Method for producing 5-aminolevulinic acid by inhibiting expression of global transcriptional regulatory factor gene mcbR in corynebacterium glutamicum

Abstract

The invention discloses a method for producing 5-aminolevulinic acid by inhibiting expression of a global transcriptional regulatory factor gene mcbR in corynebacterium glutamicum. The method comprises the following steps: (1) constructing a pCRISPRi-mid-McbR plasmid and a pCRISPRi-high-McbR plasmid; (2) respectively introducing the plasmids into corynebacterium glutamicum Cgh3 through electrotransfection, verifying by using a bacterial colony PCR (Polymerase Chain Reaction) mode to obtain an expression strain Cgh3-m-McbR or Cgh3-h-McbR for inhibiting a global transcriptional regulation factor gene mcbR in the corynebacterium glutamicum, and fermenting to obtain 5-aminolevulinic acid; experiments prove that compared with a control strain Cgh3-pXi, the strain for inhibiting the expression of the global transcriptional regulation factor gene mcbR in corynebacterium glutamicum has the advantage that the yield of 5-aminolevulinic acid is increased by 1.27 times or more after fermentation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and its applications, and particularly relates to a method for producing 5-aminolevulinic acid by inhibiting the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum Background Art

[0002] 5-aminolevulinic acid (5-ALA) is a non-protein amino acid and a key precursor for the biosynthesis of tetrapyrrole compounds such as porphyrin, chlorophyll, heme, and vitamin B12, and has wide applications in the fields of medicine, agriculture, animal husbandry, etc [1] . It has been confirmed that there are two naturally occurring biosynthetic pathways for 5-ALA in nature, the C4 pathway and the C5 pathway, both of which synthesize 5-ALA using intermediates of the tricarboxylic acid cycle (TCA cycle) as precursors [2] . The C4 pathway mainly exists naturally in certain purple non-sulfur photosynthetic bacteria and higher animals, while the C5 pathway is widely present in plants, algae, and several bacterial classes [3] . At present, the large-scale production of 5-ALA mainly relies on chemical methods, but problems such as high cost, high pollution, and low yield in the synthesis process have greatly restricted its large-scale production and application. Adopting a biosynthetic strategy to replace the chemical synthesis route and reduce production costs is the mainstream trend to promote the wide application of 5-ALA in the fields of medicine, agriculture, and livestock breeding

[0003] Corynebacterium glutamicum is considered a safe microorganism by the US Food and Drug Administration, and has a relatively clear genetic background and relatively mature gene manipulation tools, and has been applied to the industrial production of various amino acids [2] . In addition, Corynebacterium glutamicum is a Gram-positive bacterium with better acid tolerance than Escherichia coli, and it lacks the glycine cleavage system [4] . Based on the above-mentioned multiple advantages, Corynebacterium glutamicum has become an important chassis for the biosynthesis of 5-ALA

[0004] Transcription factors (TFs) are a class of protein molecules that can regulate the gene transcription process by binding to specific sequences (called cis-acting elements) on DNA [5,6] . According to different functions, transcription factors can be divided into activators and repressors. Transcription factors can simultaneously interfere with the expression of multiple genes, even hundreds of genes, at the transcriptional level, and are an important part of regulating the gene expression of microbial cells [7] .

[0005] It has been reported in the literature that the global transcriptional regulator McbR in Corynebacterium glutamicum is related to sulfur metabolism. There are 17 binding sites on the genome of Corynebacterium glutamicum ATCC 6872, and the conserved sequence is TAGAC-N6-GTCTA, which can inhibit the expression of proteins related to TauE / SafE family sulfite transport, type II methionine synthesis, DL-methionine transport, and ABC class nitrate / sulfonate / taurine / bicarbonate transport. [8] However, there has been no report on a method for producing 5-ALA by inhibiting the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for producing 5-aminolevulinic acid by inhibiting the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum.

[0007] The technical solution of the present invention is outlined as follows:

[0008] A method for producing 5-aminolevulinic acid by inhibiting the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum, wherein the nucleotide sequence of the global transcriptional regulator gene mcbR is shown in SEQ ID NO.1.

[0009] The above method is preferably:

[0010] 1) Construction of pCRISPRi-middle-McbR plasmid and construction of pCRISPRi-high-McbR plasmid:

[0011] Using pdCas9gRNA as a template and m-McbR-F1 and cas-R1 as upstream and downstream primers, perform PCR amplification to obtain a linearized vector fragment 1, which is abbreviated as fragment 1;

[0012] Using pdCas9gRNA as a template and ori-F2 and CRISPRi-R2 as upstream and downstream primers, perform PCR amplification to obtain a linearized vector fragment 2, which is abbreviated as fragment 2;

[0013] After recovering and purifying the fragment 1 and fragment 2, use 2X MultiF Seamless Assembly Mix reagent to connect fragment 1 and fragment 2 by molecular cloning method, transform them into Escherichia coli DH5α competent cells, and verify by colony PCR to obtain pCRISPRi-middle-McbR plasmid;

[0014] Using pdCas9 gRNA as a template and h-McbR-F1 and cas-R1 as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 3, which is abbreviated as fragment 3;

[0015] After recovering and purifying the fragment 3 and fragment 2, the fragment 3 and fragment 2 were ligated using the 2X MultiF Seamless Assembly Mix reagent by molecular cloning method and transformed into Escherichia coli DH5α competent cells, and the pCRISPRi-high-McbR plasmid was obtained by colony PCR verification;

[0016] The nucleotide sequence of m-McbR-F1 is shown in SEQ ID NO.16;

[0017] The nucleotide sequence of cas-R1 is shown in SEQ ID NO.11;

[0018] The nucleotide sequence of ori-F2 is shown in SEQ ID NO.12;

[0019] The nucleotide sequence of CRISPRi-R2 is shown in SEQ ID NO.13;

[0020] The nucleotide sequence of h-McbR-F1 is shown in SEQ ID NO.10;

[0021] 2) Construction and fermentation of 5-aminolevulinic acid-producing strains:

[0022] The pCRISPRi-middle-McbR plasmid or pCRISPRi-high-McbR plasmid was introduced into Corynebacterium glutamicum Cgh3 by electroporation, and the expression strains Cgh3-m-McbR or Cgh3-h-McbR that inhibit the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum were obtained by colony PCR verification, and 5-aminolevulinic acid was fermented, significantly increasing the yield of 5-aminolevulinic acid.

[0023] Beneficial effects

[0024] Experiments have proved that the strain that inhibits the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum, after fermentation, has a 5-aminolevulinic acid yield increased by more than 1.27 times compared with Cgh3-pXi. Description of the drawings

[0025] Figure 1 It is the plasmid map of pD-sucCD.

[0026] Figure 2This is the plasmid map of pCRISPRi-high-McbR.

[0027] Figure 3 This is the plasmid map of pCRISPRi-middle-McbR.

[0028] Figure 4 This is the plasmid map of pCRISPRi-low-McbR.

[0029] Figure 5 This is the effect on the yield of 5-ALA after the expression of the global transcriptional regulator gene mcbR in Corynebacterium glutamicum is inhibited. Detailed implementation manners

[0030] The following further illustrates the present invention in conjunction with specific embodiments. The following embodiments are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0031] The original strain Corynebacterium glutamicum used in the present invention, ATCC13032, is sourced from ATCC (The Global Bioresource Center, http: / / www.atcc.org / ), and was purchased in October 2012.

[0032] The starting strain used in the present invention is the Corynebacterium glutamicum recombinant strain CGL11-RM-BEI. The construction method of CGL11-RM-BEI has been described in detail in the authorized patent "Mutant of Corynebacterium glutamicum transcriptional regulator IpsA and its application" (Patent No.: ZL202010333584.X).

[0033] The genotype of the Corynebacterium glutamicum recombinant strain CGL11-RM-BEI is C.glutamicum ATCC13032ΔldhAΔackA-ptaΔpqoΔcat P pyc ::P sod P ppc ::P sod P tal ::P sod P tkt ::P sod P araE ::P tuf (del21bp)P ackA-pta ::P sod ldhA::araE ackA-pta::xylAB:C131T NCgl2538:C331T.

[0034] The specific operation methods, primers, etc. for the scarless operation technology of Corynebacterium glutamicum and the construction of the tool vector pD-sacB involved in the present invention can be referred to the authorized patent (Application No.: CN201710215459.7, Corynebacterium glutamicum strain for producing high-yield chiral D-(-)-acetoin and construction and application).

[0035] The integration vector pD-aceA::hemA involved in the present invention C132A , pD-catA::hemA C132A , pD-pqo::hemA C132A The specific operation methods, primers, etc. for the construction have been described in detail in the patent "Corynebacterium glutamicum engineering bacteria for producing 5-aminolevulinic acid and construction method" (Application No.: CN202410677943.1).

[0036] The hemA gene in the present invention C132A and the pEC-ΔlacIq-Δtrc-P sod* -hemA-C132A plasmid that can be used to amplify the gene and the P sod* promoter have been described in detail in the patent "Rhodopseudomonas palustris 5-aminolevulinic acid synthase mutant and application" (Application No.: CN202210142094.0).

[0037] The preparation of pdCas9gRNA refers to the literature [9] .

[0038] The 5-aminolevulinate standard used was purchased from sigma company (http: / / www.sigmaaldrich.com / sigma-aldrich).

[0039] The molecular biology reagents such as restriction endonucleases and DNA ligases used were purchased from Thermo company (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents used were purchased from Sangon Biotech (Shanghai)

[0040] Co., Ltd. ([[]] http: / / www.sangon.com / ).

[0041] E.coli DH5α competent cells and E.coli BL21 competent cells were prepared by the conventional CaCl 2 method;

[0042] LB liquid medium: yeast extract powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, and 2% agar powder was added to the LB solid medium.

[0043] CGIII medium: 10 g / L of yeast extract powder, 10 g / L of tryptone, 21 g / L of MOPS, 2.5 g / L of NaCl, and the pH is adjusted to 7 with 5 M NaOH aqueous solution.

[0044] BHIS liquid medium: 74 g / L of brain heart infusion broth powder, and 2% agar powder is added to the BHIS solid medium.

[0045] The antibiotic concentration is: 10 μg / mL of chloramphenicol.

[0046] Detection method of 5-ALA: Take 250 μL of 5-aminolevulinic acid standard or diluted fermentation broth, add 125 μL of sodium acetate buffer solution with pH = 4.6, then add 62.5 μL of acetylacetone, incubate in a metal bath at 100 °C for 15 min, after cooling to room temperature, add 440 μL of freshly prepared Modified Ehrlich's reagent (0.2 g of p-dimethylaminobenzaldehyde, 1 mL of glacial acetic acid, 1 mL of perchloric acid, and make up the volume to 10 mL with glacial acetic acid) and mix well. After reacting at room temperature for 20 min, measure the absorbance value of the reaction solution at a wavelength of 554 nm. Calculate the 5-ALA content of the fermentation broth using the standard curve obtained from the determination of the 5-aminolevulinic acid standard.

[0047] Example 1

[0048] Construction of strain Cgh

[0049] (1) Construction of sucCD knockout plasmid pD-sucCD

[0050] Using pD-sacB (CN201710215459.7) as a template, and up-sucCD-F (SEQ ID NO.2), up-sucCD-R (SEQ ID NO.3) as upstream and downstream primers, amplify with Phanta DNA polymerase to obtain the upstream fragment;

[0051] Using pD-sacB as a template, and down-sucCD-F (SEQ ID NO.4), down-sucCD-R (SEQ ID NO.5) as upstream and downstream primers, amplify with Phanta DNA polymerase to obtain the downstream fragment;

[0052] Using pD-sacB as a template, and pD-F (SEQ ID NO.6), pD-R (SEQ ID NO.7) as upstream and downstream primers, amplify with Phanta DNA polymerase to obtain the pD vector fragment;

[0053] After recovering and purifying the upstream fragment, downstream fragment, and pD vector fragment, the upstream fragment, downstream fragment, and pD vector fragment were ligated using the 2X MultiF Seamless Assembly Mix reagent by molecular cloning method, transformed into Escherichia coli DH5α competent cells, and the obtained transformants were verified by colony PCR using the primer pair test-pD-F (SEQ ID NO.8) / test-pD-R (SEQ ID NO.9). After correct sequencing verification, the pD-sucCD plasmid was obtained. The plasmid map of pD-sucCD is as shown in Figure 1 shown.

[0054] (2) Construction of strain Cgh

[0055] The pD-sucCD plasmid was introduced into the Corynebacterium glutamicum recombinant strain CGL11-RM-BEI (ZL202010333584.X) by electrotransformation. According to the scarless operation method described in the authorized patent CN201710215459.7, the sucCD gene in the genome of CGL11-RM-BEI was knocked out to obtain strain Cgh.

[0056] Example 2

[0057] Construction of strain Cgh3

[0058] The integration vectors pD-aceA::hemA in patent CN202410677943.1 C132A , pD-catA::hemA C132A , pD-pqo::hemA C132A were successively introduced into the Corynebacterium glutamicum recombinant strain Cgh by electrotransformation. According to the scarless operation method described in the authorized patent CN201710215459.7, 3 copies of the hemA C132A gene were integrated at the aceA, catA, and pqo loci on the chromosome of the Corynebacterium glutamicum recombinant strain Cgh to obtain strain Cgh3.

[0059] Example 3

[0060] Construction of pCRISPRi-McbR inhibition vector

[0061] (1) Construction of high-inhibition vector pCRISPRi-high-McbR

[0062] Using pdCas9gRNA as a template and h-McbR-F1 (SEQ ID NO.10) and cas-R1 (SEQ ID NO.11) as upstream and downstream primers, amplification was performed using Phanta DNA polymerase to obtain the linearized vector fragment 3, abbreviated as fragment 3;

[0063] Using pdCas9 gRNA as a template, and using ori-F2 (SEQ ID NO.12) and CRISPRi-R2 (SEQ ID NO.13) as upstream and downstream primers, perform amplification with Phanta DNA polymerase to obtain a linearized vector fragment 2; abbreviated as fragment 2;

[0064] After recovering and purifying fragment 3 and fragment 2, use 2X MultiF Seamless Assembly Mix reagent to ligate fragment 3 and fragment 2 by molecular cloning method, transform into Escherichia coli DH5α competent cells, and use primer pair CRISPRi-test-f (SEQ ID NO.14) / CRISPRi-test-r (SEQ ID NO.15) to perform colony PCR verification on the obtained transformants. After correct sequencing verification, obtain the pCRISPRi-high-McbR plasmid. The plasmid map of pCRISPRi-high-McbR is as Figure 2 shown.

[0065] Construction of the inhibitory vector pCRISPRi-middle-McbR in (2)

[0066] Using pdCas9 gRNA as a template, and using m-McbR-F1 (SEQ ID NO.16) and cas-R1 (SEQ ID NO.11) as upstream and downstream primers, perform amplification with Phanta DNA polymerase to obtain a linearized vector fragment 1, abbreviated as fragment 1;

[0067] After recovering and purifying fragment 1 and fragment 2, use 2X MultiF Seamless Assembly Mix reagent to ligate fragment 1 and fragment 2 by molecular cloning method, transform into Escherichia coli DH5α competent cells, and use primer pair CRISPRi-test-f (SEQ ID NO.14) / CRISPRi-test-r (SEQ ID NO.15) to perform colony PCR verification on the obtained transformants. After correct sequencing verification, obtain the pCRISPRi-middle-McbR plasmid. The plasmid map of pCRISPRi-middle-McbR is as Figure 3 shown.

[0068] Construction of the low-inhibitory vector pCRISPRi-low-McbR in (3)

[0069] Using pdCas9 gRNA as a template, and l-McbR-F1 (SEQ ID NO.17) and cas-R1 (SEQ ID NO.11) as upstream and downstream primers, amplify with Phanta DNA polymerase to obtain a linearized vector fragment 4, abbreviated as fragment 4;

[0070] After recovering and purifying fragment 4 and fragment 2, use 2X MultiF Seamless Assembly Mix reagent to ligate fragment 4 and fragment 2 by molecular cloning method, transform into Escherichia coli DH5α competent cells, and use primer pair CRISPRi-test-f (SEQ ID NO.14) / CRISPRi-test-r (SEQ ID NO.15) to perform colony PCR verification on the obtained transformants. After correct sequencing verification, obtain the pCRISPRi-low-McbR plasmid. The pCRISPRi-low-McbR plasmid map is as Figure 4 shown.

[0071] PCR amplification system: 2 μL of template, 2 μL each of upstream and downstream primers, 1 μL of dNTP, 25 μL of phanta Buffer, 17 μL of sterilized double-distilled water, 1 μL of DNA polymerase, with a total volume of 50 μL.

[0072] PCR reaction conditions are: 95°C for 3 min, 30 cycles (95°C for 20 s, 58°C for 20 s, 72°C for 1 min), 72°C for 10 min, 4°C for 10 min. Use a gel recovery kit to recover and purify the PCR product.

[0073] Table 1 Primer sequences used for strain construction

[0074]

[0075] Example 4: Construction of 5-ALA producing strain and its shake flask fermentation

[0076] (1) Construction of 5-ALA producing strain

[0077] The plasmids pCRISPRi-high-McbR, pCRISPRi-middle-McbR, pCRISPRi-low-McbR and pdCas9gRNA (Literature 9) were introduced into Corynebacterium glutamicum Cgh3 by electroporation and evenly spread on a solid BHIS plate with chloramphenicol resistance (final concentration 10 μg / mL). Single colonies were picked and verified by PCR with the following primers test-pX-1 (SEQ ID NO.18) / test-pX-2 (SEQ ID NO.19) to obtain 5-ALA producing strains Cgh3-h-McbR, Cgh3-m-McbR, Cgh3-l-McbR that can inhibit the expression of the global transcriptional regulator gene mcbR (SEQ ID NO.1) to different degrees and the control strain Cgh3-pXi.

[0078] (2) Shake flask fermentation of the producing strains

[0079] The strains Cgh3-pXi, Cgh3-h-McbR, Cgh3-m-McbR, and Cgh3-l-McbR were subjected to shake flask fermentation.

[0080] Inoculation method: First, Cgh3-pXi, Cgh3-h-McbR, Cgh3-m-McbR, and Cgh3-l-McbR were streaked on a solid BHIS medium and placed in an incubator at 30 °C for about 18 h. Single colonies on the plate were picked and inoculated into 5 mL of liquid BHIS medium, cultured at 30 °C and 220 rpm for about 12 h, and 1 mL was transferred to CGIII liquid medium (loading volume 50 mL / 500 mL Erlenmeyer flask) and cultured for another 12 h. With an initial OD 600 of 0.5, it was transferred to fresh CGIII liquid medium supplemented with glucose at a final concentration of 10 g / L (loading volume 50 mL / 500 mL Erlenmeyer flask), placed in a constant temperature shaker at 30 °C and 220 rmp, and cultured with shaking for 4 h until the OD 600 was about 5.0, and then the precursor glycine was added at a final concentration of 7.5 g / L. The 5-ALA production was measured during the culture.

[0081] The fermentation results are as Figure 5 shown. After 72 h of culture, the 5-ALA production of strain Cgh3-h-McbR was 4.20 g / L, which was about 1.01 times higher than that of Cgh3-pXi. The 5-ALA production of strain Cgh3-m-McbR was 4.76 g / L, which was about 1.27 times higher than that of Cgh3-pXi. The 5-ALA production of strain Cgh3-l-McbR was 3.11 g / L, which was about 0.49 times higher than that of Cgh3-pXi.

[0082] References

[0083] [1] Jiang M, Hong K, Mao Y, et al. Natural 5-aminolevulinic acid: Sources, Biosynthesis, Detection and Applications[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10.

[0084] [2] Chen J W, Yu, Pu, Wei, et al. Advances and perspective on bioproduction of 5-aminolevulinic acid[J]. Synthetic Biology Journal, 2021, 2(6): 1000 - 16.

[0085] [3] Yi Y-C, Shih IT, Yu T-H, et al. Challenges and opportunities of bioprocessing 5-aminolevulinic acid using genetic and metabolic engineering: a critical review[J]. Bioresources and Bioprocessing, 2021, 8(1).

[0086] [4] Feng Lili. Research on the production of 5-aminolevulinic acid by metabolic engineering of Corynebacterium glutamicum[D]; Tianjin University, 2017.

[0087] [5] Busby S J W. Transcription activation in bacteria: ancient and modern[J]. Microbiology, 2019, 165(4): 386 - 95.

[0088] [6] Weidemüller P, Kholmatov M, Petsalaki E, et al. Transcription factors: Bridge between cell signaling and gene regulation[J]. Proteomics, 2021, 21(23 - 24).

[0089] [7] Deng C, Wu Y, Lv X, et al. Refactoring transcription factors for metabolic engineering[J]. Biotechnology Advances, 2022, 57.

[0090] [8] Zhang H. Mining and functional analysis of transcription factor binding sites in Corynebacterium[D]; South China University of Technology, 2022.

[0091] [9] Liu J, Liu M, Shi T, et al. CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction[J]. Nature Communications, 2022, 13(1).

Claims

1. A method for producing 5-aminolevulinic acid by inhibiting the expression of the global transcriptional regulatory factor gene mcbR in Corynebacterium glutamicum, wherein the nucleotide sequence of the global transcriptional regulatory factor gene mcbR is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The steps include: 1) Construction of pCRISPRi-middle-McbR plasmid and pCRISPRi-high-McbR plasmid: Using pdCas9gRNA as a template and m-McbR-F1 and cas-R1 as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 1, which is referred to as fragment 1; Using pdCas9gRNA as a template and ori-F2 and CRISPRi-R2 as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 2, which is referred to as fragment 2; After the fragment 1 and the fragment 2 are recovered and purified, the fragment 1 and the fragment 2 are connected by molecular cloning using 2X MultiF Seamless Assembly Mix reagent, transformed into Escherichia coli DH5α competent cells, and verified by colony PCR to obtain the pCRISPRi-middle-McbR plasmid; Using pdCas9gRNA as a template and h-McbR-F1 and cas-R1 as upstream and downstream primers, PCR amplification was performed to obtain a linearized vector fragment 3, which is referred to as fragment 3; After the fragment 3 and the fragment 2 were recovered and purified, the fragment 3 and the fragment 2 were connected by molecular cloning using 2X MultiF Seamless Assembly Mix reagent, and transformed into Escherichia coli DH5α competent cells, and the pCRISPRi-high-McbR plasmid was obtained by colony PCR verification; The nucleotide sequence of m-McbR-F1 is shown in SEQ ID NO.16; The nucleotide sequence of cas-R1 is shown in SEQ ID NO.11; The nucleotide sequence of ori-F2 is shown in SEQ ID NO.12; The nucleotide sequence of the CRISPRi-R2 is shown in SEQ ID NO.13; The nucleotide sequence of h-McbR-F1 is shown in SEQ ID NO.10; 2) Construction and fermentation of 5-aminolevulinic acid production strain: The pCRISPRi-middle-McbR plasmid or the pCRISPRi-high-McbR plasmid was introduced into Corynebacterium glutamicum Cgh3 by electroporation, and the expression strain Cgh3-m-McbR or Cgh3-h-McbR, which inhibited the global transcriptional regulatory factor gene mcbR in Corynebacterium glutamicum, was obtained by colony PCR. 5-aminolevulinic acid was obtained by fermentation, and the yield of 5-aminolevulinic acid was significantly increased.

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