CN113999807A - Construction method of recombinant strain and application of recombinant strain in production of itaconic acid - Google Patents

Construction method of recombinant strain and application of recombinant strain in production of itaconic acid Download PDF

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CN113999807A
CN113999807A CN202111287718.XA CN202111287718A CN113999807A CN 113999807 A CN113999807 A CN 113999807A CN 202111287718 A CN202111287718 A CN 202111287718A CN 113999807 A CN113999807 A CN 113999807A
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cada
pcdfduet
recombinant strain
itaconic acid
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CN113999807B (en
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陈可泉
李春秋
冯娇
张锟
曾金磊
周彩莲
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Nanjing Tech University
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Abstract

The invention discloses a construction method of a recombinant strain and application thereof in producing itaconic acid, which mutates 470-position Arg of a natural aconitate decarboxylase gene cadA into Glu by site-directed mutagenesis of the aconitate decarboxylase gene cadA, and then transfers the Glu into escherichia coli for heterologous expression. The site-directed mutagenesis uses a large primer PCR method, and the method has the advantages of simple and convenient operation, high mutation rate and lower cost. Compared with conventional fermentation, the method for producing the itaconic acid uses a whole-cell catalysis method, can reduce the production cost, shorten the production time and effectively improve the yield of the itaconic acid. In addition, the substrate is easy to obtain, byproducts generated by biotransformation are less, purification is convenient, the defect of complex product of the traditional synthetic method is effectively overcome, and the method has wide application prospect in industrial production.

Description

Construction method of recombinant strain and application of recombinant strain in production of itaconic acid
Technical Field
The invention belongs to the fields of molecular biology and genetic engineering, and particularly relates to a construction method of a recombinant strain and application of the recombinant strain in production of itaconic acid.
Background
The aconitate decarboxylase can convert aconitate into itaconic acid, and plays an important role in the processes of connecting natural immunity and metabolism and producing itaconic acid by a biological method. Itaconic acid (itaconic acid and methylene succinic acid) is an unsaturated dicarboxylic acid, is an important industrial raw material and additive in the chemical industry field, and is widely applied to the aspects of producing plastics, chemical fibers, super adsorbents, latex, scale inhibitors and the like as a construction module. Itaconic acid has gained increasing attention as a renewable material. In 2004, itaconic acid was selected by the U.S. department of energy as one of the most promising 12 bio-based platform compounds. At present, the worldwide capacity of itaconic acid is 5 ten thousand tons/year, and at least 3 ten thousand tons of gaps are faced, and itaconic acid can replace acrylic acid and methacrylic acid to be used for producing degradable plastics, the future demand of itaconic acid will continue to increase rapidly, and the itaconic acid is an important organic acid from biomass with wide application prospect.
At present, the dominant method for industrially producing itaconic acid at home and abroad is an aspergillus terreus fermentation method. However, compared with other organic acid fermentation production, the fermentation unit for producing itaconic acid by fermentation of aspergillus terreus is still lower internationally at present because: 1) the fermentation time of the aspergillus terreus is long, and the space-time yield is not high; 2) acn is localized to the mitochondria and cadA is localized to the cytoplasm in fungal cells, and the difference in localization of the two enzymes makes diffusion and reaction of the substrates of the two enzymes with the intermediate more difficult. Therefore, the researchers select escherichia coli as a host bacterium for producing itaconic acid, which has many advantages, however, the pH stability of key enzyme is poor, the substrate conversion rate is low, the production efficiency is low, and the escherichia coli as a production host can not be compared with the aspergillus terreus fermentation economically in terms of titer and yield.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a construction method of a recombinant strain and application thereof in producing itaconic acid, wherein the aconitate decarboxylase is rationally analyzed, the site-specific mutagenesis is carried out by adopting a large primer PCR technology, the aconitate decarboxylase gene cadA which is subjected to site-specific modification is subjected to enzyme digestion by DpnI, after enzyme digestion is carried out for 1h at 37 ℃, the recombinant plasmid is transformed into escherichia coli BL21 (DE 3), a recombinant strain pCDFduet-cadA (R470E) is successfully obtained, and after the whole cell catalytic reaction, the itaconic acid production capacity is improved by 28.74% under the condition of pH 5.0 and by 8.43% under the condition of pH 5.5.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for constructing a recombinant strain pCDFduet-cadA (R470E), comprising the following steps:
step 1, designing primers
Designing a mutation site by rational analysis of the aconitate decarboxylase gene cadA, and designing a primer by using Snapgene software;
step 2, construction of recombinant plasmid
Carrying out site-directed mutagenesis by using the primer designed in the step 1, and carrying out mutagenesis by using pCDFduet-cadA as a template to obtain a recombinant plasmid pCDFduet-cadA (R470E);
step 3, constructing recombinant strains
3ul of the recombinant plasmid pCDFduet-cadA (R470E) constructed in the step 2 is taken and transformed into 30ul of escherichia coli competent cells, the escherichia coli competent cells are stood for 20min on ice, and immediately placed on the ice for cooling for 5min after being thermally shocked in water bath at 42 ℃ for 50s, and 1ml of LB culture medium (without adding antibiotics) is added; shaking the bacteria at 37 ℃ and 200r/min for 1 h; centrifuging at 5000R/min for 3min, discarding 900ul of supernatant, resuspending the thallus with the rest culture medium, lightly spreading on streptomycin plate with sterile coating rod, culturing overnight at 37 deg.C by inversion in incubator, and selecting positive colony to obtain recombinant strain pCDFduet-cadA (R470E).
As a modification, the primers described in step 1 are respectively as follows:
R470E-F：5’-CTGGTGCTGGGCCTGGATGAACTGACAGATATTAGC -3’
R470E-R：5’-GGTTCAGGGCAGGGTCGTTAAAT -3’。
the improvement is that the two rounds of PCR amplification conditions of the mutation in the step 2 are the same as the amplification system, and the amplification conditions are 95 ℃, 30s and 1 cycle; 95 ℃, 15s, 56 ℃, 15s, 72 ℃, 30s and 30 cycles; 72 ℃, 5min, 1 cycle; an amplification system: ddH2O20.5. mu.l, template 0.5. mu.l, corresponding primers 2. mu.l each, and 2xPhanta Max Master Mix (Dye Plus) 25. mu.l.
The recombinant strain pCDFduet-cadA (R470E) is applied to producing itaconic acid.
As an improvement, the steps of the application are as follows:
transferring the recombinant strain pCDFduet-cadA (R470E) into 10ml of liquid LB culture medium according to the inoculum size of 1%, shaking and culturing at 37 ℃ overnight, transferring the recombinant strain pCDFduet-cadA into 100ml of liquid LB culture medium according to the inoculum size of 1% the next day, shaking and culturing at 37 ℃ for 18h, centrifuging at 4 ℃ and 4500R/min for 15min, discarding the supernatant, re-suspending the thallus by PBS buffer solution, centrifuging at 4 ℃ and 5000R/min for 10min, discarding the supernatant, and preparing the thallus into OD (OD) by PBS60060, preparing bacterial liquid for later use;
step two, reaction system: 400 mul of 0.5M aconitic acid, 1000 mul of OD 60060 bacterial liquid and 600 mul of PBS are reacted for 24 hours in a shaking incubator with the temperature of 35 ℃ and the speed of 200r/min, the reaction liquid is detected by a high performance liquid chromatograph, and the itaconic acid detection method comprises the following steps: aminex HPX-87H type chromatographic column (300X 7.8 mm, 9 μm) with a mobile phase of 0.008 mol/L sulfuric acid; the flow rate is 0.6mL min < -1 >; the detection wavelength is 210 nm; the column oven temperature was 60 ℃.
After detection, the result shows that the recombinant strain pCDFduet-cadA (R470E) produces mutated aconitate decarboxylase, the itaconic acid production capacity of the enzyme is improved by 28.74% under the condition of pH 5.0 and is improved by 8.43% under the condition of pH 5.5 (as shown in figure 3, the itaconic acid yield is calculated by using an established standard curve, the molar conversion rate is the ratio of the generated itaconic acid moles to the consumed aconitate moles, and the relative yield is calculated by taking pCDFduet-cadA as a reference).
Has the advantages that:
the invention discloses a construction method of a recombinant strain and application thereof in producing itaconic acid. The in vitro site-directed mutagenesis technique is used to introduce the desired changes (usually those characterizing favorable directions) including base addition, deletion, point mutation, etc. into the DNA fragment of interest (which may be a genome or a plasmid) by means of polymerase chain reaction, etc. The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work. Compared with the conventional fermentation, the whole-cell catalysis method adopted by the invention can reduce the production cost and shorten the production time. In addition, the substrate is easy to obtain, byproducts generated by biotransformation are less, purification is convenient, the defect of complex product of the traditional synthetic method is overcome, and the method has wide application prospect in industrial production.
1. Compared with the prior key enzyme aconitate decarboxylase in the itaconic acid synthesis path, the aconitate decarboxylase modified by mutation has better catalytic activity, saves the production cost and is more suitable for the requirement of industrial production.
2. The large primer PCR method used in site-directed mutagenesis has almost no special limitation, high success rate, simplified operation process and saved time.
3. The related experiment operation process is mild and harmless to the environment, equipment and operators.
Drawings
FIG. 1 is a diagram of a large primer PCR nucleic acid electrophoresis, (a) shows a one-round PCR product, and (b) shows a two-round PCR product;
FIG. 2 is an SDS-PAGE of aconitate decarboxylase after whole cell culture of the recombinant strain pCDFduet-cadA (R470E) in example 2;
FIG. 3 is a graph showing the comparison between pCDFduet-cadA and pCDFduet-cadA (R470E) under the same conditions for the production of itaconic acid, wherein (a) shows reaction pH 5.0, and (b) shows reaction pH 5.5.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
The techniques not mentioned in the examples are conventional in the art, and further, materials such as Escherichia coli BL21 (DE 3), pCDF-duet1, 2xPHanta Max Master Mix (Dye Plus), dNTPs and the like used are commercial products and can be directly purchased.
The PBS buffer used in the examples had a pH of 7.0.
In the examples, the concentrations of the reagents such as aconitic acid refer to the final concentration in the system.
Example 1 site-directed mutagenesis of the aconitate decarboxylase Gene cadA
The aconitic acid desaturase is derived from aspergillus terreus, and the amino acid sequence is shown as SEQ NO. 1: MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV
(1) Designing a primer, namely carrying out site-directed mutagenesis by using a large primer PCR technology through rational design of a aconitate decarboxylase gene cadA, and designing the primer by utilizing Snapgene software as follows:
R470E-F：5’-CTGGTGCTGGGCCTGGATGAACTGACAGATATTAGC -3’
R470E-R：5’-GGTTCAGGGCAGGGTCGTTAAAT -3’。
EXAMPLE 2 construction of recombinant plasmid pCDFduet-cadA (R470E)
(2) PCR amplification was performed using pCDFduet-cadA as a template and primers R470E-F, R470E-R provided in example 1 under the following conditions: 95 ℃, 30s, 1 cycle; 95 ℃, 15s, 56 ℃, 15s, 72 ℃, 30s and 30 cycles; 72 ℃, 5min, 1 cycle. An amplification system: ddH220.5 mul of O, 0.5 mul of template, 2 mul of each primer R470E-F, R470E-R, and 25 mul of 2xPHANTA Max Master Mix (Dye Plus) to amplify a PCR product of 300 bp; the conditions for the two-round PCR amplification are as follows: 95 ℃, 30s, 1 cycle; 30 cycles of 95 ℃, 15s, 56 ℃, 15s, 72 ℃, 6 min; 72 ℃, 5min, 1 cycle. An amplification system: ddH2Mu.l of O23, 1. mu.l of template, 1. mu.l of PCR product, 25. mu.l of 2xPhanta Max Master Mix (Dye Plus), and PCR product pCDFduet-cadA (R470E) containing the R470E mutation site. The target gene was recovered using an agarose gel recovery cassette, and the agarose gel electrophoresis of the large primer PCR is shown in FIG. 1, wherein (a) is a PCR product of one round, i.e., 300bp large primer, and (b) is a PCR product of two rounds, i.e., recombinant plasmid pCDFduet-cadA (R470E).
EXAMPLE 2 acquisition of recombinant Strain pCDFduet-cadA (R470E)
The recombinant plasmid pCDFduet-cadA (R470E) constructed in example 1 was digested with Dpn I, and the digestion system: the recombinant plasmid pCDFduet-cadA (R470E) 8.5. mu.l, Dpn I0.5. mu.l, 10xQuick Cut Buffer 1. mu.l, was digested at 37 ℃ for 1 hour, and the digested recombinant plasmid pCDFduet-cadA (R470E) was transformed into E.coli competent cells.
The transformation method comprises the following steps: 3ul of recombinant plasmid pCDFduet-cadA (R470E) is taken and transformed into 30ul of escherichia coli competent cells, the cells are kept still on ice for 20min, and immediately placed on ice for cooling for 5min after being heat-shocked in water bath at 42 ℃ for 50s, and 1ml of LB culture medium (without antibiotics) is added; shaking the bacteria at 37 ℃ and 200r/min for 1 h; centrifuging at 5000R/min for 3min, discarding 900ul of supernatant, resuspending the thallus with the rest culture medium, lightly spreading on streptomycin plate with sterile coating rod, culturing overnight at 37 deg.C by inversion in incubator, and selecting positive colony to obtain recombinant strain pCDFduet-cadA (R470E).
EXAMPLE 3 use of recombinant Strain pCDFduet-cadA (R470E) for the production of itaconic acid
The recombinant strain pCDFduet-cadA (R470E) was inoculated into 5ml of LB liquid medium at an inoculum size of 1%, cultured overnight at 37 ℃ and then inoculated into a new 100ml of LB liquid medium at an inoculum size of 1%, and cultured at 37 ℃ to OD600When the value of (A) is 0.6, inducing with 5 ‰ IPTG, and culturing at 18 deg.C for 18 h. Centrifuging at 4500r/min for 15min, removing supernatant, resuspending the thallus with PBS buffer solution, centrifuging at 5000r/min for 10min, using the thallus for whole cell reaction, wherein the reaction system comprises: 0.5M aconitic acid 400. mu.l, OD600 Reacting 60 bacteria liquid 1000 mul and PBS 600 mul in a shaking incubator at 35 ℃ and 200r/min for 24h, detecting the reaction liquid by a high performance liquid chromatograph, and detecting itaconic acid: aminex HPX-87H type chromatographic column (300X 7.8 mm, 9 μm) with a mobile phase of 0.008 mol/L sulfuric acid; flow rate 0.6 mL/min-1(ii) a The detection wavelength is 210 nm; the column oven temperature was 60 ℃. The results showed that the recombinant strain pCDFduet-cadA (R470E) produced a mutated aconitate decarboxylase whose enzyme has an increased itaconate-producing capacity of 28.74% at pH 5.0 and 8.43% at pH 5.5 (as shown in FIG. 3).
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> Nanjing university of industry
<120> construction method of recombinant strain and application thereof in production of itaconic acid
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctggtgctgg gcctggatga actgacagat attagc 36
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggttcagggc agggtcgtta aat 23
<210> 3
<211> 490
<212> PRT
<213> amino acid Sequence (amino acid Sequence)
<400> 3
Met Thr Lys Gln Ser Ala Asp Ser Asn Ala Lys Ser Gly Val Thr Ser
1 5 10 15
Glu Ile Cys His Trp Ala Ser Asn Leu Ala Thr Asp Asp Ile Pro Ser
20 25 30
Asp Val Leu Glu Arg Ala Lys Tyr Leu Ile Leu Asp Gly Ile Ala Cys
35 40 45
Ala Trp Val Gly Ala Arg Val Pro Trp Ser Glu Lys Tyr Val Gln Ala
50 55 60
Thr Met Ser Phe Glu Pro Pro Gly Ala Cys Arg Val Ile Gly Tyr Gly
65 70 75 80
Gln Lys Leu Gly Pro Val Ala Ala Ala Met Thr Asn Ser Ala Phe Ile
85 90 95
Gln Ala Thr Glu Leu Asp Asp Tyr His Ser Glu Ala Pro Leu His Ser
100 105 110
Ala Ser Ile Val Leu Pro Ala Val Phe Ala Ala Ser Glu Val Leu Ala
115 120 125
Glu Gln Gly Lys Thr Ile Ser Gly Ile Asp Val Ile Leu Ala Ala Ile
130 135 140
Val Gly Phe Glu Ser Gly Pro Arg Ile Gly Lys Ala Ile Tyr Gly Ser
145 150 155 160
Asp Leu Leu Asn Asn Gly Trp His Cys Gly Ala Val Tyr Gly Ala Pro
165 170 175
Ala Gly Ala Leu Ala Thr Gly Lys Leu Leu Gly Leu Thr Pro Asp Ser
180 185 190
Met Glu Asp Ala Leu Gly Ile Ala Cys Thr Gln Ala Cys Gly Leu Met
195 200 205
Ser Ala Gln Tyr Gly Gly Met Val Lys Arg Val Gln His Gly Phe Ala
210 215 220
Ala Arg Asn Gly Leu Leu Gly Gly Leu Leu Ala His Gly Gly Tyr Glu
225 230 235 240
Ala Met Lys Gly Val Leu Glu Arg Ser Tyr Gly Gly Phe Leu Lys Met
245 250 255
Phe Thr Lys Gly Asn Gly Arg Glu Pro Pro Tyr Lys Glu Glu Glu Val
260 265 270
Val Ala Gly Leu Gly Ser Phe Trp His Thr Phe Thr Ile Arg Ile Lys
275 280 285
Leu Tyr Ala Cys Cys Gly Leu Val His Gly Pro Val Glu Ala Ile Glu
290 295 300
Asn Leu Gln Gly Arg Tyr Pro Glu Leu Leu Asn Arg Ala Asn Leu Ser
305 310 315 320
Asn Ile Arg His Val His Val Gln Leu Ser Thr Ala Ser Asn Ser His
325 330 335
Cys Gly Trp Ile Pro Glu Glu Arg Pro Ile Ser Ser Ile Ala Gly Gln
340 345 350
Met Ser Val Ala Tyr Ile Leu Ala Val Gln Leu Val Asp Gln Gln Cys
355 360 365
Leu Leu Ser Gln Phe Ser Glu Phe Asp Asp Asn Leu Glu Arg Pro Glu
370 375 380
Val Trp Asp Leu Ala Arg Lys Val Thr Ser Ser Gln Ser Glu Glu Phe
385 390 395 400
Asp Gln Asp Gly Asn Cys Leu Ser Ala Gly Arg Val Arg Ile Glu Phe
405 410 415
Asn Asp Gly Ser Ser Ile Thr Glu Ser Val Glu Lys Pro Leu Gly Val
420 425 430
Lys Glu Pro Met Pro Asn Glu Arg Ile Leu His Lys Tyr Arg Thr Leu
435 440 445
Ala Gly Ser Val Thr Asp Glu Ser Arg Val Lys Glu Ile Glu Asp Leu
450 455 460
Val Leu Gly Leu Asp Arg Leu Thr Asp Ile Ser Pro Leu Leu Glu Leu
465 470 475 480
Leu Asn Cys Pro Val Lys Ser Pro Leu Val
485 490

Claims (5)

1. A method for constructing a recombinant strain pCDFduet-cadA (R470E), which is characterized by comprising the following steps:
step 1, designing primers
Designing a mutation site by rational analysis of the aconitate decarboxylase gene cadA, and designing a primer by utilizing Snapgene software;
step 2, construction of recombinant plasmid
Carrying out site-directed mutagenesis by using the primer designed in the step 1, and carrying out mutagenesis by using pCDFduet-cadA as a template to obtain a recombinant plasmid pCDFduet-cadA (R470E);
step 3, obtaining recombinant strains
The recombinant plasmid pCDFduet-cadA (R470E) constructed in step 2 is transformed into competent cells of Escherichia coli, and cultured to obtain a recombinant strain pCDFduet-cadA (R470E).
2. The method for constructing a recombinant strain pCDFduet-cadA (R470E) according to claim 1, wherein the primers in step 1 are as follows:
R470E-F：5’-CTGGTGCTGGGCCTGGATGAACTGACAGATATTAGC-3’
R470E-R：5’-GGTTCAGGGCAGGGTCGTTAAAT-3’。
3. the method for constructing the recombinant strain pCDFduet-cadA (R470E) according to claim 1, wherein the two rounds of PCR amplification conditions of the mutation in step 2 are the same as the amplification system, and the amplification conditions are 95 ℃, 30s and 1 cycle; 95 ℃, 15s, 56 ℃, 15s, 72 ℃, 30s and 30 cycles; 72 ℃, 5min, 1 cycle; an amplification system: ddH2O20.5. mu.l, template 0.5. mu.l, corresponding primers 2. mu.l each, and 2xPhanta Max Master Mix (Dye Plus) 25. mu.l.
4. Use of the recombinant strain pCDFduet-cadA (R470E) obtained according to claim 1 for the production of itaconic acid.
5. The application of claim 4, comprising the following steps: firstly, transferring a recombinant strain pCDFduet-cadA (R470E) into 10ml of liquid LB culture medium according to the inoculation amount of 1%, carrying out shake culture at 37 ℃ overnight, transferring the recombinant strain pCDFduet-cadA into 100ml of liquid LB culture medium according to the inoculation amount of 1% the next day, carrying out shake culture at 37 ℃ for 18h, centrifuging at 4 ℃ and 4500R/min for 15min, discarding supernatant, re-suspending thallus by PBS buffer solution, centrifuging at 4 ℃ and 5000R/min for 10min, discarding supernatant, and configuring thallus into OD (OD) by PBS60060, preparing bacterial liquid for later use;
step two, reaction system: 0.5M aconitic acid 400. mu.l, OD600 1000 mul of 60 bacterium liquid, 600 mul of PBS and pH 5.5 are reacted for 24 hours in a shaking incubator with the temperature of 35 ℃ and the speed of 200r/min, the reaction liquid is detected by a high performance liquid chromatograph, and the method for detecting the itaconic acid comprises the following steps: aminex HPX-87H type chromatographic column (300X 7.8 mm, 9 μm) with a mobile phase of 0.008 mol/L sulfuric acid; flow rate 0.6 mL/min-1(ii) a The detection wavelength is 210 nm; the column oven temperature was 60 ℃.
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