CN111304105A - 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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- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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 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 cosubstrates, reducing the production cost to a certain extent and having 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 candida antarctica by a synthetic biological means, the methanol and 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 using methanol and xylose co-substrate is prepared by introducing methanol oxidase gene into host bacteriumaox1. Dihydroxyacetone synthase genedasCatalase genectaAnd dihydroxyacetone kinase genedak(ii) a 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 in catalasectaTo oxygen and water; formaldehyde and xylulose-5-phosphate in dihydroxyacetone synthasedasThe xylose is added externally to provide xylulose-5 phosphoric acid for the dihydroxyacetone to accelerate the production of the dihydroxyacetone; subsequent dihydroxyacetone in dihydroxyacetone kinasedakIs converted into dihydroxyacetone phosphate under the catalysis of the enzyme (2), and then is further metabolized into fructose-6-phosphate, and further enters the glycolysis pathway to participate in substance circulation and organic acid metabolism, as shown in figure 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 metabolism of methanol so as to improve the related enzyme activity of the constructed strain lipase.
Wherein the host bacterium is candida antarctica (A)Candida antarctica) ZJB09193 with preservation number of CCTCC M2010263, and the strainStrains have been disclosed in Liu et al, Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and indications in biosynthesis of vitamins A esters, microbiological research, Volume 167, Issue 8, 6 September 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 methanol oxidase geneaoxGenBank accession number of XM-002494226.1;
the dihydroxyacetone synthase genedasGenBank accession No. FJ 752551.1;
the catalase genectaGenBank accession No. AB 472085.1;
the dihydroxyacetone kinase genedakGenBank accession number is XM-002493026.1.
Wherein the methanol oxidase geneaoxDihydroxyacetone synthase genedasCatalase genectaDihydroxyacetone kinase genedakThe promoter sequence of (A) is selected from one of TEF, PGPD and PDC 1P.
Methanol oxidase geneaoxDihydroxyacetone synthase genedasCatalase genectaDihydroxyacetone kinase genedakThe terminator of (2) is selected from one of CYC1t and TDH2 t.
The construction method of the genetic engineering bacteria for producing lipase by using methanol comprises the following steps:
(1) construction of TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dakTXPR2 expression cassette, TEF-das-tCYC1、pGPD-dakThe two gene segments of TXPR2 were ligated with the 113 plasmid, TEF-aox1-CYC1t、PDC1p-ctaTwo gene fragments of-TDH 2t were ligated with Pki plasmid and transformed intoE.coli DH5αPerforming the following steps;
(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: 6 g/L of peptone, 4 g/L of hydrolyzed casein, 3 g/L of yeast powder, 1.5 g/L of beef extract and 1 g/L of glucose;
wherein the formula of the fermentation medium is as follows: 40 g/L of soybean meal, 5 ml/L of soybean oil, 4 g/L of dipotassium phosphate, 1 g/L of magnesium sulfate, 80ml/L of Tween, 5 g/L of yeast powder, 15 g/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 plasmid 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-nitrophenylacetate (p-NPA) as a substrate. The reaction system is as follows: 900 uL of 20mM Tris-HCl buffer, 50 uL of 10 mM p-NPA acetonitrile solution, and 50 uL of the enzyme solution were reacted at 30 ℃ for 2 min (in this case, within the time range of the initial reaction rate). The change in absorbance of the reaction solution at 405 nm 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 pH8.0 was defined as 1U.
Example 1: obtaining expressed genes
By Pichia pastorisPichia pastorisGenome as template, designing primer to amplify methanol oxidaseaox1. Dihydroxyacetone synthase genedasCatalase genectaDihydroxyacetone kinase genedak。
Example 2: construction by synthetic biologyCandida-aox1-das-cta-dak
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) Performing multi-fragment cloning to constitute a startExpression cassette TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dak-TXPR 2; 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) Will be provided withdas、dakTwo gene segments are connected with 113 plasmids by a multi-segment cloning method and are transformed intoE.coli DH5αIn the process, theaox1、ctaTwo gene segments are connected with Pki plasmid by a multi-segment cloning method and transformed intoE.coli DH5αAnd (4) a plasmid. As shown in FIG. 3, the plasmid with the correct verification is sent to a sequencing company for sequencing after the plasmid is cut and the colony PCR is verified.
(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 starting strain into 5 mL of seed culture medium, culturing at 24 ℃ for 12 h, transferring 500 muL of culture into 50 mL of seed culture medium, and culturing at 24 ℃ until the thallus concentration OD600=0.8～1.0。
(2) And (5) carrying out ice bath for 15 min, stopping cell growth, transferring the bacterial liquid into a 50 mL centrifuge tube, carrying out 4000 rpm for 5 min, and removing the supernatant.
(3) The cells were resuspended in 30 mL of pre-cooled sterile water, centrifuged at 4000 rpm for 5 min, and the supernatant removed.
(4) The resuspended cell pellet was washed with 20 mL of pre-cooled 1M sorbitol, 4000 rpm, 5 min, the supernatant removed, and repeated 2 times.
(5) And (4) resuspending the cells by using 200 muL-250 muL 1M sorbitol, and transferring the cells to a precooled centrifuge tube, wherein the cells are the yeast competence.
And (3) electric conversion:
(1) 10 μ L of the recombinant plasmid and 40 μ L of the competence were added to a pre-cooled 1.5 mL 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) 1 mL of seed culture medium was added to the clicked cuvette, the cells were gently suspended and transferred to a 1.5 mL 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 5 mL 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: 6 g/L of peptone, 4 g/L of hydrolyzed casein, 3 g/L of yeast powder, 1.5 g/L of beef extract and 1 g/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 250 mL triangular flask with the liquid loading capacity of 50 mL 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 250 mL triangular flask with the liquid loading amount of 50 mL, 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: 40 g/L of soybean meal, 5 ml/L of soybean oil, 4 g/L of dipotassium phosphate, 1 g/L of magnesium sulfate, 80ml/L of Tween, 5 g/L of yeast powder, 15 g/L of xylose, 0 g/L of methanol, 5 g/L of methanol, 7g/L of xylose and 15 g/L of methanol.
As shown in FIG. 5, the lipase was obtained at a methanol concentration of 7g/LThe enzyme activity is highest. The results of the gene expression level of lipase A at 7g/L as compared with other methanol concentrations are shown in FIG. 6, and it is concluded that appropriate methanol increases lipase enzyme activity,dasthe increased expression level of (D) indicates that the addition of xylose at 15 g/L provides xylulose-5 phosphate for the metabolism of methanol. 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 (10)
1. A genetically engineered bacterium for producing lipase by using a methanol and xylose cosubstrate, which is characterized in that the genetically engineered bacterium is used for producing the lipase to a hostIntroduction of methanol oxidase Gene into the bacteriumaox1. Dihydroxyacetone synthase genedasCatalase genectaAnd dihydroxyacetone kinase genedak(ii) a The host bacteria is candida antarctica which can produce lipase by using xylose.
2. The genetically engineered bacterium of claim 1, wherein the host bacterium is Candida antarcticaCandida antarctica）ZJB09193。
3. The genetically engineered bacterium of claim 1, wherein the construction method is as follows:
(1) construction of TEF-aox1-CYC1t、TEF-das-tCYC1、PDC1p-cta-TDH2t、pGPD-dakTXPR2 expression cassette, TEF-das-tCYC1、pGPD-dakThe two gene segments of TXPR2 were ligated with the 113 plasmid, TEF-aox1-CYC1t、PDC1p-ctaTwo gene fragments of-TDH 2t were ligated with Pki plasmid and transformed intoE.coli DH5αPerforming the following steps;
(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.
4. Use of the genetically engineered bacterium of any one of claims 1 to 3 in the production of lipase by fermentation.
5. Use according to claim 4, 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.
6. The use according to claim 5, 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.
7. The use according to claim 5, wherein the seed medium is formulated as follows: 6 g/L of peptone, 4 g/L of hydrolyzed casein, 3 g/L of yeast powder, 1.5 g/L of beef extract and 1 g/L of glucose.
8. The use according to claim 5, wherein the fermentation medium is formulated as follows: 40 g/L of soybean meal, 5 ml/L of soybean oil, 4 g/L of dipotassium phosphate, 1 g/L of magnesium sulfate, 80ml/L of Tween, 5 g/L of yeast powder, 15 g/L of xylose and 0-15 g/L of methanol.
9. Use according to claim 8, wherein the methanol concentration is 7 g/L.
10. The use of claim 5, 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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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565713A (en) * | 2009-06-01 | 2009-10-28 | 华南理工大学 | Candida Antarctica lipase B gene and applications thereof in yeast display |
| CN103789364A (en) * | 2013-11-19 | 2014-05-14 | 南宁市新科健生物技术有限责任公司 | Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis |
| WO2014076232A2 (en) * | 2012-11-19 | 2014-05-22 | Novozymes A/S | Isopropanol production by recombinant hosts using an hmg-coa intermediate |
| CN104480083A (en) * | 2014-10-15 | 2015-04-01 | 深圳华中科技大学研究院 | Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium |
| US20150094483A1 (en) * | 2009-05-06 | 2015-04-02 | Jon E. Ness | Biotransformation using genetically modified candida |
| CN104718280A (en) * | 2012-10-16 | 2015-06-17 | 帝斯曼知识产权资产管理有限公司 | Cells with improved pentose conversion |
| CN106047917A (en) * | 2016-07-25 | 2016-10-26 | 北京化工大学 | Building method of pichia pastoris gene engineering strain for producing lipase |
| CN107881123A (en) * | 2017-08-08 | 2018-04-06 | 南京工业大学 | One plant of genetic engineering bacterium and its construction method and application using methanol production pyruvic acid |
-
2020
- 2020-02-27 CN CN202010125659.5A patent/CN111304105B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150094483A1 (en) * | 2009-05-06 | 2015-04-02 | Jon E. Ness | Biotransformation using genetically modified candida |
| CN101565713A (en) * | 2009-06-01 | 2009-10-28 | 华南理工大学 | Candida Antarctica lipase B gene and applications thereof in yeast display |
| CN104718280A (en) * | 2012-10-16 | 2015-06-17 | 帝斯曼知识产权资产管理有限公司 | Cells with improved pentose conversion |
| WO2014076232A2 (en) * | 2012-11-19 | 2014-05-22 | Novozymes A/S | Isopropanol production by recombinant hosts using an hmg-coa intermediate |
| CN103789364A (en) * | 2013-11-19 | 2014-05-14 | 南宁市新科健生物技术有限责任公司 | Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis |
| CN104480083A (en) * | 2014-10-15 | 2015-04-01 | 深圳华中科技大学研究院 | Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium |
| CN106047917A (en) * | 2016-07-25 | 2016-10-26 | 北京化工大学 | Building method of pichia pastoris gene engineering strain for producing lipase |
| CN107881123A (en) * | 2017-08-08 | 2018-04-06 | 南京工业大学 | One plant of genetic engineering bacterium and its construction method and application using methanol production pyruvic acid |
Non-Patent Citations (2)
| Title |
|---|
| KATIYAR, M等: "Immobilization of Candida rugosa Lipase on MCM-41 for the Transesterification of Cotton Seed Oil", 《JOURNAL OF OLEO SCIENCE》 * |
| 李迅等: "南极假丝酵母脂肪酶B在毕赤酵母中的分泌型表达及酶学性质初探", 《四川师范大学学报(自然科学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107081A (en) * | 2021-11-30 | 2022-03-01 | 南京工业大学 | Recombinant yarrowia lipolytica genetic engineering bacterium utilizing methanol biotransformation and construction method and application thereof |
| CN114107081B (en) * | 2021-11-30 | 2023-05-05 | 南京工业大学 | Recombinant yarrowia lipolytica genetically engineered bacterium utilizing methanol bioconversion and construction method and application thereof |
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