CN112062822A

CN112062822A - Carbon catabolism regulatory protein CcpA mutant I42A

Info

Publication number: CN112062822A
Application number: CN202011017359.1A
Authority: CN
Inventors: 石贵阳; 李由然; 张玉鹏; 肖丰旭; 王瀚容; 张梁; 丁重阳; 徐沙; 顾正华
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2020-12-11
Anticipated expiration: 2040-09-24
Also published as: CN112062822B

Abstract

The invention discloses a carbon catabolism regulatory protein CcpA mutant I42A, and belongs to the technical field of protein engineering. The carbon catabolism regulatory protein CcpA mutant I42A obtained by mutating the CcpA gene cloned from the genome of the bacillus licheniformis has the amino acid sequence shown as SEQ ID NO.2 and the nucleotide sequence of the gene coding the protein CcpA mutant is shown as SEQ ID NO. 1. The invention constructs a recombinant expression vector containing the gene encoding the CcpA mutant I42A, and the constructed expression vector is transformed into a Bacillus licheniformis CcpA gene defective strain to obtain recombinant Bacillus licheniformis; the obtained recombinant bacillus licheniformis can obviously change the phenomenon of carbon catabolism repression generated in the presence of glucose, and can reduce the preference of the bacillus licheniformis to xylose.

Description

Carbon catabolism regulatory protein CcpA mutant I42A

Technical Field

The invention belongs to the field of protein engineering, relates to a carbon catabolism regulatory protein CcpA mutant I42A, and also relates to a recombinant expression vector and a recombinant microbial cell for expressing the mutant, and further relates to the influence of the mutant on the utilization of xylose when the xylose and glucose coexist in the fermentation process of bacillus licheniformis.

Background

Bacillus licheniformis (Bacillus licheniformis) is a gram-positive bacterium, and has the advantages of heat resistance, rich enzyme system, high enzyme yield, moderate growth rate, protein folding, whole genome information disclosure and the like. Compared with escherichia coli, bacillus licheniformis has the advantages of high heat resistance, low pH tolerance, high biomass and the like, so that the bacillus licheniformis not only can be widely used as an expression host of foreign genes, but also has great potential in the fermentation industry.

Xylose, a high-quality carbon source, is obtained by hydrolysis of lignocellulose, and with the current environmental and energy pressures, renewable carbon such as lignocellulose is becoming more and more popular. Bacillus licheniformis can utilize xylose, but its utilization of xylose is inhibited by glucose. While lignocellulose produces a large amount of xylose during hydrolysis, it also produces a large amount of glucose. The presence of glucose inhibits the use of xylose by Bacillus licheniformis due to the carbon catabolism repression effect, and in order to counteract the carbon catabolism repression effect produced by the presence of glucose, many attempts to counteract or reduce the carbon catabolism repression effect have been made in other microorganisms, mainly divided into two groups: firstly, the utilization of xylose is increased by artificially constructing a metabolic pathway; and secondly, the engineering bacteria with reduced carbon catabolism repression are constructed by intervening the own metabolic pathways of the bacteria. However, the carbon catabolism repression is inhibited by knocking out the protein CcpA of the carbon catabolism regulatory protein, the inhibition of glucose on xylose utilization is reduced, the utilization efficiency of xylose is increased, the growth of thalli is obviously inhibited, and other metabolic pathways are also obviously influenced. Therefore, the engineering bacteria constructed by gene knockout can not be put into commercial production.

Therefore, at present, it is generally considered in the field that the simple knock-out of the carbon catabolism regulatory protein causes the growth of the thallus to be limited, the metabolic pathway of the thallus is damaged, and the expected result is often not obtained. Thus, reducing the inhibition of xylose utilization due to the presence of glucose by targeted engineering of the active site of carbon catabolism regulatory proteins may be an effective means.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a carbon catabolism regulatory protein CcpA mutant I42A (Ile42Ala) and application thereof. The gene sequence of the protein CcpA of the carbon catabolism regulatory protein is obtained by cloning from the genome of the bacillus licheniformis ATCC 9945A, the amino acid of the protein CcpA, which is responsible for the DNA binding structural domain, is subjected to site-directed mutation, and heterologous expression and purification are carried out in escherichia coli; in vitro measuring the binding capacity of the CcpA protein and a cre site of a regulation site, and preliminarily screening the CcpA mutant I42A protein with obvious difference in binding capacity with the cre site; the mutant protein is expressed in a bacillus licheniformis CcpA defective strain, and the influence of the mutant on xylose utilization in the presence of glucose is verified, so that a CcpA mutant I42A capable of improving the preference of bacillus licheniformis on xylose utilization is obtained.

The technical scheme of the invention is as follows:

the invention provides a carbon catabolism regulatory protein CcpA mutant I42A, and the amino acid sequence is shown in SEQ ID NO. 2.

The invention also provides a gene for coding the carbon catabolism regulatory protein CcpA mutant I42A, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The invention also provides a recombinant expression vector containing the gene.

The invention also provides a recombinant microbial cell expressing the carbon catabolism regulatory protein CcpA mutant I42A.

Further, the recombinant microbial cell is a host of Bacillus Licheniformis (Bacillus Licheniformis).

The invention also provides application of the carbon catabolism regulatory protein CcpA mutant I42A in improving the preference of bacillus licheniformis on xylose utilization.

The invention also provides application of the gene in improving the preference of the bacillus licheniformis on xylose utilization.

The invention also provides application of the recombinant expression vector in improving the xylose utilization preference of the bacillus licheniformis.

The invention also provides application of the recombinant microbial cell in improving the preference of the bacillus licheniformis in xylose utilization.

The main ideas of the invention are as follows:

(1) carrying out site-directed mutation on amino acids according to key amino acids of the protein CcpA protein by homologous modeling of the protein;

(2) carrying out heterologous expression and purification on the obtained mutant protein in escherichia coli;

(3) screening the mutant for its ability to bind to nucleic acid in vitro;

(4) constructing a CcpA protein recombinant expression plasmid by using the screened mutant protein;

(5) the in vivo effect of the CcpA mutant I42A protein is verified, and the effect on the utilization of xylose and glucose in the thallus is verified.

The invention has the beneficial effects that:

the carbon catabolism regulatory protein CcpA mutant I42A obtained by mutating the CcpA gene cloned from the bacillus licheniformis has the amino acid sequence shown as SEQ ID NO.2 and the nucleotide sequence of the gene coding the protein CcpA mutant is shown as SEQ ID NO. 1. The invention constructs a recombinant expression vector containing the gene encoding the CcpA mutant I42A, and the constructed expression vector is transformed into a strain with the defect of the CcpA gene of the bacillus licheniformis to obtain the recombinant bacillus licheniformis; the obtained recombinant bacillus licheniformis can obviously change the phenomenon of carbon catabolism repression generated in the presence of glucose, and can reduce the preference of the bacillus licheniformis to xylose.

Drawings

FIG. 1 shows the confirmation of the site-directed mutagenesis colony PCR amplification of Bacillus licheniformis CcpA mutant I42A. Lane 1 is Marker, lanes 2-8 are PCR-verified transformants picked up after transforming the CcpA protein mutant expression vector into E.coli, the length of the target band is 1002 bp.

FIG. 2 is a SDS-PAGE gel validation of CcpA mutant I42A protein purification. Lane 1 is Marker, lane 2 is purified mutant protein, protein size 36.8 kDa.

FIG. 3 is a Bacillus licheniformis CcpA protein expression vector.

FIG. 4 shows recombinant plasmid overexpression of CcpA mutant I42A in CcpA gene-deficient strains.

FIG. 5 is a growth curve of recombinant strain I42A-BL21- Δ CcpA in a medium cultured with xylose and glucose mixed carbon source.

FIG. 6 is a graph showing the consumption of xylose and glucose by recombinant bacterium I42A-BL21- Δ CcpA in a medium containing xylose and glucose mixed carbon sources.

Detailed Description

The invention is described in one step below with reference to the examples and the figures. Bacillus licheniformis ATCC 9945A used in the present invention is a commercial strain and can be obtained commercially. The means of constructing expression vector, transformation, PCR and the like used in the invention are all conventional molecular biology methods.

The culture medium: the medium for in vivo functional verification of the CcpA protein mutant is LB medium added with 30g/L glucose and 30g/L xylose.

(II) construction method of bacillus licheniformis CcpA gene defective strain

554bp is selected as a first homology arm from 299bp upstream of a CcpA gene on a bacillus licheniformis genome, 500bp is selected as a second homology arm from 991bp downstream of the CcpA gene of the bacillus licheniformis, a Kan gene containing an FRT site is inserted between the two homology arms to form a CcpA gene knockout box, and the knockout box is connected to a pNZT1-Tet particle to construct a CcpA gene knockout plasmid. The plasmid is transformed into bacillus licheniformis to construct a recombinant bacterium containing a CcpA gene knockout box. The recombinant strain is subjected to activated culture at 30 ℃, the seed solution subjected to activated culture is cultured at 42 ℃, the seed solution is cultured on an LB (Langmuir-Blodgett) plate containing Kan, a single-double exchange strain is selected, the obtained single-exchange strain is cultured at 30 ℃, the cultured seed solution is subjected to underlined culture at 37 ℃, and the double-exchange strain is selected to serve as a bacillus licheniformis CcpA gene defective strain.

(III) method for determining contents of xylose and glucose in culture medium by high performance liquid chromatography

The bacillus licheniformis CcpA mutant I42A overexpression strain is cultured and activated in an LB culture medium, then 1mL of the strain liquid is inoculated into a 250mL shake flask, and 30g/L glucose and 30g/L xylose are added into the culture medium. Sampling every 3h to determine the consumption of carbon source in the culture medium, centrifuging the culture medium at 12000rpm for 5min, taking out the obtained supernatant, and adding 10% trichloroacetic acid with equal volume to remove impurities in the culture medium. Then, detection was performed using an amino column, and acetonitrile and water (3: 7) were used as mobile phases for detection.

Example 1 selection of key amino acids of the Bacillus licheniformis CcpA protein

The bacillus licheniformis CcpA gene is mainly combined with a nucleic acid site through protein as a global regulatory factor and then plays a regulating function, the relative position of the corresponding combined part of the CcpA protein and the nucleic acid is changed to a certain extent, and amino acids which play a role of supporting points in the conversion process are possibly positioned at two ends of an alpha spiral in the CcpA protein substructure. Meanwhile, the combination of the CcpA protein and nucleic acid requires the recognition of the protein and cre site, and the amino acids which are mainly used in the recognition of the protein and cre site are probably positioned in the middle of the alpha helix, so that the amino acid sites are mainly distributed at both ends and in the middle of the alpha helix of the CcpA protein when the amino acid sites are selected.

Example 2 construction of Bacillus licheniformis CcpA protein expression vector

The expression vector of the Bacillus licheniformis CcpA protein was constructed by amplifying the CcpA gene using the Bacillus licheniformis CcpA protein genome as a template and ligating the resulting gene to pET28a (FIG. 3). It was transformed into E.coli BL21(DE3) to construct a recombinant strain.

Example 3 site-directed mutagenesis and expression and purification of Bacillus licheniformis CcpA protein

Site-directed mutagenesis was performed on the B.licheniformis CcpA protein using the B.licheniformis CcpA protein expression vector constructed in example 2 as a template. The mutation primers used were:

I42A-F(SEQ ID NO.3)：5’aaggtgcttgaagccgccgagcgtcttggctatcgtccaaatgccgtggc3’；

I42A-R(SEQ ID NO.4)：3’agccaagacgctcggcggcttcaagcaccttctttctcgtcgtcggcttg5’。

heterologous expression and purification are carried out on the CcpA protein (the amino acid sequence is shown as SEQ ID NO.2, and the nucleotide sequence of the gene for coding the CcpA protein is shown as SEQ ID NO. 1) subjected to site-specific mutation in Escherichia coli, the colony PCR amplification verification diagram is shown as FIG. 1, and the SDS-PAGE gel verification diagram is shown as FIG. 2, so that the CcpA protein subjected to mutation is successfully transformed into the Escherichia coli and successfully expressed in the Escherichia coli.

Example 4 determination of the binding and Capacity of mutant proteins to the cre site

The mutant protein obtained in example 3 was tested for its ability to bind to the cre site in vitro using fluorescence polarization and EMSA. This result indicates that mutant I42A had significantly reduced binding and capacity to the cre site compared to the control.

Example 5 overexpression of Bacillus licheniformis CcpA mutant I42A protein

Mutant I42A was ligated to P43 promoter and then ligated to pHY300 to construct a recombinant expression vector, and as shown in FIG. 4, the recombinant expression vector was expressed in a Bacillus licheniformis CcpA-deficient strain to obtain strain I42A-BL21- Δ CcpA which recombinantly expressed the CcpA protein mutant.

Example 6 Effect of CcpA mutant I42A protein on xylose and glucose utilization by Bacillus licheniformis

The strain I42A-BL 21-. DELTA.CcpA recombinantly expressing the CcpA protein mutant obtained in example 5 was cultured at 250rpm and 37 ℃ in a medium containing a mixed carbon source of xylose and glucose (LB medium supplemented with 30g/L glucose and 30g/L xylose). Sampling and determining the contents of xylose and glucose in the culture medium by using high performance liquid chromatography. As a result, as shown in FIGS. 5 and 6, the residual xylose content was decreased from 28.9g/L to 22.9g/L after 21 hours of culture. The glucose content of the medium was still maintained at 14.6g/L at this time. The recombinant strain I42A-BL 21-delta CcpA expressing the mutant protein has no obvious growth difference compared with a control group, but has obvious improvement on the utilization of xylose in the presence of glucose.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> carbon catabolism regulatory protein CcpA mutant I42A

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aggggaatcg aagatatcgc aacgatgtac aagtacaata ttattttgag caactccgac 300

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gattatgaac aggcgattta tgatgcggct accatgctga ttgaaaaagg ccataagcgc 540

cttgcgtttg tatcaggacc tatgactgag ccggtcaatc aagcgaaaaa acttcaaggc 600

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gattatacgt acgattcagg aatggaagcg ctggaggcgc taatgaagct ggatgaaaaa 720

ccgacggccg tcctgtcagc gacagacgaa atggcactcg gcgttattca cgcagcacag 780

gataaaggac tggctattcc ggatgacctt gaagtgatcg gctttgacaa tacaaggctt 840

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Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala

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Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Ala Thr Met Leu Ile Glu Lys

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Asn Gln Ala Lys Lys Leu Gln Gly Phe Lys Arg Ala Leu Glu Asp Lys

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Gly Leu Thr Phe Lys Glu Glu Tyr Val Ala Glu Gly Asp Tyr Thr Tyr

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Asp Ser Gly Met Glu Ala Leu Glu Ala Leu Met Lys Leu Asp Glu Lys

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Pro Thr Ala Val Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Val Ile

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Claims

1. The carbon catabolism regulatory protein CcpA mutant I42A is characterized in that the amino acid sequence of the carbon catabolism regulatory protein CcpA mutant I42A is shown as SEQ ID No. 2.

2. The gene for coding the carbon catabolism regulatory protein CcpA mutant I42A of claim 1, wherein the nucleotide sequence of the gene is as shown in SEQ ID No. 1.

3. A recombinant expression vector comprising the gene of claim 2.

4. A recombinant microbial cell expressing the carbon catabolism regulatory protein CcpA mutant I42A of claim 1.

5. The recombinant microbial cell of claim 4, wherein said recombinant microbial cell is hosted by Bacillus Licheniformis (Bacillus Licheniformis).

6. The use of the carbon catabolism regulatory protein CcpA mutant I42A of claim 1 to improve the preference of bacillus licheniformis to xylose utilization.

7. Use of the gene of claim 2 to improve the xylose utilization preference of bacillus licheniformis.

8. Use of the recombinant expression vector of claim 3 to improve the xylose utilization preference of Bacillus licheniformis.

9. Use of the recombinant microbial cell of any one of claims 4-5 to improve the xylose utilization preference of Bacillus licheniformis.