CN111304105B - Genetically engineered bacterium for producing lipase by using methanol and xylose cosubstrate and application thereof - Google Patents
Genetically engineered bacterium for producing lipase by using methanol and xylose cosubstrate and application thereof Download PDFInfo
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- 241001661345 Moesziomyces antarcticus Species 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 15
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Abstract
The invention relates to a genetically engineered bacterium for producing lipase by using a methanol and xylose co-substrate and application thereof, and a methanol oxidase gene is introduced into a host bacteriumaox1. Dihydroxyacetone synthase genedasCatalase genectaAnd dihydroxyacetone kinase genedak(ii) a The host bacteria is candida antarctica which can produce lipase by using xylose. The method introduces a methanol metabolic pathway into the candida antarctica by using a synthetic biology method, so that the candida antarctica produces lipase by using non-food-grade raw materials of methanol and xylose as cosubstrates, the production cost is reduced to a certain extent, and the method has great significance and economic value.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a genetically engineered bacterium for producing lipase by using a methanol and xylose cosubstrate and application thereof.
Background
Microbial lipases are widely distributed in organisms, have the advantages of wider action pH and action temperature range than animal lipases and have the action in enzyme theory research and practical application, can catalyze various reactions such as decomposition, synthesis, ester exchange and the like of ester compounds, have high chemical, regional and stereo selectivity, and are widely applied to the fields of organic synthesis, pharmacy, detergents, biosurfactants and the like in recent years.
The candida antarctica lipase is an important lipase and has a plurality of excellent characteristics, and because the candida antarctica lipase is a novel nonspecific enzyme, the candida antarctica lipase has strong stability in solution or in an immobilized form, the enzyme activity is reduced quickly initially when heat treatment is carried out, but the enzyme activity is stabilized after a period of time, the enzyme activity is not changed obviously even if further heat treatment is carried out, the candida antarctica lipase has high stereoselectivity in hydrolysis reaction and organic synthesis reaction, and has potential application value in glycolipid synthesis. At present, the cost of a culture medium for producing the candida antarctica lipase is higher, so that the production cost of the candida antarctica lipase is higher, and therefore, if a cheap reductive substrate is used as a raw material, the cost can be reduced to a certain extent.
Methanol is an important product in the coal chemical industry, and the price of methanol is continuously lowered along with the development of a methanol process in recent years, so that the methanol is used as a fermentation raw material to become an important breakthrough in reducing the cost in a biotransformation process. Therefore, if a methanol metabolism module is introduced into the candida antarctica by a synthetic biological means, the methanol and the xylose are used as cosubstrates to produce the lipase, and a foundation is laid for the research of producing the lipase by using cheap carbon resources.
Disclosure of Invention
The invention aims to provide a strain which is constructed by using a synthetic biology method and can be metabolized by using a methanol and xylose co-substrate, and lipase is produced by using the strain through fermentation, so that the problem of high cost of the traditional lipase production is solved.
In order to solve the problems, the invention adopts the following scheme:
a genetically engineered bacterium for producing lipase by utilizing a methanol and xylose co-substrate is prepared by introducing a methanol oxidase gene aox1, a dihydroxyacetone synthase gene das, a catalase gene cta and a dihydroxyacetone kinase gene dak into a host bacterium; the host bacteria is candida antarctica which can produce lipase by using xylose.
Wherein the methanol oxidase gene oxidizes methanol into formaldehyde, consumes a large amount of oxygen and produces hydrogen peroxide; hydrogen peroxide is converted into oxygen and water under the catalysis of catalase cta; formaldehyde and xylulose-5 phosphate are converted into dihydroxyacetone under the catalysis of dihydroxyacetone synthase das, and meanwhile, xylulose-5 phosphate is provided by externally added xylose to accelerate the production of dihydroxyacetone; dihydroxyacetone is then converted into dihydroxyacetone phosphate under the catalysis of dihydroxyacetone kinase dak, and then further metabolized into fructose-6-phosphate, which in turn enters the glycolysis pathway and participates in substance circulation and organic acid metabolism, as shown in FIG. 1. The candida antarctica can utilize xylose as a carbon source, and xylulose-5 phosphate metabolized by the xylose can be used as a precursor for methanol metabolism, so that the xylose can accelerate the methanol metabolism so as to improve the related enzyme activity of lipase of the constructed strain.
Wherein the host bacterium is Candida antarctica (Candida antarctica) ZJB09193 with the preservation number of CCTCC M2010263, which is disclosed in Liu et al cloning, expression and characterization of a lipase gene from the Candida antarctica Z09193 and its application in biochemical research volume 167, Issue 8,6September 2012, Pages 452-. The applicant hereby states that it is guaranteed that the biological material of the present strain is released to the public free of charge within 20 years from the date of filing.
Wherein the GenBank accession number of the methanol oxidase gene aox is XM-002494226.1;
GenBank accession number of the dihydroxyacetone synthase gene das is FJ 752551.1;
the GenBank accession number of the catalase gene cta is AB 472085.1;
GenBank accession number of the dihydroxyacetone kinase gene dak is XM-002493026.1.
The construction method of the genetic engineering bacteria for producing lipase by using methanol comprises the following steps:
(1) constructing expression frames of TEF-aox1-CYC1t, TEF-das-tCYC1, PDC1p-cta-TDH2t and pGPD-dak-TXPR2, connecting two gene segments of TEF-das-tCYC1 and pGPD-dak-TXPR2 with a 113 plasmid by a multi-segment cloning method, connecting two gene segments of TEF-aox1-CYC1t and PDC1p-cta-TDH2t with a Pki plasmid, and transforming the gene segments into E.coli DH5 alpha;
(2) and carrying out enzyme digestion on the plasmid with correct sequencing to obtain a gene recombinant fragment, electrically transforming the gene recombinant fragment to host bacteria, and screening through G418 resistance to obtain the gene engineering bacteria.
The invention also aims to provide application of the genetically engineered bacteria in producing lipase by fermentation.
The invention provides a specific application method, which comprises the following steps:
(1a) and (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture;
(2a) and (3) seed culture in a shaking flask: inoculating the test tube seed culture solution into a seed culture medium of a shake flask for culture;
(3a) producing lipase by fermentation: inoculating the shake flask seed culture solution into a fermentation culture medium for fermentation culture to obtain the lipase.
As a further improvement of the invention, the culture temperature of the test tube seed culture and the shake flask seed culture is 24 ℃, and the culture time is 18-22 h.
Wherein the seed culture medium comprises the following formula: 6g/L of peptone, 4g/L of hydrolyzed casein, 3g/L of yeast powder, 1.5g/L of beef extract and 1g/L of glucose;
wherein the formula of the fermentation medium is as follows: 40g/L of soybean meal, 5ml/L of soybean oil, 4g/L of dipotassium phosphate, 1g/L of magnesium sulfate, 80ml/L of Tween, 5g/L of yeast powder, 15g/L of xylose and 0-15 g/L of methanol; preferably, the methanol concentration is 7 g/L.
Further, inoculating the shake flask seed culture solution into a fermentation culture medium according to the inoculation amount of 10% for fermentation culture; the incubation temperature was 24 ℃.
The invention utilizes a synthetic biology method to introduce a methanol metabolic pathway into the candida antarctica, thereby realizing that the candida antarctica produces lipase by taking non-food-grade raw materials methanol and xylose as co-substrate carbon sources, reducing the production cost to a certain extent and having great significance and economic value.
Drawings
FIG. 1 is a metabolism map of genetically engineered bacteria.
FIG. 2 shows the original plasmids 113 and pki.
FIG. 3 is a diagram showing the construction of recombinant plasmid 113-DAS-DAK, Pki-AOX-CTA.
FIG. 4 is a genomic PCR validation.
FIG. 5 shows the change in lipase activity with the addition of different concentrations of methanol.
FIG. 6 is a graph showing the effect of different concentrations of methanol on gene expression levels.
Detailed Description
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the examples, the lipase activity detection method is as follows: the activity of lipase is measured by a multifunctional microplate reader by taking p-nitrophenyl acetate (p-NPA) as a substrate. The reaction system is as follows: 900uL of 20mM Tris-HCl buffer, 50uL of 10mM p-NPA acetonitrile solution, and 50uL of the enzyme solution were reacted at 30 ℃ for 2min (in this case, within the time range of the initial reaction rate). The change in absorbance at 405nm of the reaction solution was measured. Enzyme activity unit (U): the amount of enzyme required to catalyze the formation of 1umol of p-nitrophenol in 1min at 30 ℃ and pH 8.0 was defined as 1U.
Example 1: obtaining expressed genes
A Pichia pastoris genome is taken as a template, and primers are designed to amplify methanol oxidase aox1, dihydroxyacetone synthase gene das, catalase gene cta and dihydroxyacetone kinase gene dak.
Example 2: construction of Candida-aox1-das-cta-dak by synthetic biology
In order to realize the co-expression of multiple genomes quickly and effectively and ensure the stability of gene expression, a DNA (deoxyribonucleic acid) assembly method is utilized to integrate a gene expression frame into a Candida antarctica genome.
(1) Designing primers for amplification, respectively adding homologous arms of a promoter and a terminator at two ends of each gene, designing upstream and downstream primers, and obtaining an expression frame, wherein the sequences of the genes and the primers are shown in table 1.
TABLE 1 genes and primer sequences
(2) Carrying out multi-fragment cloning to form an expression frame TEF-aox1-CYC1t, TEF-das-tCYC1, PDC1p-cta-TDH2t and pGPD-dak-TXPR2 of a promoter-gene-terminator; the expression cassette of promoter, gene and terminator is formed by combining and connecting the genes shown in the specification by using a multi-fragment cloning method to form an expression fragment.
(3) Connecting the two gene segments of das and dak with a 113 plasmid by a multi-segment cloning method, transforming the gene segments into E.coli DH5 alpha, connecting the two gene segments of aox1 and cta with a Pki plasmid by a multi-segment cloning method, and transforming the gene segments into E.coli DH5 alpha, and obtaining a plasmid. As shown in FIG. 3, the correctly verified plasmids were sent to the sequencing company for sequencing by plasmid digestion and colony PCR verification.
(4) The plasmid with correct sequencing is digested by NotI, the gene recombination fragment is electrically converted into candida antarctica after the fragment of the target gene is recovered by kit glue, and the method specifically comprises the following steps:
the preparation method of the candida antarctica competence comprises the following steps:
(1) inoculating the original strain into 5mL seed culture medium, culturing at 24 deg.C for 12 hr, transferring 500 μ L culture into 50mL seed culture medium, and culturing at 24 deg.C to thallus concentration OD600＝0.8～1.0。
(2) And (5) carrying out ice bath for 15min, stopping cell growth, transferring the bacterial liquid into a 50mL centrifuge tube, carrying out 4000rpm for 5min, and removing the supernatant.
(3) The cells were resuspended in 30mL of pre-cooled sterile water, centrifuged at 4000rpm for 5min, and the supernatant removed.
(4) The resuspended cell pellet was washed with 20mL of pre-cooled 1M sorbitol, 4000rpm, 5min, the supernatant removed, and repeated 2 times.
(5) 200-250 μ L of 1M sorbitol is used to resuspend the cells, and the cells are transferred to a pre-cooled centrifuge tube, which is the yeast competence.
And (3) electric conversion:
(1) mu.L of recombinant plasmid and 40. mu.L of competence were added to a pre-cooled 1.5mL centrifuge tube.
(2) The target fragment and competence were mixed by gentle blowing in a centrifuge tube, transferred to an electric rotor cup, and precooled in ice for 5 min.
(3) And wiping water at the periphery of the electric revolving cup with absorbent paper, and clicking with 1500V voltage.
(4) 1mL of seed culture medium was added to the clicked cuvette, the cells were gently suspended and transferred to a 1.5mL centrifuge tube, and the incubator was allowed to recover for 2h at 24 ℃.
The recovered cells were washed twice with sterile water, spread on G418-resistant selection medium, and screened at 1G/L G418 to obtain recombinant Candida antarctica, and PCR verification was performed as shown in FIG. 4. So as to realize the simultaneous expression of the genes and further realize the metabolism of the methanol.
Example 3: fermentation experiments with recombinant strains
(1) And (3) test tube seed culture: inoculating the recombinant Candida antarctica into a test tube seed culture medium from a cryopreservation tube according to the inoculation amount of 1% (v/v), filling 5mL of test tube with liquid, and carrying out aerobic culture at 24 ℃ for 18-22h to obtain a test tube seed culture solution.
Wherein the seed culture medium comprises the following formula: 6g/L of peptone, 4g/L of hydrolyzed casein, 3g/L of yeast powder, 1.5g/L of beef extract and 1g/L of glucose.
(2) And (3) seed culture in a shaking flask: inoculating the test tube seed culture solution into a shake flask seed culture medium according to the inoculation amount of 1% (v/v), carrying out aerobic culture for 18-22h at 24 ℃ in a 250mL triangular flask with the liquid loading capacity of 50mL to obtain the shake flask seed culture solution.
(3) Producing lipase by fermentation: inoculating a shake flask seed culture solution into a fermentation culture medium according to the inoculation amount of 10% (v/v), carrying out aerobic culture at 24 ℃ for 24h in a 250mL triangular flask with the liquid loading amount of 50mL, extracting RNA, and measuring the expression level of the methanol metabolism related gene and the expression level of the candida antarctica xylose expression related gene after fermenting for 10h by fluorescence quantitative PCR.
Wherein the formula of the fermentation medium is as follows: 40g/L of soybean meal, 5ml/L of soybean oil, 4g/L of dipotassium phosphate, 1g/L of magnesium sulfate, 80ml/L of Tween, 5g/L of yeast powder, 15g/L of xylose, 0g/L of methanol, 5g/L of methanol, 7g/L of xylose and 15g/L of methanol.
As shown in FIG. 5, the lipase activity was highest at a methanol concentration of 7 g/L. The results of the gene expression level of lipase A at 7g/L compared to other methanol concentrations are shown in FIG. 6, and it is concluded that appropriate methanol increases lipase enzyme activity, and that an increased expression level of das indicates that the addition of 15g/L xylose provides xylulose-5 phosphate for methanol metabolism. The addition of 7g/L of methanol can improve the gene expression of lipase A, and simultaneously, the expression of genes related to xylose metabolism is also improved, so that the introduction of a metabolic module of methanol promotes the utilization of xylose, and the expression of genes related to lipase production is improved.
Sequence listing
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Claims (9)
1. A genetically engineered bacterium for producing lipase by using a methanol and xylose co-substrate is characterized in that a methanol oxidase gene aox1, a dihydroxyacetone synthase gene das, a catalase gene cta and a dihydroxyacetone kinase gene dak are introduced into a host bacterium; the host bacteria is Candida antarctica (Candida antarctica) ZJB 09193.
2. The genetically engineered bacterium of claim 1, wherein the construction method is as follows:
(1) constructing expression frames of TEF-aox1-CYC1t, TEF-das-tCYC1, PDC1p-cta-TDH2t and pGPD-dak-TXPR2, connecting two gene segments of TEF-das-tCYC1 and pGPD-dak-TXPR2 with a 113 plasmid by a multi-segment cloning method, connecting two gene segments of TEF-aox1-CYC1t and PDC1p-cta-TDH2t with a Pki plasmid, and transforming the gene segments into E.coli DH5 alpha;
(2) and carrying out enzyme digestion on the plasmid with correct sequencing to obtain a gene recombinant fragment, electrically transforming the gene recombinant fragment to host bacteria, and screening through G418 resistance to obtain the gene engineering bacteria.
3. Use of the genetically engineered bacterium of any one of claims 1 to 2 in the production of lipase by fermentation.
4. Use according to claim 3, characterized in that it comprises the following steps:
(1a) and (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture;
(2a) and (3) seed culture in a shaking flask: inoculating the test tube seed culture solution into a seed culture medium of a shake flask for culture;
(3a) producing lipase by fermentation: inoculating the shake flask seed culture solution into a fermentation culture medium for fermentation culture to obtain the lipase.
5. The use according to claim 4, wherein the culture temperature of the test tube seed culture and the shake flask seed culture is 24 ℃; the culture time is 18-22 h.
6. The use according to claim 4, wherein the seed culture medium is formulated as follows: 6g/L of peptone, 4g/L of hydrolyzed casein, 3g/L of yeast powder, 1.5g/L of beef extract, 1g/L of glucose and the balance of water.
7. The use according to claim 4, wherein the fermentation medium is formulated as follows: 40g/L of soybean meal, 5ml/L of soybean oil, 4g/L of dipotassium phosphate, 1g/L of magnesium sulfate, 80ml/L of Tween, 5g/L of yeast powder, 15g/L of xylose, 0-15 g/L of methanol and the balance of water.
8. Use according to claim 7, wherein the methanol concentration is 7 g/L.
9. The use of claim 4, wherein the shake flask seed culture is inoculated into the fermentation medium at an inoculum size of 10% for fermentation culture.
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