A crp mutant gene and its use

Abstract

The application discloses a CRP mutant gene and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the CRP mutant gene is shown in SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9, the CRP mutant gene can significantly eliminate the 'glucose inhibition' effect, activate glycolysis and TCA cycle in a basic medium with glucose as a carbon source, strengthen the utilization and biosynthesis of glucose, and promote the growth of a strain, and the CRP mutant gene with the amino acid sequence shown in SEQ ID NO. 7, 8 or 9 also has the effect of improving the expression amount of vanillin or naringenin of the strain.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a CRP mutant gene and its application. Background Technology

[0002] Bacterial heterologous expression systems have significant advantages such as short growth time, high cell density, low substrate cost, clear genetic background, and suitability for engineered strain modification. In particular, Escherichia coli has become the most widely used host bacterium, and is widely used in food, pharmaceutical and other production fields.

[0003] To achieve maximum biosynthetic capacity, recombinant expression levels and cell growth should be optimized. Glucose is the primary carbon source in high-density fermentation, but in glucose-supplemented media, commonly used promoters in recombinant expression in *E. coli* cells (such as the lactose promoter) are in a state of "glucose inhibition." Studies show that glucose inhibition is regulated by the 3′,5′ cyclic adenosine monophosphate (cAMP) receptor protein (CRP)–cAMP complex. The specific process of glucose inhibition is as follows: in the presence of readily metabolizable carbon source glucose, the intracellular concentrations of cAMP and the cAMP-CRP complex are low, inhibiting the catabolism of carbon sources other than glucose; in the absence of glucose, the expression of adenylate cyclase is activated, catalyzing the conversion of ATP to cAMP. The CRP-cAMP complex activates catabolism operons involved in alternative carbon source metabolism under various conditions, such as the lactose operon and the maltose operon. As a global transcriptional regulator that senses cellular energy status through cAMP levels, CRP can regulate more than 400 genes, including those related to important physiological activities such as the central metabolic pathway tricarboxylic acid cycle (TCA cycle) and glycolysis. Summary of the Invention

[0004] The present invention provides a CRP mutant gene that relieves the glucose inhibition effect of a bacterial strain, the amino acid sequence of which is shown in SEQ ID NO.7, SEQ ID NO.8, or SEQ ID NO.9.

[0005] The present invention also provides a recombinant vector containing the above-mentioned CRP mutant gene.

[0006] Preferably, the vector is the pSB1k plasmid.

[0007] The present invention also provides a recombinant strain containing the above-mentioned CRP mutant gene.

[0008] Preferably, the strain is Escherichia coli BW25113.

[0009] The present invention also provides the application of the above-mentioned CRP mutant gene or the above-mentioned recombinant vector in relieving the glucose inhibition effect of the strain.

[0010] Preferably, the strain is Escherichia coli.

[0011] The present invention also provides the application of the CRP mutant gene with the amino acid sequence shown in SEQ ID NO.7, 8, 9 or the vector containing the CRP mutant gene with the amino acid sequence shown in SEQ ID NO.7, 8, 9 in promoting the growth of bacterial strains, wherein the bacterial strain is Escherichia coli or Pseudomonas putida.

[0012] The present invention also provides the application of the CRP mutant gene with the amino acid sequence shown in SEQ ID NO.7, 8, 9 or the vector containing the CRP mutant gene with the amino acid sequence shown in SEQ ID NO.7, 8, 9 in increasing the expression level of vanillin or naringenin in strains.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention provides a CRP mutant gene that can relieve the "glucose inhibition" effect, and at the same time, in a basic culture medium with glucose as the carbon source, it activates glycolysis and the TCA cycle, enhances glucose utilization and biosynthesis, and promotes strain growth. Attached Figure Description

[0015] Figure 1 The figure shows the results of the expression of red fluorescent protein regulated by the lactose promoter in M9 basic medium with 2% glucose as the sole carbon source in Example 2, where the global transcription factors CRP wild-type, CRP*, CRP mutants CRP mu2, CRP mu9, and CRP mu12 regulated the strains.

[0016] Figure 2 The image shows the growth results of strains regulated by global transcription factor CRP wild-type, CRP*, CRP mutants CRP mu2, CRP mu9, and CRP mu12 in Example 2 in M9 basic medium with 2% glucose as the sole carbon source (after 9 hours of culture).

[0017] Figure 3 The figure shows the effect of CRP mu9 on the growth of Escherichia coli and the expression of the lactose promoter (which induces the expression of red fluorescent protein) in Example 2.

[0018] Figure 4 The figure shows the effect of wild-type CRP and CRP mu9 on the growth of *Pseudomonas putida* in Example 2.

[0019] Figure 5The graph shows the regulatory effect of CRP mu9 on several CRP-regulated promoters in Example 3.

[0020] Figure 6 This is a graph comparing the relative contents of major metabolites of CRP mu9 strain and wild-type CRP strain in Example 4.

[0021] Figure 7 The graphs show the expression levels of vanillin and naringenin in the biosynthesis of Example 5, with the upper graph showing the expression level of vanillin and the lower graph showing the expression level of naringenin. Detailed Implementation

[0022] Example 1: Obtaining the CRP mutant, a global transcriptional regulator, and its encoding gene.

[0023] Genomic DNA was extracted from *Escherichia coli* MC1061 (ATCC 53338) and used as a template for PCR amplification with primers of 5'-AATGTGC CTGTCAAATGGACAGAGTACGCGTACTAACCAA-3' (SEQ ID NO.1) and 5'-TGATTT AATCTGTAAGATCTTTAACGAGTGCCGTAAACG-3' (SEQ ID NO.2). The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 4 min, followed by 30 cycles of 95℃ for 45 s, 55℃ for 30 s, and 72℃ for 30 s; and a final extension at 72℃ for 5 min.

[0024] The PCR reaction products were recovered and detected by agarose gel electrophoresis, yielding a 630bp band (wild-type CRP gene).

[0025] Using pSB1k plasmid (plasmid source: L.Wang, X.Piao, S.Cui, M.Hu, Y.Tao, Enhanced production of β-alanine through co-expressing two different subtypes of L-aspartate-α-decarboxylase. J.Ind. Microbiol. Biotechnol. 47, 465-474 (2020).) as a template, with 5'

[0026] PCR amplification was performed using primers 5'-AGATCTTACAGATTAAATCAGA-3' (SEQ ID NO.3) and 5'-GTCCATTTGACAGGCACATT-3' (SEQ ID NO.4). The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 4 min, followed by 30 cycles of 95℃ for 45 s, 55℃ for 30 s, and 72℃ for 3 min; and finally extension at 72℃ for 5 min.

[0027] The PCR reaction products were recovered and detected by agarose gel electrophoresis, yielding a 3656bp band.

[0028] The two PCR products were assembled using the Gibson Assembly kit (produced by Shanghai Lingsheng Company, and the operation was performed according to the kit instructions) to obtain the plasmid pSB1k-CRP, which is the wild-type CRP plasmid.

[0029] Mutations were introduced using the HieffMut™ Multi Site-Directed Mutagenesis Kit (manufactured by Shanghai Lingsheng Co., Ltd., and operated according to the kit instructions) to obtain mutants CRP* ​​(CRP mutants have been reported in the literature, with mutation sites E72A and A144T. Reference: H. Tagami, T. Inada, T. Kunimura, H. Aiba, Glucoselowers CRP* ​​levels resulting in repression of the lac operon in cellslacking cAMP. Mol. Microbiol. 17, 251-258 (1995).), CRP mu2, CRP mu9, and CRP mu12.

[0030] The amino acid sequence of wild-type CRP protein in *E. coli* (mutated amino acids are indicated by underscores) SEQ ID NO. 5: MVLGKPQTDPTLEWFLSHCHIHKYPSKSTLIHQGEKAETLYYIVKGSVAVLIKDEEGKEMIL

[0031] SYLNQGDFIGELGLFEEGQE R

[0032] SAWVRAKTACEVAEISYKKFRQLIQVNPDILMRLSAQMARRLQVTSEKVGNLAFLDVT G

[0033] RIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR。

[0034] CRP * Amino acid sequence SEQ ID NO.6:

[0035] MVLGKPQTDPTLEWFLSHCHIHKYPSKSTLIHQGEKAETLYYIVKGSVAVLIKDEEGK

[0036] EMILSYLNQGDFIG A

[0037] LGLFEEGQERSAWVRAKTACEVAEISYKKFRQLIQVNPDILMRLSAQMARRLQVTSEKVG

[0038] NLAFLDVTGRI T

[0039] QTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR。

[0040] CRP mu2 Amino acid sequence SEQ ID NO.7:

[0041] MVLGKPQTDPTLEWFLSHCHIHKYPSKSTLIHQGEKAETLYYIVKGSVAVLIKDEEGK

[0042] EMILSYLNQGDFIG G LGLFEEGQE L

[0043] SAWVRAKTACEVAEISYKKFRQLIQVNPDILMRLSAQMARRLQVTSEKVGNLAFLDVT H

[0044] RIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR。

[0045] CRP mu9 Amino acid sequence SEQ ID NO.8:

[0046] MVLGKPQTDPTLEWFLSHCHIHKYPSKSTLIHQGEKAETLYYIVKGSVAVLIKDEEGK

[0047] EMILSYLNQGDFIGELGLFEEGQE F

[0048] SAWVRAKTACEVAEISYKKFRQLIQVNPDILMRLSAQMARRLQVTSEKVGNLAFLDVT P

[0049] RIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR.

[0050] CRP mu12 amino acid sequence SEQ ID NO.9:

[0051] MVLGKPQTDPTLEWFLSHCHIHKYPSKSTLIHQGEKAETLYYIVKGSVAVLIKDEEGK

[0052] EMIL

[0053] SYLNQGDFIG D LGLFEEGQE V

[0054] SAWVRAKTACEVAEISYKKFRQLIQVNPDILMRLSAQMAR

[0055] RLQVTSEKVGNLAFLDVT P

[0056] RIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR.

[0057] Example 2: CRP mutant, a global transcription factor, relieves the "glucose effect".

[0058] The above plasmid pSB1k-CRP wild-type and mutant were co-transformed with the commercial vector DsRed (replication origin pBR322, ampicillin resistant, including red fluorescent protein regulated by lactose promoter) into Escherichia coli BW25113Δcrp (i.e., the crp gene knocked out in the genome). Single colonies were picked and cultured overnight at 37°C in LB medium (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, ampicillin to 100 μg / mL, kanamycin 50 μg / mL) until saturation. The seed culture was then transferred to M9 basal medium (12.8 g / L Na₂PO₄·7H₂O, 3.0 g / L KH₂PO₄, 0.5 g / L NaCl, 1.0 g / L NH₄Cl, 2 mM MgSO₄, 0.1 mM MgSO₄) at a concentration of 0.1 mM MgSO₄. Add CaCl2, 2% glucose (w / v), and antibiotics ampicillin to 100 μg / mL and kanamycin to 50 μg / mL, along with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). The induction time is generally 10-12 h, and the culture conditions are 37℃ with a shaker (250 rpm). At the end of the induction time, remove the culture, centrifuge to collect the bacterial cells and discard the supernatant (3000 g, 10 min). Resuspend the bacterial cells in phosphate buffer (100 mM, pH 6.0), and take 200 μL of the bacterial cells to test the bacterial concentration (OD 600) and red fluorescent protein reading using a microplate reader.

[0059] See test results Figure 1 ,Depend on Figure 1 It can be seen that the CRP mutant relieves the glucose effect of the lactose promoter, resulting in an increase in the red fluorescent protein reading of the reporter gene.

[0060] Among them, CRP mu9 showed the most outstanding performance in relieving the glucose effect, exceeding that of wild-type CRP by 130 times. It was also found that CRP mu9 not only relieved the glucose effect of the lactose promoter throughout the entire growth cycle, but also significantly promoted the growth of the strain, especially in the early growth stage. Figure 3 ).

[0061] Similarly, the above plasmid pSB1k-CRP wild-type and mutant were co-transformed with the commercial vector DsRed (replication origin pBR322, ampicillin resistant, including red fluorescent protein regulated by lactose promoter) into Escherichia coli BL21(DE3)Δcrp or DH5aΔcrp (i.e., strains with the crp gene knocked out in the genome). Single colonies were picked and cultured overnight at 37°C in LB medium (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, ampicillin to 100 μg / mL, kanamycin 50 μg / mL) until saturation. The culture was then transferred to M9 basal medium (12.8 g / L Na₂PO₄·7H₂O, 3.0 g / L KH₂PO₄, 0.5 g / L NaCl, 1.0 g / L NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂) at a concentration of 0.1 g / L. 2. Add 2% glucose (w / v), ampicillin to 100 μg / mL, and kanamycin to 50 μg / mL, along with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). The induction time is generally 10-12 h, and the culture conditions are 37℃ on a shaker (250 rpm). At the end of the induction time, remove the culture, centrifuge to collect the bacterial cells and discard the supernatant (3000 g, 10 min). Resuspend the bacterial cells in phosphate buffer (100 mM, pH 6.0), and take 200 μL of the bacterial cells to test the bacterial concentration (OD600) and red fluorescent protein reading using a microplate reader (see results below). Figure 1 and Figure 2 ).

[0062] Meanwhile, *Pseudomonas putida* KT2440 is another microbial host bacterium commonly used for biosynthesis. Therefore, this study also involves investigating the growth of *Pseudomonas putida* using the CRP mutant CRP mu9 in a basal medium with glucose as the sole carbon source, compared to wild-type CRP. The experimental protocol is as follows:

[0063] Using pSB1k-CRP and pSB1k-CRP mu9 plasmids as templates, respectively, at a 5'...

[0064] -GAGGAGAAATACTAGATGGTGCTTGGCAAACCG-3' (SEQ ID NO. 10) and 5'

[0065] PCR amplification was performed using primers -GATGCCTGGCTTATCAACGAGTGCCGTAAACGAC-3' (SEQ ID NO.11). The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 4 min, followed by 30 cycles of 95℃ for 45 s, 55℃ for 30 s, and 72℃ for 30 s; and finally extension at 72℃ for 5 min.

[0066] The PCR reaction products were recovered and detected by agarose gel electrophoresis, yielding a 630bp band (wild-type CRP gene or CRPmu9 mutant gene fragment).

[0067] Using p2015a-p23107-LacI-Ptac-sfGFP plasmid (plasmid source: Y. Xue; T. Qiu; Z. Sun; X. Liu; B. Yu, Mercury bioremediation by engineered Pseudomonas putida KT2440 with adaptively optimized biosecurity circuit. Environ. Microbiol. 24(7), 3022-3036(2022).) as a template, with 5'

[0068] -CGGTTTGCCAAGCACCCATCTAGTATTTCTCCTC-3' (SEQ ID NO. 12) and 5'

[0069] PCR amplification was performed using primers -GTCGTTTACGGCACTCGTTGATAAGCCAGGCATC-3' (SEQ ID NO.13). The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 4 min, followed by 30 cycles of 95℃ for 45 s, 55℃ for 30 s, and 72℃ for 6 min; and a final extension at 72℃ for 10 min.

[0070] The PCR reaction products were recovered and detected by agarose gel electrophoresis, yielding a 5434bp band.

[0071] The two PCR products were assembled using the Gibson Assembly kit (produced by Shanghai Lingsheng Company, operated according to the kit instructions) to obtain plasmids p2015a-p23107-LacI-Ptac-CRP wild-type or p2015a-p23107-LacI-Ptac-CRP mu9, which can overexpress wild-type CRP and mutant CRP mu9 plasmids in Pseudomonas putida, respectively.

[0072] The p2015a-p23107-LacI-Ptac-CRP wild-type or p2015a-p23107-LacI-Ptac-CRP mu9 plasmids were co-transfected into *Pseudomonas putida*. After culturing in LB medium supplemented with ampicillin (μg / mL) at 30°C and 220 rpm for 15 h, the culture medium was inoculated with 2% glucose basal medium (with ampicillin, 50 μg / mL) at an OD600 of 0.01 and cultured at 30°C and 220 rpm. Samples were taken at different time points for testing. The results are shown in [link to results]. Figure 4 .

[0073] Example 3 verifies the deactivation effect of CRP mu9 on a series of glucose-regulated promoters.

[0074] Promoters Plac, Ptac, Ptrc, Pgal, and Pgap are all CRP-activating promoters, while PfadR is a CRP-repressing promoter. The expression of the fluorescent protein RFP was regulated using these promoters, and promoter function was assessed. The wild-type and mutant pSB1k-CRP strains, along with the aforementioned promoter plasmids, were co-transformed into *E. coli* BW25113Δcrp. Single colonies were picked and cultured overnight at 37°C in LB medium (tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, ampicillin to 100 μg / mL, kanamycin 50 μg / mL) until saturation. The culture was then transferred at a concentration of 0.1 to M9 basal medium (Na₂PO₄·7H₂O 12.8 g / L, KH₂PO₄ 3.0 g / L, NaCl 0.5 g / L, NH₄Cl 1.0 g / L, 2 mM MgSO₄, 0.1 mM CaCl₂, glucose 2% (w / v), ampicillin to 100 μg / mL, kanamycin 50 μg / mL) (for Plasmacluster I, Plasmacluster II, Plasmacluster III). TRC requires the simultaneous addition of 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). Culture conditions are 37°C, shaker (250 rpm), for 10-12 h. At the end of the induction or culture period, remove the culture, centrifuge to collect the cells and discard the supernatant (3000 g, 10 min), resuspend the cells in phosphate buffer (100 mM, pH 6.0), and use a 200 μL sample to test the bacterial concentration (OD 600) and red fluorescent protein reading using a microplate reader. The CRPmu9 mutant can relieve the "glucose effect" of the above promoter, activating or inhibiting the expression of fluorescent proteins regulated by this promoter (see...). Figure 5 ).

[0075] Example 4

[0076] For ease of operation, the CRP mu9 mutant was inserted in situ into the chromosome of *E. coli* BW25113 using the CRISPR-Cas9 method to replace wild-type CRP, thus obtaining the basal strain Mu9. The glycolytic and tricarboxylic acid cycle intermediate metabolites of the CRP-expressing strain Mu9 and the wild-type BW25113 were measured. Figure 6 The results showed that most intermediates of the tricarboxylic acid cycle and glycolysis pathway were upregulated, indicating that the metabolic flux of these two pathways was significantly enhanced, thus accelerating the growth rate of the strain and enhancing glucose utilization.

[0077] Example 5: The vanillin synthesis pathway and naringenin synthesis pathway were introduced into CRP strain mu9.

[0078] The biosynthesis of aromatic compounds, such as vanillin and naringenin, has long been a research hotspot. This study transformed CRP-expressing strain mu9 with either a vanillin synthesis pathway plasmid or a naringenin synthesis plasmid, and then detected the yields of vanillin and naringenin. The results are shown below. Figure 7 The results showed that the CRP mu9 strain could also enhance the biosynthesis of aromatic compounds and express wild-type...

[0079] CRP (CRPwild-type) strains were lower than strains expressing the CRPmu9 mutant.

[0080] The reference for constructing the vanillin synthesis pathway plasmid is as follows:

[0081] X. Zhang, Y. He, Z. Wu, G. Liu, Y. Tao, J.-M. Jin, W. Chen, S.-Y. Tang, Whole-cell biosensors aid exploration of vanillin transmembrane transport. J. Agric. Food Chem. 69, 3114-3123 (2021). The specific details of the vanillin biosynthesis experiment are as follows: Single colonies of *E. coli* BW25113 / pGAP-EFG and Mu9 / pGAP-EFG were inoculated into LB medium and cultured at 37°C, 220 rpm for 12 h. Then, they were inoculated into 2% glucose basal medium with an OD of 6000.01 and cultured at 37°C, 220 rpm for 14 h on a shaker. Ferulic acid was then added to a final concentration of 66.5 mM, and the culture was transferred to 30°C and cultured on a shaker at 220 rpm for further incubation. Samples were taken at different times for analysis.

[0082] The reference for constructing plasmids for the naringin synthesis pathway is as follows:

[0083] D.Xiong, S.Lu, J.Wu, C.Liang, W.Wang, W.Wang, J.-M.Jin, S.-Y.Tang, Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor. Metab. Eng. 40, 115-123 (2017). The biosynthesis experiment of naringenin was carried out as follows: Escherichia coli BW25113 / pGAP-CHS-4CL and Mu9 / pGAP-CHS-4CL single colonies were inoculated into LB medium and cultured at 37℃, 220rpm for 12h. Then, they were inoculated into 2% glucose basal medium with OD 6000.01, and the substrate p-coumaric acid was added to a final concentration of 4.5mM. The culture was carried out at 30℃, 220rpm in a shaker, and samples were taken at different times for analysis.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A CRP mutant gene capable of relieving glucose inhibition in bacterial strains, characterized in that, The amino acid sequence encoded by the CRP mutant gene is shown in SEQ ID NO.7, SEQ ID NO.8, or SEQ ID NO.

9.

2. A recombinant vector comprising the CRP mutant gene as described in claim 1.

3. The recombinant vector according to claim 2, characterized in that, The vector is the pSB1k plasmid.

4. A recombinant strain comprising the CRP mutant gene as described in claim 1; The strain in question is Escherichia coli BW25113.

5. The use of the CRP mutant gene as described in claim 1 or the recombinant vector as described in claim 2 in relieving the glucose inhibition effect of the strain; The strain in question is Escherichia coli.

6. The application of the CRP mutant gene encoding the amino acid sequence shown in SEQ ID NO. 7, 8, 9 as described in claim 1 or the vector containing the CRP mutant gene encoding the amino acid sequence shown in SEQ ID NO. 7, 8, 9 as described in claim 2 in promoting the growth of a bacterial strain, wherein the bacterial strain is Escherichia coli or Pseudomonas putida.

7. The use of the CRP mutant gene encoding the amino acid sequence as shown in SEQ ID NO. 7, 8, 9 as described in claim 1, or the vector containing the CRP mutant gene encoding the amino acid sequence as shown in SEQ ID NO. 7, 8, 9 as described in claim 2, in increasing the expression level of vanillin or naringenin in strains.

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