluble protein produced in the culture system is relatively small. When the bacterial solution is washed with 20% glycerol, almost no specific protein bands can be observed in the SDS-PAGE gel. It may be that YCB Gal The culture system effectively inhibits the production of insoluble enzyme protein.Gal The total protein yield of the culture system was relatively low (Table 7), which slowed down the rate of enzyme protein synthesis in the bacteria and reduced the misfolding rate of enzyme protein.

[0200] Table 7 Comparison of total protein production in culture medium between YCB culture and segmented culture

[0201]

[0202] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The use of engineered bacteria in the preparation of lignin decomposing enzymes, characterized in that: The following steps are involved: After pre-culturing the engineered bacteria in a YCB medium with acetamide as a nitrogen source for 12 to 48 hours, the pre-cultured engineered bacteria are cultured in an induction medium containing galactose, and the culture solution after the culture is centrifuged to obtain a supernatant and a precipitate; the supernatant contains a soluble lignin decomposing enzyme; The precipitate is mixed with an extract to obtain a lignin decomposing enzyme and a bacterial precipitate dissolved in the extract; The extract comprises glycerol and a buffer; the buffer comprises a sodium tartrate buffer having a pH value of 2.7 or 4.5 or a sodium succinate buffer having a pH value of 4.5; the volume concentration of the glycerol in the extract is 20%; the pH value of the sodium tartrate buffer is 2.7 or 4.5; the pH value of the sodium succinate buffer is 4.5; The construction method of the engineering bacteria comprises the following steps: The transformation vector was cut with SacⅡ enzyme to obtain a linear expression frame containing the lignin decomposing enzyme gene; The linear expression frame was inserted into the LAC4 promoter homology frame of the Kluyveromyces lactis genome, and the engineered bacteria secreting lignin decomposing enzymes were screened and named GG799-TgN (pKLAC1-lipH8)-LCEL1, GG799-TgN (pKLAC1-lip2)-LCEL1, GG799-TgN (pKLAC1-mnp3)-LCEL1, and GG799-TgN (pKLAC1-mnp2)-LCEL1, respectively; The basic vector of the transformation vector includes a pKLAC1 vector; the lignin decomposing enzyme gene includes a lipH8 gene, a lip2 gene, a mnp3 gene or a mnp2 gene; When the lignin decomposing enzyme gene is the lipH8 gene, the lipH8 gene is located between the BglⅡ and StuⅠ restriction sites of the basic vector; When the lignin decomposing enzyme gene is the lip2 gene, the lip2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector; When the lignin decomposing enzyme gene is the mnp2 gene, the mnp2 gene is located between the BglⅡ and KpnⅠ restriction sites of the basic vector; When the lignin decomposing enzyme gene is the mnp3 gene, the mnp3 gene is located between the AvrⅡ and StuⅠ restriction sites of the basic vector; The induction medium comprises a carbon source basal medium, 5 mmol / L acetamide, 20 g / L galactose, 33-100 mg / L hemin and 70 μL / L compound protease inhibitor, or a YP medium, 20 g / L galactose, 33-100 mg / L hemin and 70 μL / L compound protease inhibitor; The carbon source basic culture medium includes YCB culture medium, YCB Gal Culture medium; The YCB Gal The culture medium is prepared by replacing the glucose in the YCB medium with an equal mass of galactose; The YP culture medium includes: 10 g / L yeast extract and 20 g / L peptone.

2. The use according to claim 1, characterized in that: The screening culture medium includes: YCB culture medium with acetamide as nitrogen source.

3. The use according to claim 1, characterized in that: The culture temperature is 20-30° C., and the culture time is 24-60 hours.

4. The use according to claim 1, characterized in that: The lignin decomposing enzyme gene is cloned from Phanerochaete chrysosporium (commonly known as white rot fungus).

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