CN114395518A - Recombinant escherichia coli and construction method and application thereof - Google Patents

Recombinant escherichia coli and construction method and application thereof Download PDF

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CN114395518A
CN114395518A CN202111485206.4A CN202111485206A CN114395518A CN 114395518 A CN114395518 A CN 114395518A CN 202111485206 A CN202111485206 A CN 202111485206A CN 114395518 A CN114395518 A CN 114395518A
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escherichia coli
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章文明
姜岷
陶雨萱
信丰学
王含笑
蒋羽佳
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Abstract

The invention discloses a recombinant escherichia coli, which improves the stress resistance and product synthesis performance of escherichia coli based on cell membrane modification, and particularly introduces and expresses a gene of an oxidative stress regulating enzyme from corynebacterium glutamicumwhiB(NCBI ID: AKL 15-01645) and unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosacti（NCBI ID:CWI20_17990）To be implemented. The recombinant strain can tolerate the inhibition of high-concentration substrates and products under the anaerobic condition, and can improve the growth amount of the strain and the accumulation of succinic acid under the anaerobic condition of a 5L fermentation tank.

Description

Recombinant escherichia coli and construction method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering and fermentation thereof, and relates to recombinant escherichia coli, a construction method and application thereof.
Background
Succinic acid, also known as succinic acid, is a common natural organic acid that is widely found in humans, animals, plants and microorganisms. Succinic acid is not only an intermediate product of the TCA cycle, but also one of the terminal reduction products of anaerobic metabolism, and has a very important role in the biological metabolic process. The succinic acid and derivatives thereof are widely applied, can be used as a C4 platform compound to synthesize 1, 4-butanediol, tetrahydrofuran and poly (butylene succinate) (PBS) biodegradable polyesters, and are widely applied to various industries such as food, medicine, printing and the like. In recent years, with the increasing conflict between fossil resource utilization and environmental protection, the adoption of a biological method for preparing succinic acid meets the environment-friendly production regulations of green, renewable and the like, and has a wide prospect.
Compared with the defects of complex process, harsh conditions, dependence on petroleum and the like of the traditional chemical synthesis method, the biological method has the advantages of low raw material price, small pollution and the like. However, the fermentation strain is one of the key points of succinic acid biosynthesis, and only a part of strains can produce high-concentration succinic acid. And the biosynthesis method needs to continuously optimize the synthesis path and modify engineering strains so as to achieve the purposes of industrial high yield and high efficiency. The Escherichia coli has clear genetic background, simple gene modification, high growth speed and simple and cheap culture medium, and becomes a research hotspot for preparing succinic acid by a biological method in recent years.
Due to the continuous accumulation of products during the fermentation process of the strains, alkali is added to adjust the pH value to maintain the optimal growth of the strains. However, in the later stage of fermentation, the growth of the strain is still greatly inhibited, and the inhibition brought by high-concentration substrates and high-concentration products cannot be completely relieved by the regulation of alkali liquor. Therefore, the improvement of the stress resistance of the strain becomes a new breakthrough.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides a recombinant escherichia coli and application thereof.
In order to achieve the above object, the present invention provides a recombinant Escherichia coli into which genes whiB (NCBI ID: AKL 15-01645) expressing an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum and cti (NCBI ID: CWI 20-17990) expressing an unsaturated fatty acid cis-trans isomerase derived from Pseudomonas aeruginosa are introduced, wherein the sequence number of the gene WhiB is NCBI ID: AKL 15-01645; the sequence number of the gene cti is NCBI ID: CWI20_ 17990; the host bacteria have a preservation number of CCTCC NO: escherichia coli (Escherichia coli) BER208 of M2012351. The preservation date of the Escherichia coli BER208 is 9/14 days 2012, the preservation unit is totally called China center for type culture Collection and the address is Wuhan, Wuhan university, and the details thereof are disclosed in patent CN 102864113A.
The invention further provides a construction method of the recombinant escherichia coli, which constructs a gene whiB for expressing the oxidative stress regulating enzyme from corynebacterium glutamicum and a gene cti for expressing the unsaturated fatty acid cis-trans isomerase from pseudomonas aeruginosa into a recombinant escherichia coli with a preservation number of CCTCCNO: m2012351, in Escherichia coli (Escherichia coli) BER 208.
Specifically, the construction method comprises the following steps:
(1) obtaining target gene fragments whiB and cti;
(2) cloning a target gene fragment to a starting plasmid by using a one-step cloning method to obtain a recombinant plasmid;
(3) and (3) converting the positive cloning plasmid with correct sequencing into a host bacterium Escherichia coli (Escherichia coli) BER208, and screening by Amp resistance to obtain the succinic acid-producing recombinant Escherichia coli with improved anti-reverse performance.
Preferably, the starting plasmid is the pTrc99A plasmid.
Specifically, in the step (2), primers carrying enzyme cutting sites Hind III and homologous arms of a vector pTrc99A are designed and inserted into linearized gene fragments whiB and ct to obtain a cloning plasmid pTrc 99A-whiB-cti.
The invention further provides application of the recombinant escherichia coli in preparation of succinic acid.
Furthermore, the invention provides the inhibition effect of the recombinant escherichia coli on the high-concentration substrate and high-concentration product in the succinic acid produced by the fermentation method.
The recombinant escherichia coli can be used for producing succinic acid by fermentation in a fermentation system in which 90g/L of substrate is put into glucose or 30g/L of sodium succinate in a product and 60g/L of glucose are used as carbon sources. The continuous accumulation of succinic acid in the later fermentation period causes the adverse environment of hypertonic and high-yield product inhibition, influences the growth and production of the strain, relieves the inhibition by improving the metabolic flux, improves the strength of cell membranes by improving the proportion of unsaturated fatty acid of the cell membranes, improves the tolerance performance of the strain and ensures the growth.
The invention further provides a method for improving the stress resistance of escherichia coli and the synthesis performance of products, which is characterized in that a gene whiB for expressing oxidative stress regulating enzyme from corynebacterium glutamicum and a gene cti for expressing unsaturated fatty acid cis-trans isomerase from pseudomonas aeruginosa are introduced into the escherichia coli, wherein the sequence number of the gene whiB is NCBI ID: AKL15_ 01645; the sequence number of gene cti is NCBI ID: CWI 20-17990.
Preferably, the preservation number of the escherichia coli is CCTCC NO: escherichia coli (Escherichia coli) BER208 of M2012351.
Has the advantages that: compared with the prior art, the pTrc99A plasmid is introduced into host bacteria, and the oxidative stress regulating enzyme whiB from corynebacterium glutamicum and the unsaturated fatty acid cis-trans isomerase cti from pseudomonas aeruginosa are expressed to carry out cell membrane modification, so that the recombinant microorganism can resist the inhibition of high-concentration substrates and products under anaerobic conditions. The strain can improve the growth amount of the strain and the accumulation of succinic acid under the anaerobic condition of a 5L fermentation tank. In the process of adjusting pH by using sodium salt and carrying out anaerobic fermentation for 180 hours, the final succinic acid yield of the modified strain is 78.05g/L, and compared with the original strain, the succinic acid yield is improved by 41.91%, so that the modified strain has great economic value.
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FIG. 1 shows the results of colony PCR verification;
FIG. 2 shows the stress resistance results of the strains under the inhibition of a high concentration of substrate;
FIG. 3 shows the strain stress resistance results under the inhibition of high concentration products;
FIG. 4 shows the results of the increase in the productivity of the fermenter strains.
Detailed Description
The present invention will be described in further detail with reference to specific examples, which will help understanding the present invention, but the scope of the present invention is not limited to the following examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: and (3) constructing recombinant Escherichia coli.
The plasmid pTrc99A-whiB-cti was constructed by a synthetic biological method and introduced into the target strain.
(1) Designing upstream and downstream primers, connecting the whiB and cti gene fragments with the digested linear pTrc99A plasmid by a multi-fragment one-step cloning method, and transforming the fragments into E.coli DH5 alpha. After plasmid cutting and colony PCR verification, the correctly verified plasmid is sent to a sequencing company for sequencing. The sequences of the upstream and downstream primers are shown in Table 1. The restriction site is Hind III. The PCR conditions are shown in Table 2. The results of colony PCR validation are shown in FIG. 1. For preparation of competent cells, gene extraction, and extraction of plasmids such as pTrc99A, please refer to the third edition of molecular cloning Experimental guidelines.
TABLE 1
Figure BDA0003397291710000041
TABLE 2
Temperature of Time
95℃ 5min
95℃ 15s
56℃ 15s
72℃ 2min30s
72℃ 10min
16℃ 1h (for temporary preservation only)
(2) Mixing the recombinant plasmid with correct sequencing with the competent cells of a target strain (Escherichia coli BER208 with the preservation number of CCTCC NO: M2012351), and carrying out ice bath for 30 min; then putting the mixture into a 42 ℃ water bath kettle, performing heat shock for 90s, and performing ice bath for 2 min; adding 900 mu L of sterile LB culture medium, mixing uniformly, and putting in a shaking table for resuscitation for 1 h; finally, 150. mu.L of the bacterial solution was pipetted and spread on LB solid medium containing ampicillin in a sterile operating table. The plate was inverted and placed in a 37 ℃ incubator. To screen out positive single colonies from hundreds of single clones on the plate, sequencing was performed.
Example 2: and constructing stress resistance fermentation experiments of the strains in adverse environment.
(1) And (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture. The culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 12 h.
Wherein the test tube seed culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride. Amp 100. mu.g/ml was added at the time of inoculation.
(2) 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, wherein the liquid loading of the shake flask is 50 mL. When inoculating, 100 mug/ml Amp and 30g/L glucose are added, and the inoculation amount is 2%. The culture temperature is 37 ℃, the rotation speed is 200r/min, and the culture time is 18-24 h.
Wherein the formula of the shake flask culture medium is as follows: betaine 0.12g/L, KH2PO4 3.5g/L，K2HPO4·3H2O 6.54g/L，(NH4)2HPO4 3.5g/L，MgSO4·7H2O 0.25g/L，CaCl2·2H2O15 mg/L, and adding trace element FeCl3·6H2O 1.6mg/L，CoCl2·2H2O 0.2mg/L，CuCl2·2H2O 0.1mg/L，ZnCl2·4H2O 0.2mg/L，Na2MoO4·2H2O 0.2mg/L，H3BO3 0.05mg/L。
(3) Reverse-environment fermentation: inoculating the shake flask seed culture solution into an anaerobic flask in a fermentation culture medium for fermentation culture under the conditions of high-concentration substrate and high-concentration product. When in inoculation, 100 mu g/mL Amp, 0.5mmol/L IPTG and 10% of the inoculation amount of an anaerobic bottle are added, the liquid loading amount of a 100mL anaerobic shaking bottle is 30mL, basic magnesium carbonate is added as a pH regulator, and carbon dioxide is added for 3 min; the culture temperature is 37 ℃, 200r/min, and the fermentation time is 48 h.
Wherein the anaerobic bottle culture medium comprises the following components in parts by weight: betaine 0.12g/L, (NH)4)2HPO4 2.6g/L，NH4H2PO40.87g/L，KCl 0.15g/L，MgSO4·7H2O0.37 g/L, and trace element FeCl3·6H2O 2.4mg/L，CoCl2·2H2O 0.3mg/L，CuCl2·2H2O 0.15mg/L，ZnCl2·4H2O 0.5mg/L，Na2MoO4·2H2O 0.5mg/L，MnCl2·4H2O0.5 mg/L and H3BO3 0.075g/L。
The fermentation result under the high substrate concentration inhibition is shown in figure 3, compared with a control strain G1, the OD growth amount of the strain G2 is obviously improved, the succinic acid yield is improved to some extent, the expression of whiB successfully improves the glucose utilization rate of the strain, the high sugar inhibition is relieved, and the expression of cti resists the hypertonic environment caused by high sugar, the growth of the strain is ensured, and the stress resistance of the strain is improved.
The fermentation results under the inhibition of high product concentration are shown in fig. 4, and compared with a control strain G1, the OD (optical density) growth amount of the strain G2 is obviously improved under the stress of adding 30G/L sodium succinate, and the succinic acid yield is obviously improved, so that the stress resistance of the strain is obviously improved by modifying cell membranes.
Example 3: fermentation experiments and yield improvement of the constructed strain under a 5L fermentation tank.
(1) And (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture. The culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 12 h.
Wherein the test tube seed culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride. Amp 100. mu.g/ml was added at the time of inoculation.
(2) 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, wherein the liquid loading of the shake flask is 50 mL. When inoculating, 100 mug/ml Amp and 30g/L glucose are added, and the inoculation amount is 2%. The culture temperature is 37 ℃, the rotation speed is 200r/min, and the culture time is 18-24 h.
(3) Fermentation in a fermentation tank: inoculating the shake flask seed culture solution into a 5L fermentation tank filled with 2L fermentation medium for fermentation culture. The inoculum size was 10%, Amp 100. mu.g/ml and IPTG 0.5mmol/L were added at the time of inoculation, and glucose was added so that the initial sugar concentration was 50 g/L. And continuously introducing sterile carbon dioxide gas to maintain the anaerobic environment of fermentation. The fermentation temperature is maintained at 37 ℃, and the stirring speed is 200 r/min. The pH value of the fermentation is kept to be 6.75-6.80 by using mixed sodium salt, sampling is carried out once every 12h, and the fermentation time is 180 h.
The fermentation result of the fermentation tank is shown in figure 4, compared with a control strain G1, the total sugar consumption of the strain G2 is obviously improved, the succinic acid yield is improved to 78.05G/L, and the fermentation tank has an industrial prospect.
The present invention provides a new idea and method for recombinant escherichia coli, and a number of methods and ways for implementing the technical solution, and the above description is only a preferred embodiment of the present invention, it should be noted that, for those skilled in the art, many modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
<110> Nanjing university of industry
<120> recombinant escherichia coli and construction method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> DNA
<213> upstream primer of gene whiB (Artificial Sequence)
<400> 1
gtcgacctgc aggcatgcaa gcttggatgg aattcatgac gtctgtgatt ccagagca 58
<210> 2
<211> 42
<212> DNA
<213> downstream primer of whiB Gene (Artificial Sequence)
<400> 2
ccaaaacagc caagctttca gaggttctcg tagcgattca tg 42
<210> 3
<211> 47
<212> DNA
<213> upstream primer of Gene cti (Artificial Sequence)
<400> 3
ccattaacgt ggtcagcttt ttgaatgttg ccaagaccgt tggttgg 47
<210> 4
<211> 42
<212> DNA
<213> downstream primer of Gene cti (Artificial Sequence)
<400> 4
ccaaaacagc caagctttca gaggttctcg tagcgattca tg 42

Claims (10)

1. A recombinant Escherichia coli into which a gene expressing an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum has been introducedwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiWherein, the genewhiBThe sequence number of (1) is NCBI ID: AKL15_ 01645; genectiCWI20_ 17990; the host bacteria have a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli）BER208。
2. The method for constructing recombinant Escherichia coli according to claim 1, wherein the expression of a gene encoding an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum is usedwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiConstructing the culture medium with a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli) BER 208.
3. The construction method according to claim 2, characterized by comprising the steps of:
(1) target gene fragmentwhiBAndctiobtaining;
(2) cloning a target gene fragment to a starting plasmid by using a one-step cloning method to obtain a recombinant plasmid;
(3) transforming the positive cloning plasmid with correct sequencing into the host bacterium Escherichia coli (Escherichia coli) In BER208, the succinic acid-producing recombinant Escherichia coli with improved stress resistance is obtained by Amp resistance screening.
4. The method according to claim 3, wherein the starting plasmid is pTrc99A plasmid.
5. The method of claim 3, wherein in step (2), primers carrying HindIII cleavage sites and homology arms of pTrc99A vector are designed and inserted into the linearized gene fragmentwhiB、ctiTo obtain the cloning plasmid pTrc99A-whiB-cti。
6. Use of the recombinant E.coli of claim 1 or claim 5 for the preparation of succinic acid.
7. The use of claim 6, wherein the recombinant E.coli reduces inhibition of high substrate and high product concentrations in the fermentative production of succinic acid.
8. The use of claim 7, wherein the recombinant Escherichia coli can be used for producing succinic acid by fermentation in a fermentation system with 90g/L glucose as substrate or 30g/L sodium succinate and 60g/L glucose as carbon source.
9. A method for improving the stress resistance of Escherichia coli and the synthesis performance of products, which is characterized in that the method comprises expressing a gene of an oxidative stress regulating enzyme from Corynebacterium glutamicumwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiIntroduced into Escherichia coli, wherein the genewhiBThe sequence number of (1) is NCBI ID: AKL15_ 01645; genectiCWI20_ 17990.
10. The method of claim 9, wherein the E.coli has a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli）BER208。
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864113A (en) * 2012-10-16 2013-01-09 南京工业大学 Bacterial strain for producing succinic acid, method for producing succinic acid by using bacterial strain and application of bacterial strain

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864113A (en) * 2012-10-16 2013-01-09 南京工业大学 Bacterial strain for producing succinic acid, method for producing succinic acid by using bacterial strain and application of bacterial strain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUNG HO AHN等: "Membrane engineering via trans-unsaturated fatty acids production improves succinic acid production in Mannheimia succiniciproducens" *
SOON-CHUN CHUNG等: "Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum" *

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