A recombinant corynebacterium glutamicum and a method for producing spermidine by fermenting glucose as raw material

Abstract

The present application relates to a kind of recombinant coryneform bacteria and its method for fermenting production of spermidine with glucose as raw material.The present application first knocks out snaA gene in the genome of coryneform bacteria, constructs knockout strain SP-1.Spermidine transporter gene cluster, S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase, S-adenosylmethionine decarboxylase derived from Escherichia coli are expressed in previously constructed knockout strain SP-1 using shuttle plasmid pEC-XK99E and pXMJ19 plasmid between Escherichia coli and coryneform bacteria as expression vector.The recombinant coryneform bacteria of the present application can directly utilize glucose to directly produce spermidine.In the absence of exogenous methionine, 2.61g / L spermidine can be accumulated in 250mL shake flask fermentation for 72h.Spermidine yield can reach 45.2g / L in 5L fermentor by substrate feeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant corynebacterium glutamicum and a method for producing spermidine by fermenting glucose. BACKGROUND

[0002] Spermidine (also known as trihydrochloride spermidine) contains three positively charged amino groups and is a low molecular weight natural polyamine widely distributed in various organisms. As an autophagy inducer, spermidine has various health effects related to autophagy, including prolonging life, inhibiting tumors, protecting the cardiovascular system, regulating nerves, and anti-inflammatory effects. In addition, spermidine plays an important role in the fields of agriculture, industry, and medicine and health.

[0003] At present, the synthesis method of spermidine mainly uses 1,4-butanediamine and acrylonitrile as raw materials, first undergoes an addition reaction, and then the cyano group is reduced by hydrogenation to obtain spermidine. Although this synthesis method has only two reaction steps, the conversion rate of the reaction is low, a large amount of butanediamine remains, which is difficult to remove, increases the difficulty of purification, and greatly affects the quality of spermidine products. In addition, the purification cost is high, which is not conducive to industrial production. Especially, spermidine itself is often used as a nutritional ingredient, so people require that the source of spermidine be non-toxic.

[0004] At present, the intake of spermidine is mainly obtained from food. The existing spermidine functional food is processed from wheat germ, but the resource of wheat germ is limited, which restricts the wide application of spermidine related products. Therefore, a method is needed that does not use harmful or polluting chemical intermediates and can obtain spermidine on an industrial scale. Therefore, microbial synthesis method as a green and sustainable means of producing bioactive ingredients is worth further studying.

[0005] Although in the existing reports, more and more research groups try to biosynthesize spermidine by metabolic engineering and synthetic biology methods, according to Qin et al. in Nature Catalysis, by engineering the natural metabolism of Saccharomyces cerevisiae to overproduce the precursor putrescine, after deleting OAZ1, overexpressing ODC gene and systemically optimizing the ornithine synthesis pathway, the putrescine titer is improved to 255.9 mg / L. In addition, Qin et al. also carried out in-depth research on the metabolic pathway of SAM, and adjusted the supply of SAM by rewiring the methionine and adenine salvage pathways. After fed-batch fermentation, the spermidine titer reached 2.3 g / L. However, the fermentation titer of this method is relatively low, and the fermentation time is long, reaching 120 h, which does not meet the requirements of commercial production, and the need for exogenous addition of intermediate methionine has become an urgent problem for the large-scale production of spermidine. Therefore, how to construct a recombinant strain that efficiently utilizes glucose, and under the condition of reducing or even not adding exogenous methionine, to ferment and synthesize spermidine has become a research difficulty. SUMMARY

[0006] To solve the above technical problems, the present application provides a recombinant corynebacterium glutamicum and a method for fermenting and producing spermidine with glucose as raw material. The present application provides a recombinant corynebacterium glutamicum which simultaneously expresses spermidine transporter, S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase and S-adenosylmethionine decarboxylase, realizes the production of spermidine under the condition of shake flask fermentation, and finally realizes the high production of spermidine in a 5L fermenter.

[0007] The present application is realized by the following technical solutions:

[0008] The first object of the present application is to provide a recombinant corynebacterium glutamicum, which is taken as an expression host by corynebacterium glutamicum; the putrescine degradation gene is knocked out, and the spermidine transporter gene cluster, S-adenosylmethionine synthetase gene, spermidine synthase gene, ornithine decarboxylase gene and S-adenosylmethionine decarboxylase gene are expressed heterologously.

[0009] In an embodiment of the present application, the ornithine decarboxylase and S-adenosylmethionine decarboxylase are located on the pXMJ19 plasmid; the S-adenosylmethionine synthetase, spermidine synthase and spermidine transporter are located on the pEC-XK99E plasmid.

[0010] In an embodiment of the present application, the nucleotide sequence of the putrescine degradation gene is shown as SEQ ID NO 1; the nucleotide sequence of the spermidine transporter gene cluster is shown as SEQ ID NO 2; the nucleotide sequence of the S-adenosylmethionine synthetase gene is shown as SEQ ID NO. 3; the nucleotide sequence of the spermidine synthase gene is shown as SEQ ID NO. 4; the nucleotide sequence of the ornithine decarboxylase gene is shown as SEQ ID NO. 5; and the nucleotide sequence of the S-adenosylmethionine decarboxylase gene is shown as SEQ ID NO. 6.

[0011] The amino acid sequence of the sanA gene is shown as SEQ ID NO 7; the amino acid sequence of the spermidine transporter is shown as SEQ ID NO 8 and SEQ ID NO 9; the amino acid sequence of the S-adenosylmethionine synthetase is shown as SEQ ID NO 10; the amino acid sequence of the spermidine synthase is shown as SEQ ID NO 11; the amino acid sequence of the ornithine decarboxylase is shown as SEQ ID NO 12; and the amino acid sequence of the S-adenosylmethionine decarboxylase is shown as SEQ ID NO 13.

[0012] In an embodiment of the present application, the Corynebacterium glutamicum is Corynebacterium glutamicum var. CCTCC AB 2021051.

[0013] In an embodiment of the present application, the spermidine transporter, the S-adenosylmethionine synthetase, the spermidine synthase, the ornithine decarboxylase and the S-adenosylmethionine decarboxylase are derived from Escherichia coli MG1655.

[0014] A second object of the present application is to provide a microbial preparation comprising the recombinant Corynebacterium glutamicum.

[0015] A third object of the present application is to provide a method for fermentatively producing spermidine, comprising the following steps: using glucose as a substrate, and adding the recombinant Corynebacterium glutamicum or the microbial preparation into a fermentation system to produce spermidine.

[0016] In an embodiment of the present application, the fermentation medium in the fermentation system comprises: glucose 100 g / L-150 g / L, ammonium sulfate 5 g / L-15 g / L, yeast powder 5 g / L-10 g / L, potassium dihydrogen phosphate 1 g / L-2 g / L, potassium chloride 1 g / L-2 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.02 g / L, calcium carbonate 20 g / L, and antifoam agent.

[0017] In one embodiment of the present application, the fermentation conditions are as follows: 28-32 DEG C, 200-250 rpm, and 72-96 h of fermentation culture.

[0018] A fourth object of the present application is to provide the use of the recombinant C. glutamicum, the microbial preparation or the method in the preparation of spermidine.

[0019] The above technical solution of the present application has the following advantages compared with the prior art:

[0020] The present application provides a recombinant C. glutamicum and a method for fermenting spermidine with glucose as raw material. The present application overexpresses S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase, S-adenosylmethionine decarboxylase of different sources in different C. glutamicum, produces spermidine (SPD) through fermentation, and selects a C. glutamicum C.g AB2021051 / pEC-XK99E-metk-speE, pXMJ19-speC-speD with high SPD production, which has a yield of 0.35 g / L, breaking through the low efficiency of original C. glutamicum fermentation method for producing spermidine. Further optimization of the expression vector and arrangement order of each gene, optimization of the expression amount of each enzyme and the medium component, enhances the production of spermidine, and the yield reaches more than 0.49 g / L. Knocking out snaA eliminates the degradation of intermediate product putrescine, and overexpressing spermidine transporter mdtJI starting from a strong promoter, the recombinant bacteria SPD yield is 2.61 g / L. The final obtained recombinant bacteria are used for fermenting and producing spermidine, through enhancing the utilization of carbon and nitrogen sources by the production strain, optimizing the addition time and amount of substrate, and optimizing the dissolved oxygen control, 45.2 g / L of spermidine is produced in 5L fermentation tank for 72 h. The method of the present application produces spermidine by one-step fermentation with glucose as substrate, has the advantages of high efficiency and no need of adding exogenous intermediate products such as arginine and methionine, can reduce production cost and improve production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, wherein,

[0022] Figure 1 is a recombinant plasmid pEC-XK99E-metk-speE map in the embodiment of the present application;

[0023] Figure 2 is a recombinant plasmid pXMJ19-speC-speD map in the embodiment of the present application;

[0024] Figure 3The influence of different series sequences on the expression amount of genes in the embodiment of the application; wherein, 1: BL21 / pEC-XK99E control; 2: BL21 / pEC-XK99E-speE-metk; 3: BL21 / pEC-XK99E-metk-speE; 4: BL21 / pXMJ19 control; 5: BL21 / pXMJ19-speD-speC; 6: BL21 / pXMJ19-speC-speD;

[0025] Figure 4 The liquid phase detection spectrum of spermidine standard (1g / L) in the embodiment of the application;

[0026] Figure 5 The liquid phase detection spectrum of the 40-fold diluted sample of the recombinant bacteria C.gAB-AJMECD after 72h fermentation in the fermentation tank in the embodiment of the application;

[0027] Figure 6 The fermentation parameter diagram of the recombinant bacteria C.gAB-AJMECD in the embodiment of the application;

[0028] Figure 7 The fermentation parameter diagram of the recombinant bacteria C.gAB-AJMECD in the embodiment of the application;

[0029] Figure 8 The spermidine metabolic pathway involved in the embodiment of the application. DETAILED DESCRIPTION

[0030] The application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.

[0031] The recombinant Corynebacterium glutamicum in the application takes Corynebacterium glutamicum var.CCTCC AB 2021051 (also named Corynebacterium crenatum SYPA5-5) as an expression host, knocks out snaA, and integrates and expresses spermidine transporter protein, takes plasmid pEC-XK99E and pXMJ19 as expression vectors, and overexpresses S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase. Among them, the recombinant Corynebacterium glutamicum expresses ornithine decarboxylase and S-adenosylmethionine decarboxylase by using pXMJ19 plasmid; expresses S-adenosylmethionine synthetase and spermidine synthase by using pEC-XK99E plasmid.

[0032] The first object of the application is to provide a recombinant Corynebacterium glutamicum, which takes Corynebacterium glutamicum as a host and is modified as follows:

[0033] (a) overexpressing S-adenosylmethionine synthetase metK;

[0034] (b) overexpressing spermidine synthase SpeE;

[0035] (c) overexpressing ornithine decarboxylase SpeC

[0036] (d) overexpressing S-adenosylmethionine decarboxylase SpeD

[0037] (e) on the basis of (a), (b), (c) and (d), knocking out snaA and integrating expression of spermidine transporter mdtJI.

[0038] Further, the metk, SpeE, SpeC and SpeD are all derived from Escherichia coli MG1655.

[0039] Further, the spermidine transporter mdtJI is expressed by a strong promoter.

[0040] Further, after knocking out snaA, the ammonium transporter mdtJI is integrated at the snaA point.

[0041] Further, the strong promoter includes tac promoter and trc promoter.

[0042] Further, the spermidine transporter nucleotide sequence is shown in SEQ ID NO 2.

[0043] Further, the S-adenosylmethionine synthetase nucleotide sequence is shown in SEQ ID NO 3.

[0044] Further, the spermidine synthase nucleotide sequence is shown in SEQ ID NO 4.

[0045] Further, the ornithine decarboxylase nucleotide sequence is shown in SEQ ID NO 5.

[0046] Further, the S-adenosylmethionine decarboxylase nucleotide sequence is shown in SEQ ID NO 6.

[0047] Further, the Corynebacterium glutamicum is selected from Corynebacterium glutamicum var. CCTCC AB 2021051 (C.g AB2021051).

[0048] Further, C.g AB2021051 is used as the host.

[0049] Further, pXMJ19 and pEC-XK99E plasmids are used as the expression vectors.

[0050] A second object of the present application is a method for fermentatively producing spermidine, which is fermentatively producing spermidine by using the above-mentioned recombinant bacteria.

[0051] Further, the method comprises the following steps:

[0052] (1) activating and culturing the recombinant bacteria to obtain a seed liquid of the recombinant bacteria,

[0053] (2) transferring the seed liquid to a fermentation medium for fermentation culture to prepare spermidine.

[0054] Further, the method for preparing the seed liquid of the recombinant Corynebacterium glutamicum is as follows: inoculating the above-mentioned recombinant Corynebacterium glutamicum into a seed culture medium, and culturing at 28-32°C and 180-250 rpm for 24 h to obtain the seed liquid.

[0055] Further, the fermentation medium is as follows: glucose 100-150 g / L, ammonium sulfate 5-15 g / L, yeast powder 5-10 g / L, potassium dihydrogen phosphate 1-2 g / L, potassium chloride 1-2 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.02 g / L, calcium carbonate 20 g / L, and antifoam agent.

[0056] Further, the seed liquid of the recombinant Corynebacterium glutamicum is added to the fermentation medium at a ratio of 10-20% (v / v) for fermentation to prepare spermidine.

[0057] Further, the obtained seed liquid is inoculated into the fermentation medium at an inoculation amount of 10% (v / v), and is subjected to fermentation culture at 28-32°C and 180-250 rpm for 72-96 h.

[0058] Further, when the seed liquid is inoculated into the fermentation medium for fermentation culture, IPTG is added after 24 h of inoculation, and the final concentration is 0.5 mM.

[0059] The present application also provides the use of the above-mentioned recombinant Corynebacterium glutamicum or the above-mentioned method for preparing spermidine in the preparation of a product containing spermidine.

[0060] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0061] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0062] In the quantitative experiments in the following examples, three repeated experiments were set, and the average value was taken.

[0063] The host used for constructing recombinant plasmid in the following examples is E. coli BL21 (DE3) purchased from Benao Bio, pXMJ19 plasmid and pK18moacB plasmid are purchased from BioVector Plasmid Bank Cell Gene Preservation Center.

[0064] The host used for expressing vector in the following examples is Corynebacterium glutamicum ATCC 13032 (C.g ATCC 13032) purchased from China Industrial Microbial Strain Preservation and Management Center; Corynebacterium glutamicum var. CCTCC AB 2021051 (C.g AB 2021051) is described in the paper “Improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply”.

[0065] The culture medium involved in the following examples is as follows:

[0066] LB liquid medium: yeast powder 5.0 g / L, protein peptone 5.0 g / L, sodium chloride 10 g / L.

[0067] LB solid medium: yeast powder 5.0 g / L, protein peptone 5.0 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0068] LBGS liquid sucrose medium: protein peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sucrose 100 g / L.

[0069] BHI liquid medium: brain heart infusion broth 38.5 g / L.

[0070] BHI solid medium: brain heart infusion broth 38.5 g / L, agar 20 g / L.

[0071] Seed medium: glucose 50 g / L, yeast extract 20 g / L, ammonium sulfate 20 g / L, potassium dihydrogen phosphate 15 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium carbonate 5 g / L.

[0072] Fermentation medium: glucose 150 g / L, ammonium sulfate 50 g / L, yeast powder 5 g / L, corn syrup 10 g / L, potassium dihydrogen phosphate 1.5 g / L, potassium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate heptahydrate 0.02 g / L, ferrous sulfate heptahydrate 0.02 g / L, calcium carbonate 20 g / L, antifoam agent.

[0073] The detection method involved in the following examples is as follows:

[0074] Detection of spermidine content: high performance liquid chromatography; Agilent C18, 5 μm, 4.6 x 250 mm column; flow rate 1.0 mL / min; column temperature 40 °C; detection wavelength 338 nm; mobile phase: phase A: 8.0 g of sodium acetate (13.3 g of sodium acetate trihydrate) is dissolved in 1000 mL of water, 225 μL of triethylamine is added, the pH is adjusted to 7.20 ± 0.05 with 5% acetic acid, and finally 5 mL of tetrahydrofuran is added and mixed; phase B: 6.0 g of sodium acetate is weighed into 200 mL of water, the pH is adjusted to 7.20 ± 0.05 with 5% acetic acid, and the solution is added to 400 mL of HPLC grade methanol and 400 mL of HPLC grade acetonitrile and mixed.

[0075] Example 1: Construction of recombinant bacteria C. g ATCC13032-MECD and C. g AB2021051-MECD

[0076] (1) Construction of recombinant plasmid

[0077] According to the gene sequences of spermidine transporter (nucleotide sequence as shown in SEQ ID NO 2), S-adenosylmethionine synthetase (metk, nucleotide sequence as shown in SEQ ID NO 3), spermidine synthase (speE, nucleotide sequence as shown in SEQ ID NO 4), ornithine decarboxylase (speC, nucleotide sequence as shown in SEQ ID NO 5), S-adenosylmethionine decarboxylase (speD, nucleotide sequence as shown in SEQ ID NO 6) in Escherichia coli MG1655 whole genome nucleic acid sequence in NCBI, the PCR primers F1, R1, F2, R2 of plasmid pEC-XK99E-metk-speE and the PCR primers F3, R3, F4, R4 of plasmid pXMJ19-speC-speD were designed.

[0078] F1: 5'-ATGTATATTTATTGGATTTTATTAGGTCTGGCTATTGC-3'

[0079] R1: 5'-GAATGATCATGGTGAAACTTGCCTGA-3'

[0080] F2: 5'-ACAGACCATGGAATTCTTGACAATTAATCA-3'

[0081] R2: 5'-GATGCTGCCGGTCTGAAGTAA-3'

[0082] F3: 5'-AGAGGATCCAAAGGAGGACAACCATGAAATCA-3'

[0083] R3: 5'-CGGTTGTACGGTTATGTGTTGAAGTAA-3'

[0084] F4: 5'-ATGGAAACAATGGGGCGTCAC-3'

[0085] R4: 5'-GCAAGCGAAAGTACTATAAGAATTCAGCt-3'

[0086] PCR amplification was performed using the above primers with Escherichia coli MG1655 whole genome as template, and the amplification conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 120 s, 30 cycles, and final extension at 72°C for 10 min. The PCR amplification system was as follows: 1 μL of template, 1 μL of each of the upper and lower primers, 22 μL of sterilized double distilled water, and 25 μL of 2x PhantaMax MasterMix. The PCR products were purified and recovered using a gel recovery kit, and the concentration of the recovered products was detected by electrophoresis. The recovered products were stored in 1.5 mL centrifuge tubes and kept in a refrigerator at -20°C for standby use.

[0087] The plasmids pEC-XK99E and pXMJ19 stored in E. coli JM109 were extracted, double-digested with Bam H I and Eco R I, and the S-adenosylmethionine synthetase and spermidine synthase gene fragments were recovered using a gel recovery kit after fusion PCR. The fusion fragments were connected to the expression vector pEC-XK99E with an inducible promoter trc. The ornithine decarboxylase and S-adenosylmethionine decarboxylase gene fragments were subjected to fusion PCR, and the fusion fragments were connected to the expression vector pXMJ19 with an inducible promoter trc to construct recombinant plasmids. The connection system was as follows: 2 μL of Exnase II, 4 μL of 5x CE II Buffer, and the vector and fragments were added according to the instructions of the Exnase II, and sterilized double distilled water was added to make up the total volume to 20 μL, followed by enzyme connection at 37°C for 30 min.

[0088] The connected recombinant plasmid was transformed into E. coli BL21 competent cell respectively, and the positive transformants were screened by LB solid medium with 50 μg / mL kanamycin and LB solid medium with 10 μg / mL chloramphenicol respectively.

[0089] The correct transformants were inoculated into 10 mL LB liquid medium with 50 μg / mL kanamycin and 10 mL LB liquid medium with 10 μg / mL chloramphenicol respectively, and then cultured at 37°C overnight. After plasmid extraction, enzyme digestion verification and sequencing, the correct recombinant strain Escherichia coli BL21 was obtained. The recombinant plasmid was extracted, and the correct recombinant strain was added with glycerol to a final concentration of 15% to 20% (v / v) and stored in a refrigerator at -80°C for standby. The recombinant plasmids pEC-XK99E-metk-speE (as shown in Figure 1 Figure 2 The construction of pXMJ19-speC-speD (as shown in

[0090] (2) Construction of recombinant bacteria

[0091] Preparation of competent cells: C. g ATCC13032 and C. g AB2021051 were inoculated into 10 mL BHI liquid medium respectively and cultured at 30°C for 24 h. The bacterial liquid after culture was transferred into 100 mL liquid BHI medium containing 3 g / L glycine and 0.1% Tween-80. The culture was carried out at 30°C and 200 rpm until the OD 600 of the cells reached 0.9. After the cell culture was completed, the bacterial liquid was pre-cooled for 30 min, and then the bacterial cells were collected by centrifugation. The bacterial cells were washed with pre-cooled 10% glycerol for 3 times, and finally resuspended with 0.2 mL 10% glycerol. The cells were divided into 1.5 mL tubes, each tube containing 80 μL, which were directly used for electroporation.

[0092] Electroporation: 1850V electroporation for 5 ms, and then 800 μL BHI medium was added and the culture was carried out at 30°C and 200 rpm for 2-3 h.

[0093] ​Recombinant bacteria were obtained: the recombinant plasmid extracted in step (1) was transformed into C. g ATCC13032 and C. g AB2021051 competent cells, respectively, to obtain transformants, and the transformants were spread on BHI solid medium containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin double resistance, and cultured at 30°C. Positive colonies were picked and colony PCR was performed to obtain recombinant bacteria C. g ATCC13032 / pEC-XK99E-metk-speE, pXMJ19-speC-speD (named C. g ATCC13032-MECD) and C. g AB2021051 / pEC-XK99E-metk-speE, pXMJ19-speC-speD (named C. g AB2021051-MECD). The correct recombinant bacteria were added to glycerol to a final concentration of 15%-20% (v / v), and stored in a -80°C refrigerator for standby.

[0094] Example 2: Shake flask fermentation of recombinant bacteria to produce spermidine

[0095] (1) The recombinant bacteria C. g ATCC13032-MECD and C. g AB2021051-MECD constructed in Example 1 and the original strains C. g ATCC13032 and C. g AB2021051 were respectively streaked on BHI solid medium containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin double resistance and non-resistant BHI solid medium, and single colonies were picked and inoculated in seed culture medium for 24 h to prepare seed liquid;

[0096] (2) The prepared seed liquid was transferred to a 250 mL flask containing 30 mL of fermentation medium prepared in step (1) at a transfer amount of 10% (v / v), and cultured in a reciprocating shaker at 30°C and 220 rpm for 96 h. IPTG was added after 24 h of fermentation of the recombinant bacteria, with a final concentration of 0.5 mM. The fermentation time was 72-96 h, and the fermentation liquid was collected after fermentation. The content of spermidine was detected by HPLC, as shown in Table 1 below:

[0097] Table 1: Content of spermidine produced by different recombinant bacteria in shake flask fermentation

[0098]

[0099] It is found through fermentation that C.g ATCC13032 can only produce 0.52 g / L arginine, while C.g AB202105 can produce 16.8 g / L arginine, and arginine, as a precursor for SPD synthesis, is crucial for the synthesis of spermidine. When the four enzymes metk, SpeC, SpeD and SpeE are overexpressed, they all have the ability to produce GAA, and the spermidine yield of the recombinant bacteria C.g AB2021051 / pEC-XK99E-metk-speE and pXMJ19-speC-speD is higher than that of C.g ATCC13032 / pEC-XK99E-metk-speE and pXMJ19-speC-speD. At the same time, it is found through fermentation results (as shown in Table 1) that when pXMJ19 plasmid is used to express SpeC and SpeD and pEC-XK99E plasmid is used to express metk and SpeE, the spermidine yield is the highest. In summary, C.g AB202105 is selected as the production strain of spermidine, and the recombinant bacteria C.g AB2021051 / pEC-XK99E-metk-speE and pXMJ19-speC-speD are used for subsequent research, which is named C.g AB-MECD here. Figure 3

[0100] The determination method of the single enzyme activity of the several key enzymes metk, SpeC, SpeD and SpeE in the application is high in cost and complicated in determination conditions, so the expression amount is directly determined by using the method of determining the overall enzyme activity, the reaction conditions are pH 7.0, 30 DEG C reaction for 8h, and the reaction system is shown in Table 2:

[0101] Table 2 Reaction system for determining overall enzyme activity

[0102]

[0103]

[0104] Among them, by replacing adenosine and sodium hexametaphosphate with S-adenosyl methionine (SAM), or replacing ornithine with butanediamine hydrochloride, it is confirmed that the rate-limiting enzyme for the reaction to generate SPD is metk, and the addition ratio of each enzyme metk, SpeC, SpeD and SpeE is 4:3:3:3, which is the best, and the reduction of the addition amount of any enzyme will cause the SPD yield to be greatly reduced, so all the enzymes use high-strength promoters.

[0105] Since the gene arrangement order on the same plasmid will affect the expression amount of each key enzyme during fermentation, and the expression intensity of different enzymes will also affect the final SPD yield, the effect of each enzyme in different tandem orders during fermentation of the strain C.g AB-MECD for producing SPD is directly selected.​

[0106] Table 3: Effect of different key enzyme tandem sequences on the production of SPD in shake flask fermentation

[0107]

[0108] It was determined that the strain C.gAB-MECD expressed each key enzyme gene at high strength, and the tandem sequence was pEC-XK99E-metk-speE, pXMJ19-speC-speD, which had the highest SPD production.

[0109] Example 3: Optimization of fermentation conditions to promote the production of SPD

[0110] The specific steps are as follows:

[0111] 1. Optimization of medium components

[0112] Since the original medium has a high ratio of carbon source and nitrogen source, the amount and source of nitrogen source are optimized, and the type and content of each component such as the defoaming agent in the fermentation system are also optimized. Then, the ability of C.gAB-MECD to produce SPD in fermentation is verified according to the method of shake flask fermentation in Example 2. After 72 h of fermentation, the strain grows normally, and the SPD production is 0.49 g / L after the fermentation is completed, which is 40% higher than before optimization.

[0113] 2. Knocking out snaA and in situ integrating spermidine transporter mdtJI

[0114] Under the condition of exogenous addition of methionine, the fermentation broth of strain C.gAB-MECD is centrifuged after 72 h of fermentation, and the supernatant is the extracellular content. The solution obtained by breaking the centrifuged bacterial cells and resuspending them can roughly estimate the intracellular substance level.

[0115] Table 4: Intracellular and extracellular substance content levels of strain C.gAB-MECD after 72 h of fermentation

[0116]

[0117] The above data show that the intracellular substrate is not completely converted, and the product is not timely transported and released. By improving the transport and release of spermidine, on the one hand, the extracellular yield can be increased, and on the other hand, the forward production of spermidine reaction can be promoted.

[0118] To promote the production of bacteria to transport intracellular spermidine to the extracellular, a single copy of the ammonium transporter gene mdtJI is integrated under the tac promoter, and the degradation of the intermediate product putrescine is reduced by knocking out the putrescine degradation gene snaA. Therefore, mdtJI is integrated at the snaA site to realize the integration of mdtJI and the knockout of snaA.

[0119] Firstly, the mdtJI gene fragment was obtained by PCR using Escherichia coli MG1655 genome as a template with primer pair F5 and R5, and then the pXMJ19-tac-mdtJI was obtained by connecting the linearized vector pXMJ19 digested with EcoR I and Hind III with the mdtJI gene fragment. The tac-mdtJI fragment was obtained by PCR using pXMJ19-tac-mdtJI as a template with primer pair F6 and R6, and then the fragment with integration site upstream and downstream 500bp homologous arm was obtained by PCR using primer pair F7 and R7, F8 and R8 with the genome of Corynebacterium glutamicum as a template. The tac-mdtJI fragment with integration site upstream and downstream 500bp homologous arm was obtained by three-fragment fusion PCR using primer pair F7 and R8, and then the obtained fragment and pK18moacB vector were simultaneously digested with EcoR I and Hind III, and then the homologous recombination enzyme was used for 37°C connection after gel purification. The connection product was transformed into E. coli BL21(DE3), and then the transformant was picked and cultured for 12h, and then the plasmid was extracted for PCR verification. The plasmid was sent to Suzhou JUNWIZ company for sequencing, and the sequence was correct after DNAMAN software analysis, and then the pK18-mdtJI was successfully constructed.

[0120] F5: 5'-AAAGGAGGACAACCATGTATATTTATTGGATTT-3'

[0121] R5: 5'-GGAATGATCATGGTGAAACTTGCCTGA-3'

[0122] F6:

[0123] 5'-TGACATGATTACGAATTCTTGACAATTAATCATCCGGCTCGTATA-3'

[0124] R6: 5'-GGAATGATCATGGTGAAACTTGCCTGAGGCACTGGCCGTCGTTTTAC-3'

[0125] F7: 5'-GAATTCCCCTTTCCAACCTTTATCTAAA-3'

[0126] R7: 5'-CGCTGTCGCCAACTGCTAATTGACAATT-3'

[0127] F8:

[0128] 5'-GAAACTTGCCTGAGGAGAAATAATGGGAATTGTAATCATGCT-3'

[0129] R8: 5'-CCTTGGATACCTGGAGCAATCCGAAGCTTGGCACTG-3'

[0130] The pK18-mdtJI plasmid was electroporated into C.gAB2021051, and after the colonies grew, they were identified and expanded, then transferred to LBGS liquid sucrose medium for screening. The screened bacterial liquid was first streaked on an antibiotic-free BHI plate, then the colonies grown on the antibiotic-free plate were streaked on a BHI plate containing Kan, and the colonies that did not grow on the antibiotic-free BHI plate were selected for culture. Genomic sequencing was successful, and the strain C.gAB-AJ was obtained. The plasmids pEC-XK99E-metk-speE, pXMJ19-speC-speD described in Example 1 were introduced into C.gAB-AJ to obtain the strain C.gAB-AJMECD.

[0131] The ability of C.gAB-AJMECD to produce SPD was verified according to the method of the shake flask fermentation in Example 2. The fermentation was carried out for 72 h, and the bacteria grew normally. During the fermentation process, the SPD yield was 1.39 g / L with exogenous addition of methionine, and the SPD yield was 2.61 g / L without exogenous addition of methionine, which was 4.3 times higher than that of C.gAB-MECD.

[0132] 3. Effect of substrate methionine addition on SPD yield

[0133] Since methionine has low solubility, and high concentration in the fermentation system can be toxic to cell growth, and low concentration can result in insufficient substrate and reduced SPD accumulation. Therefore, determining the most appropriate amount of methionine addition is very important for SPD accumulation.

[0134] In order to explore the effect of methionine addition time and amount on SPD yield, 5 g / L methionine was added at 0 h, 6 h, 12 h, and 24 h of shake flask fermentation medium transfer. Every 12 h, the fermentation-related parameters were measured. The absorbance value of the bacterial liquid at OD 600 nm was measured by ultraviolet spectrophotometer, the residual glucose amount was measured by SBA biosensor, and the residual amount of methionine and the yield of spermidine in the fermentation broth were measured by HPLC method.

[0135] The results showed (as shown in Figure 6 When 5 g / L methionine was added twice at 24 h and 48 h, the SPD yield of C.gAB-MECD strain was the highest at 0.49 g / L, while the SPD yield of recombinant strain C.gAB-AJMECD reached 2.61 g / L without exogenous addition of methionine. At the same time, the residual sugar amount and OD600 It was found that its growth condition was good, while the yield and growth condition were poor in the case of exogenous addition of methionine, and the highest yield was less than 1.4 g / L.

[0136] Through detection of the levels of various substances in the cell of the strain C.gAB-AJMECD without exogenous addition of methionine at 72 h of fermentation, it was found that the contents of arginine, putrescine and spermidine in the cell were extremely low, indicating that the intermediate product in the cell was fully converted, and the product was timely released to the outside of the cell, and the intermediate product methionine which was not beneficial to the growth of the cell did not need to be added exogenously to maintain the pressure of spermidine production in the cell.

[0137] Example 4: Production of spermidine by recombinant strain C.gAB-AJMECD in a 5L fermenter

[0138] The specific steps are as follows:

[0139] 1. Dissolved oxygen control optimization

[0140] The dissolved oxygen (DO) level of the fermenter affects the growth of the bacterial cells and the synthesis of SPD, and the rotation speed and the aeration amount are two main factors affecting the dissolved oxygen level. It is reported that the optimal dissolved oxygen control level for the fermentation of amino acids by Corynebacterium glutamicum is 30%-40%. Therefore, in the present application, the rotation speed coupled with the dissolved oxygen and the manual increase of the aeration amount are used to maintain the dissolved oxygen level in the fermentation process at 30%-40%, so as to explore the influence of the dissolved oxygen control on the growth of the bacterial cells and the synthesis of SPD.

[0141] 2. Fermentation of the engineering bacteria in a 5L fermenter to produce spermidine

[0142] Seed culture: a single colony of the recombinant strain C.gAB-AJMECD prepared in Example 3 was picked from an activated plate and inoculated into 10 mL of BHI culture medium, and cultured at 30℃ and 200 rpm for 24 h, then transferred to a 250 mL flask containing 30 mL of seed culture medium at an inoculation amount of 2% (v / v), and cultured at 30℃ and 200 rpm for 24 h to prepare a primary seed liquid. The primary seed liquid was transferred to a 1000 mL flask containing 200 mL of seed culture medium at an inoculation amount of 10% (v / v), and cultured at 30℃ and 200 rpm for 20 h to prepare a secondary seed liquid.

[0143] 5L fermenter fermentation culture: the above cultured secondary seed liquid was transferred to a 5L fermenter containing 2L of fermentation culture medium, and cultured at 30℃ under the condition that the dissolved oxygen level in the fermentation process was maintained at 30%-40% by using the rotation speed coupled with the dissolved oxygen and manually increasing the aeration amount for 96 h; wherein IPTG was added at a final concentration of 0.5 mM at 24 h of culture for induction, and the fermentation was performed for 72 h without exogenous addition of the intermediate product methionine.

[0144] After the fermentation, the fermentation liquor was collected, diluted 40 times, and then the contents of arginine, putrescine and spermidine were detected by HPLC (the chromatogram of spermidine is shown in Fig. 2). Figure 5

[0145] The results show that the recombinant bacteria C.gAB-AJMECD can accumulate 45.2 g / L of spermidine (as shown in Fig. 2) under the above fermentation process and conditions, and the methionine is not added, and the one-step fermentation of the sugar raw material to produce spermidine is realized. Figure 7

[0146] The results show that the present application realizes the direct synthesis of spermidine from glucose by one-step fermentation method, and the intermediate product methionine does not need to be added externally, and there is basically no residual arginine or putrescine after fermentation.

[0147] Obviously, the above examples are only examples for clearly illustrating, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments cannot be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.​​

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that, The recombinant Corynebacterium glutamicum is Corynebacterium glutamicum as an expression host, knock-out of putrescine degradation gene, and heterologous expression of spermidine transporter gene cluster, S-adenosylmethionine synthetase gene, spermidine synthase gene, ornithine decarboxylase gene and S-adenosylmethionine decarboxylase gene; The ornithine decarboxylase and S-adenosylmethionine decarboxylase are sequentially located on the pXMJ19 plasmid; and the S-adenosylmethionine synthetase and spermidine synthase are sequentially located on the pEC-XK99E plasmid; Knock-out of putrescine degradation gene snaA in situ integration expression of spermidine transporter; The coryneform bacterium is Corynebacterium glutamicum var. CCTCC AB 2021051.

2. The recombinant C. glutamicum according to claim 1, characterized in that, The nucleotide sequence of the putrescine degradation gene is shown as SEQ ID NO 1; the nucleotide sequence of the spermidine transporter gene cluster is shown as SEQ ID NO 2; the nucleotide sequence of the S-adenosylmethionine synthetase gene is shown as SEQ ID NO. 3; the nucleotide sequence of the spermidine synthase gene is shown as SEQ ID NO. 4; the nucleotide sequence of the ornithine decarboxylase gene is shown as SEQ ID NO. 5; and the nucleotide sequence of the S-adenosylmethionine decarboxylase gene is shown as SEQ ID NO.

6.

3. The recombinant C. glutamicum according to claim 1, characterized in that, The spermidine transporter, S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase and S-adenosylmethionine decarboxylase are derived from Escherichia coli MG1655.

4. A microbial preparation comprising the recombinant Corynebacterium glutamicum according to any one of claims 1-3.

5. A method for the fermentative production of spermidine, characterized in that, comprising the following steps: Spermidine is produced by fermentation of a fermentation system with glucose as a substrate and the recombinant Corynebacterium glutamicum according to any one of claims 1-3 or the microbial preparation according to claim 4.

6. The method of claim 5, wherein, The fermentation medium in the fermentation system comprises: glucose 100 g / L-150 g / L, ammonium sulfate 5 g / L-15 g / L, yeast powder 5 g / L-10 g / L, potassium dihydrogen phosphate 1 g / L-2 g / L, potassium chloride 1 g / L-2 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.02 g / L, calcium carbonate 20 g / L, and defoaming agent.

7. The method of claim 5, wherein, The fermentation conditions are: 28°C-32°C, 200 rpm-250 rpm, and fermentation for 72 h-96 h.

8. Use of the recombinant Corynebacterium glutamicum according to any one of claims 1-3, the microbial preparation according to claim 4, or the method according to any one of claims 5-7 in the preparation of spermidine.

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