CN114426983B

CN114426983B - Method for producing 5-aminolevulinic acid by knocking out transcription regulatory factor Ncgl0580 in corynebacterium glutamicum

Info

Publication number: CN114426983B
Application number: CN202210127664.9A
Authority: CN
Inventors: 王智文; 姜玫如; 何桂美; 孙曦; 杨秋雨; 陈涛
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-06-13
Anticipated expiration: 2042-02-11
Also published as: CN114426983A

Abstract

The invention discloses a method for producing 5-aminolevulinic acid by knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum, wherein the nucleotide sequence of the transcription regulatory factor Ncgl0580 is shown as SEQ ID NO. 1. The knockout of the transcription regulatory factor Ncgl0580 in corynebacterium glutamicum improves the yield of 5-aminolevulinic acid in the corynebacterium glutamicum engineering strain CB6 by 2.49 times, and simultaneously improves the transcription level of genes such as hemA, sucA, sucB, ncgl0282 in the 5-ALA synthesis pathway and the glycolysis pathway by 29.96 times, 38.41 times, 41.12 times and 31.16 times respectively. Has guiding significance for improving the synthesis of target metabolites such as 5-aminolevulinic acid and the like on the transcriptional control level through metabolic engineering strategies.

Description

Method for producing 5-aminolevulinic acid by knocking out transcription regulatory factor Ncgl0580 in corynebacterium glutamicum

Technical Field

The invention belongs to the field of bioengineering technology and application, and particularly relates to a method for synthesizing 5-aminolevulinic acid by knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum.

Background

5-Aminolevulinic acid (5-aminolevulinic acid, 5-ALA), which is a vitamin B in vivo synthesis ₁₂ Important precursors of tetrapyrroles such as heme and chlorophyll are widely present in microorganisms and animal and plant cells. Because of the characteristics of green, non-toxicity, easy degradation and no residue, the composition can be widely applied to the fields of medicine, agriculture and the like as photodynamic medicaments, biodegradable herbicides, plant growth regulators, feed additives and the like. At present, the large-scale production of 5-ALA is mainly based on a chemical method, but the problems of high cost, high pollution, low yield and the like in the synthesis process greatly limit the large-scale production and application of the 5-ALA. In view of this, microbiological synthesis has been developed and, with the advent of synthetic biology, particularly metabolic engineering, the biological synthesis of 5-ALA has been further developed. However, most biosynthetic methodsThe yield is low, the requirement of industrial production cannot be met, and large-scale application is difficult to realize.

The microorganism realizes the growth of self cells and the synthesis of target metabolites by regulating the expression of a series of genes in the process of synthesizing target products. And is usually regulated by a specific transcription regulator in the process of gene expression, which regulates gene expression by binding to the DNA sequence of a specific gene. Many transcription regulators are involved in 5-ALA synthesis, but currently, transcription regulators which play a role in regulating 5-ALA synthesis have not been reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum to produce 5-aminolevulinic acid.

The technical scheme of the invention is summarized as follows:

a method for knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum to produce 5-aminolevulinic acid, wherein the nucleotide sequence of the transcription regulatory factor Ncgl0580 is shown as SEQ ID NO. 1.

The invention has the advantages that:

the knockout of the transcription regulatory factor Ncgl0580 in corynebacterium glutamicum improves the yield of 5-aminolevulinic acid in the corynebacterium glutamicum engineering strain CB6 by 2.49 times, and simultaneously improves the transcription level of genes such as hemA, sucA, sucB, ncgl0282 in the 5-ALA synthesis pathway and the glycolysis pathway by 29.96 times, 38.41 times, 41.12 times and 31.16 times respectively.

Drawings

FIG. 1 is a map of a pD-sacB knockout vector.

FIG. 2 is a map of the pD-Ncgl0580 knockout vector.

FIG. 3 is a map of the pXK expression vector.

FIG. 4 is a map of the pXP expression vector.

FIG. 5 is a schematic diagram of fermentation of strains CGJ2 and CGJ3 under shake flask conditions.

FIG. 6 is a schematic representation of the transcriptional level changes of the genes in the 5-ALA synthesis, downstream heme synthesis and central carbon metabolic pathway in strain CGJ 3.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to enable those skilled in the art to better understand the present invention, but are not intended to be limiting.

The original strain Corynebacterium glutamicum Corynebacterium glutamicum ATCC13032 used in the present invention was obtained from ATCC (The Global Bioresource Center, http:// www.atcc.org /), and was purchased 10 in 2012.

The recombinant plasmids pK18mobsacB, the expression plasmids pXMJ19, pECXK99E and pET28a referred to in this example are purchased from Biovector NTCC (http:// www.biovector.net /).

5-aminolevulinic acid standard is purchased from sigma (http:// www.sigmaaldrich.com/sigma aldrich).

The molecular biological reagents used were purchased from thermo (http:// www.thermoscientificbio.com/ferrons) and the other biochemical reagents were purchased from Biotechnology (Shanghai) Inc. (http:// www.sangon.com /).

E.coli DH 5. Alpha. Competent cells and E.coli BL21 competent cells were obtained by conventional CaCl ₂ Preparing by a method;

LB liquid medium: yeast extract powder 5g/L, tryptone 10g/L, naCl 10g/L, and LB solid medium with 2% agar powder.

CGIII medium: yeast extract 10g/L, tryptone 10g/L, MOPS 21g/L, naCl 2.5g/L, pH=7 with 5M NaOH aqueous solution.

BHIS liquid medium: brain heart infusion broth powder 74g/L. 2% agar powder was added to the BHIS solid medium.

The antibiotic concentration is: kanamycin 40. Mu.g/mL, 15. Mu.g/mL, chloramphenicol 10. Mu.g/mL.

Example 1: construction of pD-sacB knockout vector and construction of Gene Ncgl0580 knockout Strain

(1) pD-sacB knockout vector construction

The sacB gene was first amplified to about 1.6kb using the HindIII-cut pK18mobsacB linear fragment as a template and the primers sacB-1 (SEQ ID NO. 3)/sacB-2 (SEQ ID NO. 4) shown in Table 1. The MunI/EcoRV double digested sacB gene was ligated with EcoRI/SmaI double digested pECXK99E to give a pECXK99E-sacB plasmid, and then a trcsacB fragment containing the trc promoter was amplified to about 1.8kb using trcsacB-1 (SEQ ID NO. 5)/trcsacB-2 (SEQ ID NO. 6) as the upstream and downstream primers, and the pECXK99E-sacB plasmid as the template. The pD fragment containing kanamycin resistance and E.coli replicon was amplified with the primers pD-1 (SEQ ID NO. 7)/pD-2 (SEQ ID NO. 8) using the pK18mobsacB plasmid as a template, about 2.6kb. Finally, connecting the AatII digested fragments trcsacB and pD to obtain pD-sacB. The final pD-sacB plasmid map is shown in FIG. 1.

(2) pD-Ncgl0580 knockout vector construction

The upstream fragment of the Ncgl0580 gene was amplified using the C.glutamicum ATCC13032 genome as a template and pD-Ncgl05801 (SEQ ID NO. 9)/pD-Ncgl 05802 (SEQ ID NO. 10) as primers shown in Table 1,

the downstream fragment of the gene was amplified using pD-Ncgl05803 (SEQ ID NO. 11)/pD-Ncgl 05804 (SEQ ID NO. 12) as primers.

After the upstream and downstream fragments were excised and recovered, the fusion product of the two fragments was amplified to about 2.0kb using equimolar proportions of fragments as templates and pD-Ncgl05801 (SEQ ID NO. 9)/pD-Ncgl 05804 (SEQ ID NO. 12) as primers.

The fused fragment was ligated with pD-sacB after the same double cleavage with SmaI/BamHI, and subjected to transformation, and the single colony obtained was subjected to PCR verification using pD-Ncgl05801 (SEQ ID NO. 9)/pD-Ncgl 05804 (SEQ ID NO. 12) as a primer. Transferring the colony with the same strip size to LB liquid culture medium, culturing, extracting plasmid, and obtaining knockout plasmid pD-Ncgl0580 of Ncgl0580 gene after correct sequencing. The plasmid map is shown in FIG. 2.

(3) Knock-out of the gene Ncgl0580

Plasmid pD-Ncgl0580 with correct sequencing result is electrically transduced into Corynebacterium glutamicum CB6 (the source of Corynebacterium glutamicum CB6 is shown in Chinese patent CN 106636171A, the name of which is Corynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction), uniformly coated on BHIS solid plate containing kanamycin and sucrose, and placed in a 30 ℃ incubator for culturing for 24 hours. Positive clone strains were obtained by screening with kanamycin and Sucrose, single colonies were picked up and inoculated into 5mL of BHIS liquid medium, cultured overnight at 30℃and 220rpm, the bacterial liquid was diluted 100-fold, and plated on BHIS-Sucrose solid plates (10% (W/V) of added Sucrose). Colonies grown on the plates were individually plated on BHIS solid plates without resistance and BHIS solid plates with kanamycin (final concentration 15. Mu.g/mL). Colonies growing on a non-antibiotic plate and not growing on a kanamycin plate are selected and inoculated into 5mL of BHIS liquid culture medium, so that the genome of the colonies is extracted after the colonies do not grow in a kanamycin test tube, and PCR amplification is carried out by using primers pD-Ncgl05801 (SEQ ID NO. 9)/pD-Ncgl 05804 (SEQ ID NO. 12), wherein the final obtained Corynebacterium glutamicum engineering strain CGJ1 with the Ncgl0580 gene knocked out is the fragment size of the strain, and the nucleotide sequence of a transcription regulatory factor Ncgl0580 is shown as SEQ ID NO. 1.

The corynebacterium glutamicum CB6 is constructed by the following steps: knocking out the lactate dehydrogenase-encoding gene ldhA and the acetate-producing genes pta-ackA, pqo and cat in Corynebacterium glutamicum (C.glutamicum) ATCC13032, the resulting strain was designated as CB4; inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase encoding gene ppc in the CB4 strain to obtain a strain CB5; knocking out a phosphoenolpyruvate carboxykinase coding gene pck in the CB5 strain to obtain a strain CB6.

TABLE 1 primer sequences for construction of strains

Example 2: construction of pXMJ19 overexpression vector

(1) Construction of pXK basic vector

Corynebacterium glutamicum itself does not contain the key enzyme 5-aminolevulinic acid synthase in the C4 pathway and requires the introduction of an exogenous gene to synthesize 5-ALA. The invention constructs a basic plasmid vector pXK for knocking out tac and lacIq.

The backbone fragment of plasmid pXMJ19 was amplified using plasmid pXMJ19 as a template and pK-1 (SEQ ID NO. 13)/pK-2 (SEQ ID NO. 14) as a primer shown in Table 1 to obtain a PCR product of about 5.3 kb. The fragment is cut by HindIII through single enzyme, and plasmid pXK is obtained through the steps of enzyme ligation, transformation and the like. The plasmid map is shown in FIG. 3.

(2) Construction of pXP overexpression vector

The invention selects the reported 5-aminolevulinic acid synthase mutant R365K/C75A from the rhodopseudomonas palustris (Rhodopseudomonas palustris) ^[1] (the amino acid sequence is shown as SEQ ID NO. 21), and codon optimization is carried out according to the codon preference of corynebacterium glutamicum, and the optimized gene is synthesized by a total gene synthesis method (the nucleotide sequence is shown as SEQ ID NO. 2). The plasmid pXP overexpressing hemA was constructed with sodx as promoter and plasmid pXK as the primary vector. The primers hemA-1 (SEQ ID NO. 15)/hemA-2 (SEQ ID NO. 16) shown in Table 1 are respectively used as upstream and downstream primers for amplification to obtain an optimized hemA fragment and a promoter sodx fragment, and the primer sodx-1 (SEQ ID NO. 17)/hemA-2 (SEQ ID NO. 16) is used as upstream and downstream primers for amplification to obtain a fusion product of the two fragments, namely about 1.4kb. The fused product was digested with XbaI/HindIII and ligated with the shuttle plasmid pXK digested with the same double digestion to give pXP plasmid, the map of which is shown in FIG. 4.

Example 3: construction of 5-ALA production strain and shake flask fermentation thereof

(1) Construction of 5-ALA producing Strain

The plasmid pXP with correct sequencing result is electrically transduced into Corynebacterium glutamicum CB6 and Corynebacterium glutamicum engineering strain CGJ1 with Ncgl0580 gene knocked out, and uniformly coated on a BHIS solid plate with chloramphenicol resistance (final concentration 10 mug/mL). Single colonies were picked separately and PCR verified with the following primers test-pXP-1 (SEQ ID NO. 19)/test-pXP-2 (SEQ ID NO. 20). Sequencing to obtain the 5-ALA production strains CGJ2 and CGJ3 inserted with plasmid pXP, wherein CGJ2 is a control strain and CGJ3 is a 5-ALA production strain for knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum.

(2) Shake flask fermentation of production strains

Shake flask fermentation was performed on strains CGJ2, CGJ 3.

The inoculation mode is as follows: the CGJ2 and CGJ3 were streaked on BHIS solid medium, and cultured in an incubator at 30℃for about 18 hours. Single colonies on the plates were picked and inoculated into 5mL of BHIS liquid medium, incubated at 30℃and 220rpm for about 12h, and 1mL was transferred to CGIII liquid medium for further incubation for 12h. At an initial OD ₆₀₀ Transferring about 0.5 g/L glucose, placing into a constant temperature shaking table at 30deg.C and 220rmp, and shake culturing for 4 hr to OD ₆₀₀ About 5.0 g/L glycine, the precursor, was added at about 7.5 g/L. Its 5-ALA yield was measured during the culture (see FIG. 5). After 48h of cultivation, the strain CGJ3 had an approximately 2.49-fold increase in 5-ALA production over CGJ 2.

Example 4: analysis of transcriptional level

(1) Total RNA extraction and cDNA Synthesis

CGJ3 was fermented according to the method described in the above examples using strain CGJ2 as a control strain. Culturing for about 12h, and extracting total RNA from cells in logarithmic growth phase, wherein the method comprises the following specific steps:

1. about 300. Mu.L of the bacterial liquid was introduced into an EP tube to give a cell number of not more than 1X 10 ⁹ Centrifuging at 12000rpm at 4 ℃ for 1min to collect thalli, and pouring out the supernatant;

2. resuspension the thalli with 10mg/mL lysozyme solution, and standing at 37 ℃ for 10min;

3. taking 300 mu L of lysate A into a centrifuge tube, and fully blowing with a gun to ensure that thalli are completely cracked;

4. adding 400 mu L of chloroform into a centrifuge tube, fully shaking and uniformly mixing for about 30 seconds on a shaker, and centrifuging at the room temperature of 12000rpm for 3 minutes;

5. transferring the supernatant to a centrifugal adsorption column for preparation, wherein the supernatant is taken to avoid DNA and protein in the middle layer and organic phase in the lower layer, so as to avoid pollution of extracted RNA;

6. adding an equal volume (about 400 mu L) of solution B, fully mixing, loading on a column, centrifuging at 12000rpm at room temperature for 0.5min, and discarding the supernatant;

7. adding 700 mu L of general column washing liquid into a centrifugal adsorption column, centrifuging at room temperature for 0.5min, and discarding the supernatant;

8. to ensure that the impurities are sufficiently removed, the sample OD _260/280 The ratio is 1.80-2.00, and the step 7 is repeated;

9. centrifuging at 12000rpm for 0.5min at room temperature, pouring out waste liquid, transferring the adsorption column into an RNase-free collecting tube prepared in advance, adding 30 μl of RNA eluent, and standing at room temperature for 2min;

centrifuging at 10.12000rpm for 0.5min, and collecting the solution in the centrifuge tube as the extracted RNA sample, and storing in a-80deg.C ultralow temperature refrigerator.

11. After RNA concentration was measured by a micro-spectrophotometer, a reverse transcription system was prepared as shown in the following table, cDNA was obtained by reverse transcription and its concentration was measured for RT-PCR reaction.

TABLE 2 reverse transcription reaction system

(2) RT-PCR reaction and data analysis

The transcription level of 16s ribosomal RNA in Corynebacterium glutamicum was used as an internal reference during the RT-PCR reaction. The reaction system was configured according to the following table.

TABLE 3RT-PCR reaction System

In this study, the results of RT-PCR were analyzed by Ct value comparison. Ct value comparison method is also called 2 ^{-[△][△]Ct} The method is to compare the Ct values of a target sample and a control sample, wherein the Ct values of the target sample and the control sample are subjected to homogenization operation relative to a specific endogenous calibration gene (the experiment adopts the 16sRNA of corynebacterium glutamicum). The following formula is specifically calculated:

Fold Enrichment＝2 ^-△△Ct

wherein ΔΔct= [ Δ ] Ct (sample group) - [ Δ ] Ct (control group). It should be noted that, [ DELTA ] Ct (sample group) represents the difference between the Ct value of the target gene minus the endogenous calibration gene in the sample group, and [ (DELTA ] Ct (control group) represents the difference between the Ct value of the target gene minus the endogenous calibration gene in the control group.

The transcriptional level of each gene in the 5-ALA synthesis, downstream heme synthesis and central carbon metabolic pathway was changed significantly in the strain CGJ3, compared to the strain CGJ2, as shown in FIG. 6. Wherein hemA, sucA, sucB, ncgl0282 isogenic transcription levels are increased by 29.96, 38.41, 41.12 and 31.16 times respectively. succinyl-CoA, one of the 5-ALA precursors, is catalyzed by the alpha-ketoglutarate dehydrogenase encoded by the gene sucAB. The apparent change in transcription levels of the two genes in strain CGJ3 further explains that the increase in transcription levels of the two genes increases the supply of precursor substances, thereby directly causing the increase in transcription level of the hemA key gene for 5-ALA synthesis and promoting the synthesis of 5-ALA.

The strain codes such as CGJ1 to CGJ3 and the like in the present invention are for convenience of description, but should not be construed as limiting the present invention.

The order of the steps of the construction of the strain of the present invention is not limited, and it is within the scope of the present invention for a person skilled in the art to achieve the object of the present invention according to the present disclosure.

[1] Zheng Ping, zhao Jing, tan Zi et al 5-aminolevulinic acid synthase mutants and host cells and uses thereof 6Jul 2018,PAT:CN 108251396A.

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Glu Gly Arg Tyr Arg Val Phe Ala Asp Leu Glu Arg His Ala Gly Ser

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Phe Pro Arg Ala Thr His His Arg Pro Glu Gly Ala Gly Asp Val Thr

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Val Trp Cys Ser Asn Asp Tyr Leu Gly Met Gly Gln His Pro Ala Val

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Leu Thr Ala Met His Glu Ala Leu Asp Ser Ala Gly Ala Gly Ala Gly

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Gly Thr Arg Asn Ile Ala Gly Thr Asn His Tyr His Val Leu Leu Glu

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Gln Glu Leu Ala Ala Leu His Gly Lys Glu Ser Ala Leu Leu Phe Thr

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Ser Gly Tyr Val Ser Asn Trp Ala Ser Leu Ser Thr Leu Ala Ser Arg

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Met Pro Gly Cys Val Ile Leu Ser Asp Glu Leu Asn His Ala Ser Met

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Ile Glu Gly Ile Arg His Ser Arg Ser Glu Thr Arg Ile Phe Ala His

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Asn Asp Pro Arg Asp Leu Glu Arg Lys Leu Ala Asp Leu Asp Pro His

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Ala Pro Lys Leu Val Ala Phe Glu Ser Val Tyr Ser Met Asp Gly Asp

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Ile Ala Pro Ile Ala Glu Ile Cys Asp Val Ala Asp Ala His Asn Ala

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Met Thr Tyr Leu Asp Glu Val His Gly Val Gly Leu Tyr Gly Pro Asn

210 215 220

Gly Gly Gly Ile Ala Asp Arg Glu Gly Ile Ser His Arg Leu Thr Ile

225 230 235 240

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

245 250 255

Ala Gly Ser Ser Ala Val Cys Asp Phe Val Arg Ser Phe Ala Ser Gly

260 265 270

Phe Ile Phe Ser Thr Ser Pro Pro Pro Ala Val Ala Ala Gly Ala Leu

275 280 285

Ala Ser Ile Arg His Leu Arg Ala Ser Ser Ala Glu Arg Glu Arg His

290 295 300

Gln Asp Arg Val Ala Arg Leu Arg Ala Arg Leu Asp Gln Ala Gly Val

305 310 315 320

Ala His Met Pro Asn Pro Ser His Ile Val Pro Val Met Val Gly Asp

325 330 335

Ala Ala Leu Cys Lys Gln Ile Ser Asp Glu Leu Ile Ser Arg Tyr Gly

340 345 350

Ile Tyr Val Gln Pro Ile Asn Tyr Pro Thr Val Pro Lys Gly Thr Glu

355 360 365

Arg Leu Arg Ile Thr Pro Ser Pro Gln His Thr Asp Ala Asp Ile Glu

370 375 380

His Leu Val Gln Ala Leu Ser Glu Ile Trp Thr Arg Val Gly Leu Ala

385 390 395 400

Lys Ala Ala

Claims

1. A method for knocking out a transcription regulatory factor Ncgl0580 in corynebacterium glutamicum to produce 5-aminolevulinic acid is characterized in that the nucleotide sequence of the transcription regulatory factor Ncgl0580 is shown as SEQ ID NO. 1; the corynebacterium glutamicum takes corynebacterium glutamicum CB6 as an initial strain and overexpresses a 5-aminolevulinic acid synthase mutant R365K/C75A from rhodopseudomonas palustris (Rhodopseudomonas palustris), and the nucleotide sequence of the mutant is shown as SEQ ID NO. 2;