A method for co-fermentation of corynebacterium ammoniagenes and escherichia coli to produce putrescine

Abstract

This invention discloses a method for producing butanediamine through co-fermentation of Corynebacterium tumefaciens and Escherichia coli, belonging to the field of microbial fermentation technology. The method provided by this invention is as follows: (1) preparing activated seed culture of Corynebacterium tumefaciens CGMCC NO.0890; (2) preparing activated seed culture of recombinant Escherichia coli overexpressing arginine decarboxylase and guanidinobutanase; (3) co-fermentation: inoculating the Corynebacterium tumefaciens seed culture at a volume ratio of 4-10% into the co-fermentation medium, and culturing it with shaking at a temperature of 25-40℃ and a rotation speed of 100-130 r / min for 72-120 h; then transferring it to a 5L fermenter containing a certain amount of recombinant Escherichia coli seed culture at a certain ratio, and continuing fermentation under certain conditions for 24-120 h, ending the fermentation to obtain a fermentation broth containing butanediamine. This co-culture liquid fermentation process can significantly improve the production efficiency of butanediamine, reduce production costs, and meet the needs of industrial development.

Description

Technical Field

[0001] This invention relates to a method for producing butanediamine by co-fermentation of Corynebacterium tumefaciens and Escherichia coli, belonging to the field of microbial fermentation technology. Background Technology

[0002] Putrescine (Ph,N-butylenediamine), as a precursor for the synthesis of high-performance plastic nylon 4,6, has significant industrial value. Currently, large-scale production of Ph,N-butylenediamine mainly relies on chemical synthesis. Although the process is mature, the raw materials are non-renewable petroleum products, which does not meet the requirements of sustainable development. Furthermore, chemical reactions require expensive catalysts and relatively harsh reaction conditions. For example, patent CN106220512A requires dehydration, reduced pressure reaction, and hydrogen gas introduction, and the reaction system also involves various organic solvents and expensive catalysts. Therefore, there is a need to find a green and efficient method for synthesizing Ph,N-butylenediamine using renewable resources as raw materials. Biochemical methods have received widespread attention due to their advantages of being green and sustainable, having mild reaction conditions, and low environmental pollution. However, biochemical methods require expensive arginine or ornithine as substrates, resulting in high costs, which significantly hinders the industrialization of Ph,N-butylenediamine production. For example, in patent CN105925629A, a recombinant bacterium overexpressing ornithine decarboxylase was constructed to convert ornithine into butanediamine using ornithine as a substrate, with a yield of only 0.7 g / L. Summary of the Invention

[0003] To address the aforementioned deficiencies in existing technologies, the present invention aims to provide a method for producing butanediamine through co-fermentation of Corynebacterium tumefaciens and Escherichia coli. This co-culture liquid fermentation process significantly improves the butanediamine production level of Escherichia coli and eliminates the need for expensive arginine as a substrate, thus meeting the requirements for industrial-scale production.

[0004] The first objective of this invention is to provide a microbial preparation comprising microbial preparation A and microbial preparation B, wherein microbial preparation A is Corynebacterium crenatum JN-1 and microbial preparation B is recombinant Escherichia coli.

[0005] In one embodiment, the Corynebacterium obliterans JN-1 is described in patent publication number CN1441055A, with accession number CGMCC NO.0890.

[0006] In one embodiment, the recombinant Escherichia coli overexpresses arginine decarboxylase and guanidinobutanase.

[0007] In one embodiment, the amino acid sequence of the arginine decarboxylase can be any amino acid sequence with arginine decarboxylase activity from any source, and the amino acid sequence of the guanidinobutamine enzyme can be any amino acid sequence with guanidinobutamine enzyme activity from any source.

[0008] In one embodiment, the amino acid sequence of the arginine decarboxylase is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the guanidinobutamine enzyme is shown in SEQ ID NO.3.

[0009] In one embodiment, the recombinant Escherichia coli is expressed using a pETDuet-1 expression vector, with E. coli JM109 as the host cell.

[0010] A second object of the present invention is to provide the use of the microbial preparation in the preparation of products containing butanediamine.

[0011] The third objective of this invention is to provide a method for producing butanediamine by fermentation. The method involves first inoculating the seed culture of microbial preparation A from the above-mentioned microbial preparation into a co-fermentation medium, fermenting and culturing it, and then inoculating it into a co-fermentation medium containing the seed culture of microbial preparation B, and continuing fermentation and culturing to produce butanediamine.

[0012] In one embodiment, the specific steps of the method are as follows:

[0013] (1) The above-mentioned microbial preparation A was inoculated into seed culture medium A and fermented to obtain seed liquid A;

[0014] (2) Inoculate the above-mentioned microbial preparation B into seed culture medium B and ferment for 10-30 hours to obtain seed liquid B;

[0015] (3) Inoculate the seed liquid A obtained in step (1) into the co-fermentation medium at an inoculation amount of 4-10% by volume, and ferment for 72-120 hours to obtain fermentation liquid A;

[0016] (4) Inoculate the fermentation broth A from step (3) into a co-fermentation medium containing seed liquid B, and ferment for 2 to 5 days to obtain a fermentation broth containing butanediamine.

[0017] In one embodiment, the fermentation culture temperature in step (1) is 25-35°C and the rotation speed is 110-150 r / min.

[0018] In one embodiment, the fermentation temperature in step (2) is 35-40°C and the rotation speed is 225-275 r / min.

[0019] In one embodiment, the fermentation temperature in step (3) is 25–40°C and the rotation speed is 100–130 r / min.

[0020] In one embodiment, the seed liquid B in step (4) is inoculated into the co-fermentation medium at an inoculation amount of 1.0 to 1.5% by volume.

[0021] In one embodiment, the fermentation broth A in step (4) is inoculated into the co-fermentation medium at an inoculation amount of 2 to 3% by volume.

[0022] In one embodiment, the fermentation temperature in step (4) is 30-40°C and the rotation speed is 300-350 r / min.

[0023] In one embodiment, the co-fermentation culture medium comprises, in g / L, 25-35 g of ammonium sulfate, 55-65 g of sucrose, 3-7 g of MgSO4·7H2O, 25-35 g of KH2PO4, 0.02 g of MnSO4·H2O, 0.02 g of FeSO4·7H2O, 0.00045 g of VB1, 0.00005 g of biotin, 25-35 g of calcium carbonate, 3-7 g of yeast extract, 15-25 g of peptone, 0.5 g of NaCl, 0.95 g of MgCl2, 0.186 g of KCl, with the remainder being water.

[0024] In one embodiment, the seed culture medium A in step (1) consists of 15-25 g / L glucose, 8-12 g / L peptone, 3-7 g / L yeast extract, 8-12 g / L beef extract, 2-4 g / L NaCl, and the remainder is water.

[0025] In one embodiment, the seed culture medium B in step (2) consists of 3-7 g / L yeast extract, 8-12 g / L peptone, 8-12 g / L NaCl, and the remainder is water.

[0026] The present invention also provides the application of the method in the preparation of products containing butanediamine.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This invention is the first to apply the co-fermentation technology of Corynebacterium crenatum JN-1 and recombinant Escherichia coli JM109 / pETDuet-speA-speB to the production of butanediamine, proving that under certain process conditions, Corynebacterium crenatum and Escherichia coli can co-produce butanediamine. The method for producing butanediamine provided by this invention uses ammonium sulfate as a substrate and does not require the addition of arginine. Compared with the traditional fermentation process using arginine as a substrate, the cost is reduced by 100%.

[0029] 2. This invention can produce up to 55g / L of butanediamine using a 5L fermenter, with a conversion rate as high as 76%. Compared with traditional fermentation processes, the conversion rate is increased by 90%. The method provided by this invention meets the needs of industrial production and provides a new idea and approach for the industrial production of butanediamine by microbial fermentation. Detailed Implementation

[0030] The present invention will be specifically described below through examples.

[0031] Unless otherwise specified, all raw materials and reagents involved in the following examples are domestically produced or imported analytical grade products, and all experimental methods involved are conventional methods in the field.

[0032] The biomaterials involved in the following embodiments:

[0033] 1. Corynebacterium crenatum JN-1: It is described in the patent with publication number CN1441055A and accession number CGMCC NO.0890 (the strain number in the patent application text is SDNN403, which the inventor renumbered as JN-1 during the experiment).

[0034] 2. *Escherichia coli*: Recombinant strain *E. coli* JM109 / pETDuet-speA-speB. Using the commercially available plasmid pETDuet-1 as the expression vector, after double digestion with Nco I and EcoRI, the plasmid was ligated with the double-digested arginine decarboxylase gene *speA* and guanidinobutyrate enzyme gene *speB* to obtain the recombinant plasmid pETDuet-speA-speB. This recombinant plasmid was then transformed into *E. coli* JM109 to obtain the recombinant strain *E. coli* JM109 / pETDuet-speA-speB.

[0035] The culture media involved in the following examples:

[0036] 1. Seed culture medium for Corynebacterium tumefaciens: The components (in g / L) are: glucose 20, peptone 10, yeast extract 5, beef extract 10, NaCl 3, and the remainder is water. The pH is 7.0-7.2. The medium is sterilized at 121℃ for 20 min.

[0037] 2. Escherichia coli seed culture medium: The components, in g / L, are: yeast extract 5g, peptone 10g, NaCl 10g, and the remainder is water, pH 7.0, sterilized at 121℃ for 20min.

[0038] 3. Co-fermentation medium: The components (in g / L) are: ammonium sulfate 30, sucrose 60, MgSO4·7H2O 5, KH2PO4 30, MnSO4·H2O 0.02, FeSO4·7H2O 0.02, VB1 0.00045, biotin 0.00005, calcium carbonate 30, yeast extract 5, peptone 20, NaCl 0.5, MgCl2 0.95, KCl 0.186, and the remainder is water. The initial pH is 7.0–7.2. The medium is autoclaved at 115℃ for 30 min.

[0039] The following embodiments involve methods:

[0040] 1. Culture methods for the strain:

[0041] (1) Preparation of seed culture of Corynebacterium tumefaciens JN-1: Pick a loop of bacteria from the slant and inoculate it into the seed culture medium of Corynebacterium tumefaciens. Incubate at 30℃ with shaking for 22-24h at a shaking speed of 120r / min to obtain activated seed culture of Corynebacterium tumefaciens.

[0042] (2) Preparation of Escherichia coli seed culture: The recombinant strain E. coli JM109 / pETDuet-speA-speB was inoculated into the Escherichia coli seed culture medium and cultured at 37℃ with shaking at 250 r / min to obtain the activated Escherichia coli seed culture.

[0043] 2. The specific method for determining the butanediamine content is as follows:

[0044] In this study, dansyl chloride pre-column derivatization combined with HPLC was selected for the quantitative detection of the product butanediamine.

[0045] High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30℃ and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6.7 min, 70% B; 6.7–12 min, 70%–95% B; 12–12.6 min, 95% B; 12.6–13.5 min, 95%–55% B; 13.5–16 min, 55% B. The total flow rate was set to 0.7 mL / min. -1 .

[0046] Example 1

[0047] 2.5 mL of activated seed culture of *Corynebacterium tumefaciens* JN-1 was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 96 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 25 mL of *E. coli* seed culture incubated for 24 h. The fermentation was continued at 37 °C with shaking at 320 rpm. Fermentation was stopped on day 7 from the date of inoculation with *Corynebacterium tumefaciens*, yielding the co-culture broth of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 22 g / L, 55 g / L, and 42 g / L, respectively.

[0048] Example 2

[0049] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 96 h. Then, the culture was transferred to a 5 L fermenter containing 30 mL of *E. coli* seed culture (included with 2 L of co-fermentation medium) and incubated at 37 °C with 320 rpm. Fermentation was stopped on the 9th day from the date of *Corynebacterium tumefaciens* inoculation into the flask, yielding the co-culture broth of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 29 g / L, 54 g / L, and 55 g / L, respectively.

[0050] Example 3

[0051] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 96 h to obtain the *Corynebacterium tumefaciens* fermentation broth. This broth was then inactivated and transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 35 mL of *E. coli* seed culture cultured for 24 h. The fermentation was continued at 37 °C with shaking at 330 rpm for 8 days, ending the fermentation on the 8th day from the date of inoculation. This yielded the co-culture broth of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 15 g / L, 45 g / L, and 31 g / L, respectively.

[0052] Example 4

[0053] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 96 h to obtain the *Corynebacterium tumefaciens* fermentation broth. This broth was then inactivated and transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 20 mL of *E. coli* seed culture cultured for 12 h. The fermentation was continued at 37 °C with shaking at 350 rpm for 7 days, ending the fermentation process on day 7 from the time of inoculation. The resulting fermentation broth was a co-culture of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yields were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 16 g / L, 40 g / L, and 38 g / L, respectively.

[0054] Example 5

[0055] Two mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30°C with shaking at 115 rpm for 96 h to obtain the *Corynebacterium tumefaciens* fermentation broth. This broth was then inactivated and transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 25 mL of *E. coli* seed culture cultured for 12 h. The fermentation was continued at 37°C with shaking at 320 rpm for 7 days, ending the fermentation process on day 7 from the time of inoculation. The resulting fermentation broth was a co-culture of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yields were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 14 g / L, 35 g / L, and 22 g / L, respectively.

[0056] Example 6

[0057] Three mL of activated *Corynebacterium obliterans* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30°C with shaking at 115 rpm for 72 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 25 mL of *E. coli* seed culture cultured for 24 h. The fermentation was continued at 37°C with shaking at 330 rpm. Fermentation was stopped on day 7 from the date of *Corynebacterium obliterans* inoculation into the flask, yielding the fermentation broth co-cultured with *Corynebacterium obliterans* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 18 g / L, 25 g / L, and 30 g / L, respectively.

[0058] Example 7

[0059] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 102 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 30 mL of *E. coli* seed culture cultured for 12 h. The fermentation was continued at 37 °C with shaking at 320 rpm. Fermentation was stopped on the 8th day from the date of *Corynebacterium tumefaciens* inoculation into the flask, yielding the fermentation broth co-cultured with *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 22 g / L, 51 g / L, and 43 g / L, respectively.

[0060] Example 8

[0061] Five mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30°C with shaking at 115 rpm for 108 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 35 mL of *E. coli* seed culture cultured for 16 h. The fermentation was continued at 37°C with shaking at 325 rpm. Fermentation was stopped on the 9th day from the date of *Corynebacterium tumefaciens* inoculation into the flask, yielding the co-culture broth of *Corynebacterium tumefaciens* and *E. coli*. Samples were taken periodically to detect OD600 and butanediamine production. The results showed that the OD600, maximum arginine production, and maximum butanediamine content in the fermentation broth were 21 g / L, 50 g / L, and 48 g / L, respectively.

[0062] Example 9

[0063] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 115 rpm for 96 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 25 mL of *E. coli* seed culture cultured for 26 h. The fermentation was continued at 37 °C with shaking at 320 rpm. Fermentation was stopped on the 9th day from the date of *Corynebacterium tumefaciens* inoculation into the flask, yielding the fermentation broth co-cultured with *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 20 g / L, 46 g / L, and 39 g / L, respectively.

[0064] Example 10

[0065] 2.5 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 250 mL flask containing 50 mL of co-fermentation medium. The flask was incubated at 30 °C with shaking at 315 rpm for 120 h. Then, the culture was transferred to a 5 L fermenter (containing 2 L of co-fermentation medium) containing 25 mL of *E. coli* seed culture cultured for 28 h. The fermentation was continued at 37 °C with shaking at 315 rpm. Fermentation was stopped on day 7 from the date of *Corynebacterium tumefaciens* inoculation into the flask, yielding the co-culture broth of *Corynebacterium tumefaciens* and *E. coli*. OD600 and butanediamine yield were measured periodically. The results showed that the OD600, maximum arginine yield, and maximum butanediamine content in the fermentation broth were 25 g / L, 45 g / L, and 29 g / L, respectively.

[0066] Table 1. Fermentation conditions and results of Examples 1-10

[0067]

[0068]

[0069] The above experimental data prove that the co-culture liquid fermentation process of the present invention can significantly improve the production efficiency of butanediamine, reduce production costs, and meet the needs of industrialization development.

[0070] Comparative Example 1

[0071] 25 mL of activated *Corynebacterium tumefaciens* seed culture and 30 mL of *Escherichia coli* seed culture cultured for 24 h were inoculated into a 5 L fermenter (containing 2 L of co-fermentation medium). The fermentation was carried out at 37 °C with shaking at 220 rpm for 7 days from the time of seed culture inoculation, thus obtaining the fermentation broth of *Corynebacterium tumefaciens* and *Escherichia coli* co-culture. Samples were taken periodically to detect OD600 and butanediamine yield. The butanediamine content in the fermentation broth was found to be 15 g / L.

[0072] Comparative Example 2

[0073] 25 mL of activated *Corynebacterium tumefaciens* seed culture was inoculated into a 5 L fermenter (containing 2 L of co-fermentation medium) and cultured at 37 °C with shaking at 220 rpm. Fermentation was terminated on day 7 from the time of seed culture inoculation. OD600 and butanediamine yield were measured periodically. The arginine yield in the fermentation broth was found to be 25 g / L.

[0074] Comparative Example 3

[0075] 35 mL of *E. coli* seed culture, after 24 h of incubation, was inoculated into a 5 L fermenter (containing 2 L of co-fermentation medium). 50 g / L arginine was added, and the fermentation was carried out at 37 °C with shaking at 330 rpm. Fermentation was terminated on day 8 from the time of inoculation. OD600 and butanediamine yield were measured periodically. The butanediamine content in the fermentation broth was found to be 20 g / L.

[0076] Table 2 Cost Accounting

[0077] Butanediamine production (g / L) 42 20 Arginine content (g / L) 55 50 (additional) Conversion rate (%) 76 40 Fermentation time (d) 7 8 Fermentation cost (ten thousand yuan / ton) 5 10

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> A method for producing butanediamine by co-fermentation of Corynebacterium tumefaciens and Escherichia coli <130> BAA211434A <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 658 <212> PRT <213> Artificial sequence <400> 1 Met Ser Asp Asp Met Ser Met Gly Leu Pro Ser Ser Ala Gly Glu His 1 5 10 15 Gly Val Leu Arg Ser Met Gln Glu Val Ala Met Ser Ser Gln Glu Ala 20 25 30 Ser Lys Met Leu Arg Thr Tyr Asn Ile Ala Trp Trp Gly Asn Asn Tyr 35 40 45 Tyr Asp Val Asn Glu Leu Gly His Ile Ser Val Cys Pro Asp Pro Asp 50 55 60 Val Pro Glu Ala Arg Val Asp Leu Ala Gln Leu Val Lys Thr Arg Glu 65 70 75 80 Ala Gln Gly Gln Arg Leu Pro Ala Leu Phe Cys Phe Pro Gln Ile Leu 85 90 95 Gln His Arg Leu Arg Ser Ile Asn Ala Ala Phe Lys Arg Ala Arg Glu 100 105 110 Ser Tyr Gly Tyr Asn Gly Asp Tyr Phe Leu Val Tyr Pro Ile Lys Val 115 120 125 Asn Gln His Arg Arg Val Ile Glu Ser Leu Ile His Ser Gly Glu Pro 130 135 140 Leu Gly Leu Glu Ala Gly Ser Lys Ala Glu Leu Met Ala Val Leu Ala 145 150 155 160 His Ala Gly Met Thr Arg Ser Val Ile Val Cys Asn Gly Tyr Lys Asp 165 170 175 Arg Glu Tyr Ile Arg Leu Ala Leu Ile Gly Glu Lys Met Gly His Lys 180 185 190 Val Tyr Leu Val Ile Glu Lys Met Ser Glu Ile Ala Ile Val Leu Asp 195 200 205 Glu Ala Glu Arg Leu Asn Val Val Pro Arg Leu Gly Val Arg Ala Arg 210 215 220 Leu Ala Ser Gln Gly Ser Gly Lys Trp Gln Ser Ser Gly Gly Glu Lys 225 230 235 240 Ser Lys Phe Gly Leu Ala Ala Thr Gln Val Leu Gln Leu Val Glu Thr 245 250 255 Leu Arg Glu Ala Gly Arg Leu Asp Ser Leu Gln Leu Leu His Phe His 260 265 270 Leu Gly Ser Gln Met Ala Asn Ile Arg Asp Ile Ala Thr Gly Val Arg 275 280 285 Glu Ser Ala Arg Phe Tyr Val Glu Leu His Lys Leu Gly Val Asn Ile 290 295 300 Gln Cys Phe Asp Val Gly Gly Gly Leu Gly Val Asp Tyr Glu Gly Thr 305 310 315 320 Arg Ser Gln Ser Asp Cys Ser Val Asn Tyr Gly Leu Asn Glu Tyr Ala 325 330 335 Asn Asn Ile Ile Trp Ala Ile Gly Asp Ala Cys Glu Glu Asn Gly Leu 340 345 350 Pro His Pro Thr Val Ile Thr Glu Ser Gly Arg Ala Val Thr Ala His 355 360 365 His Thr Val Leu Val Ser Asn Ile Ile Gly Val Glu Arg Asn Glu Tyr 370 375 380 Thr Val Pro Thr Ala Pro Ala Glu Asp Ala Pro Arg Ala Leu Gln Ser 385 390 395 400 Met Trp Glu Thr Trp Gln Glu Met His Glu Pro Gly Thr Arg Arg Ser 405 410 415 Leu Arg Glu Trp Leu His Asp Ser Gln Met Asp Leu His Asp Ile His 420 425 430 Ile Gly Tyr Ser Ser Gly Ile Phe Ser Leu Gln Glu Arg Ala Trp Ala 435 440 445 Glu Gln Leu Tyr Leu Ser Met Cys His Glu Val Gln Lys Gln Leu Asp 450 455 460 Pro Gln Asn Arg Ala His Arg Pro Ile Ile Asp Glu Leu Gln Glu Arg 465 470 475 480 Met Ala Asp Lys Met Tyr Val Asn Phe Ser Leu Phe Gln Ser Met Pro 485 490 495 Asp Ala Trp Gly Ile Asp Gln Leu Phe Pro Val Leu Pro Leu Glu Gly 500 505 510 Leu Asp Gln Val Pro Glu Arg Arg Ala Val Leu Leu Asp Ile Thr Cys 515 520 525 Asp Ser Asp Gly Ala Ile Asp His Tyr Ile Asp Gly Asp Gly Ile Ala 530 535 540 Thr Thr Met Pro Met Pro Glu Tyr Asp Pro Glu Asn Pro Pro Met Leu 545 550 555 560 Gly Phe Phe Met Val Gly Ala Tyr Gln Glu Ile Leu Gly Asn Met His 565 570 575 Asn Leu Phe Gly Asp Thr Glu Ala Val Asp Val Phe Val Phe Pro Asp 580 585 590 Gly Ser Val Glu Val Glu Leu Ser Asp Glu Gly Asp Thr Val Ala Asp 595 600 605 Met Leu Gln Tyr Val Gln Leu Asp Pro Lys Thr Leu Leu Thr Gln Phe 610 615 620 Arg Asp Gln Val Lys Lys Thr Asp Leu Asp Ala Glu Leu Gln Gln Gln 625 630 635 640 Phe Leu Glu Glu Phe Glu Ala Gly Leu Tyr Gly Tyr Thr Tyr Leu Glu 645 650 655 Asp Glu <210> 2 <211> 755 <212> PRT <213> Artificial sequence <400> 2 Met Lys Val Leu Ile Val Glu Ser Glu Phe Leu His Gln Asp Thr Trp 1 5 10 15 Val Gly Asn Ala Val Glu Arg Leu Ala Asp Ala Leu Ser Gln Gln Asn 20 25 30 Val Thr Val Ile Lys Ser Thr Ser Phe Asp Asp Gly Phe Ala Ile Leu 35 40 45 Ser Ser Asn Glu Ala Ile Asp Cys Leu Met Phe Ser Tyr Gln Met Glu 50 55 60 His Pro Asp Glu His Gln Asn Val Arg Gln Leu Ile Gly Lys Leu His 65 70 75 80 Glu Arg Gln Gln Asn Val Pro Val Phe Leu Leu Gly Asp Arg Glu Lys 85 90 95 Ala Leu Ala Ala Met Asp Arg Asp Leu Leu Glu Leu Val Asp Glu Phe 100 105 110 Ala Trp Ile Leu Glu Asp Thr Ala Asp Phe Ile Ala Gly Arg Ala Val 115 120 125 Ala Ala Met Thr Arg Tyr Arg Gln Gln Leu Leu Pro Pro Leu Phe Ser 130 135 140 Ala Leu Met Lys Tyr Ser Asp Ile His Glu Tyr Ser Trp Ala Ala Pro 145 150 155 160 Gly His Gln Gly Gly Val Gly Phe Thr Lys Thr Pro Ala Gly Arg Phe 165 170 175 Tyr His Asp Tyr Tyr Gly Glu Asn Leu Phe Arg Thr Asp Met Gly Ile 180 185 190 Glu Arg Thr Ser Leu Gly Ser Leu Leu Asp His Thr Gly Ala Phe Gly 195 200 205 Glu Ser Glu Lys Tyr Ala Ala Arg Val Phe Gly Ala Asp Arg Ser Trp 210 215 220 Ser Val Val Val Gly Thr Ser Gly Ser Asn Arg Thr Ile Met Gln Ala 225 230 235 240 Cys Met Thr Asp Asn Asp Val Val Val Val Asp Arg Asn Cys His Lys 245 250 255 Ser Ile Glu Gln Gly Leu Met Leu Thr Gly Ala Lys Pro Val Tyr Met 260 265 270 Val Pro Ser Arg Asn Arg Tyr Gly Ile Ile Gly Pro Ile Tyr Pro Gln 275 280 285 Glu Met Gln Pro Glu Thr Leu Gln Lys Lys Ile Ser Glu Ser Pro Leu 290 295 300 Thr Lys Asp Lys Ala Gly Gln Lys Pro Ser Tyr Cys Val Val Thr Asn 305 310 315 320 Cys Thr Tyr Asp Gly Val Cys Tyr Asn Ala Lys Glu Ala Gln Asp Leu 325 330 335 Leu Glu Lys Thr Ser Asp Arg Leu His Phe Asp Glu Ala Trp Tyr Gly 340 345 350 Tyr Ala Arg Phe Asn Pro Ile Tyr Ala Asp His Tyr Ala Met Arg Gly 355 360 365 Glu Pro Gly Asp His Asn Gly Pro Thr Val Phe Ala Thr His Ser Thr 370 375 380 His Lys Leu Leu Asn Ala Leu Ser Gln Ala Ser Tyr Ile His Val Arg 385 390 395 400 Glu Gly Arg Gly Ala Ile Asn Phe Ser Arg Phe Asn Gln Ala Tyr Met 405 410 415 Met His Ala Thr Thr Ser Pro Leu Tyr Ala Ile Cys Ala Ser Asn Asp 420 425 430 Val Ala Val Ser Met Met Asp Gly Asn Ser Gly Leu Ser Leu Thr Gln 435 440 445 Glu Val Ile Asp Glu Ala Val Asp Phe Arg Gln Ala Met Ala Arg Leu 450 455 460 Tyr Lys Glu Phe Thr Ala Asp Gly Ser Trp Phe Phe Lys Pro Trp Asn 465 470 475 480 Lys Glu Val Val Thr Asp Pro Gln Thr Gly Lys Thr Tyr Asp Phe Ala 485 490 495 Asp Ala Pro Thr Lys Leu Leu Thr Thr Val Gln Asp Cys Trp Val Met 500 505 510 His Pro Gly Glu Ser Trp His Gly Phe Lys Asp Ile Pro Asp Asn Trp 515 520 525 Ser Met Leu Asp Pro Ile Lys Val Ser Ile Leu Ala Pro Gly Met Gly 530 535 540 Glu Asp Gly Glu Leu Glu Glu Thr Gly Val Pro Ala Ala Leu Val Thr 545 550 555 560 Ala Trp Leu Gly Arg His Gly Ile Val Pro Thr Arg Thr Thr Asp Phe 565 570 575 Gln Ile Met Phe Leu Phe Ser Met Gly Val Thr Arg Gly Lys Trp Gly 580 585 590 Thr Leu Val Asn Thr Leu Cys Ser Phe Lys Arg His Tyr Asp Ala Asn 595 600 605 Thr Pro Leu Ala Gln Val Met Pro Glu Leu Val Glu Gln Tyr Pro Asp 610 615 620 Thr Tyr Ala Asn Met Gly Ile His Asp Leu Gly Asp Thr Met Phe Ala 625 630 635 640 Trp Leu Lys Glu Asn Asn Pro Gly Ala Arg Leu Asn Ala Ala Tyr Ser 645 650 655 Gly Leu Pro Val Ala Glu Val Thr Pro Arg Glu Ala Tyr Asn Ala Ile 660 665 670 Val Asp Asn Asn Val Glu Leu Val Ser Ile Glu Asn Leu Pro Gly Arg 675 680 685 Ile Ala Ala Asn Ser Val Ile Pro Tyr Pro Pro Gly Ile Pro Met Leu 690 695 700 Leu Ser Gly Glu Asn Phe Gly Asp Lys Asn Ser Pro Gln Val Ser Tyr 705 710 715 720 Leu Arg Ser Leu Gln Ser Trp Asp His His Phe Pro Gly Phe Glu His 725 730 735 Glu Thr Glu Gly Thr Glu Ile Ile Asp Gly Ile Tyr His Val Met Cys 740 745 750 Val Lys Ala 755 <210> 3 <211> 306 <212> PRT <213> Artificial sequence <400> 3 Met Ser Thr Leu Gly His Gln Tyr Asp Asn Ser Leu Val Ser Asn Ala 1 5 10 15 Phe Gly Phe Leu Arg Leu Pro Met Asn Phe Gln Pro Tyr Asp Ser Asp 20 25 30 Ser Asp Trp Val Ile Thr Gly Val Pro Phe Asp Met Ala Thr Ser Gly 35 40 45 Arg Ala Gly Gly Arg His Gly Pro Ala Ala Ile Arg Gln Val Ser Thr 50 55 60 Asn Leu Ala Trp Glu His Asn Arg Phe Pro Trp Asn Phe Asp Met Arg 65 70 75 80 Glu Arg Leu Asn Val Val Asp Cys Gly Asp Leu Val Tyr Ala Phe Gly 85 90 95 Asp Ala Arg Glu Met Ser Glu Lys Leu Gln Ala His Ala Glu Lys Leu 100 105 110 Leu Ala Ala Gly Lys Arg Met Leu Ser Phe Gly Gly Asp His Phe Val 115 120 125 Thr Leu Pro Leu Leu Arg Ala His Ala Lys His Phe Gly Lys Met Ala 130 135 140 Leu Val His Phe Asp Ala His Thr Asp Thr Tyr Ala Asn Gly Cys Glu 145 150 155 160 Phe Asp His Gly Thr Met Phe Tyr Thr Ala Pro Lys Glu Gly Leu Ile 165 170 175 Asp Pro Asn His Ser Val Gln Ile Gly Ile Arg Thr Glu Phe Asp Lys 180 185 190 Asp Asn Gly Phe Thr Val Leu Asp Ala Cys Gln Val Asn Asp Arg Ser 195 200 205 Val Asp Asp Val Ile Ala Gln Val Lys Gln Ile Val Gly Asp Met Pro 210 215 220 Val Tyr Leu Thr Phe Asp Ile Asp Cys Leu Asp Pro Ala Phe Ala Pro 225 230 235 240 Gly Thr Gly Thr Pro Val Ile Gly Gly Leu Thr Ser Asp Arg Ala Ile 245 250 255 Lys Leu Val Arg Gly Leu Lys Asp Leu Asn Ile Val Gly Met Asp Val 260 265 270 Val Glu Val Ala Pro Ala Tyr Asp Gln Ser Glu Ile Thr Ala Leu Ala 275 280 285 Ala Ala Thr Leu Ala Leu Glu Met Leu Tyr Ile Gln Ala Ala Lily Lily 290 295 300 Gly Glu 305

Claims

1. A microbial preparation, characterized in that, The microbial preparation consists of microbial preparation A and microbial preparation B, wherein microbial preparation A is Corynebacterium obliterans (…). Corynebacterium crenatum The microbial preparation B is recombinant *Escherichia coli* JN-1; the *Corynebacterium tumefaciens* JN-1 has the accession number CGMCC NO. 0890; the recombinant *E. coli* overexpresses arginine decarboxylase and guanidinobutamine enzyme, the amino acid sequence of the arginine decarboxylase is shown in SEQ ID NO. 1 or SEQ ID NO. 2, the amino acid sequence of the guanidinobutamine enzyme is shown in SEQ ID NO. 3, and the expression vector of the recombinant *E. coli* is plasmid pETDuet-1. E. coli JM109 is the host cell.

2. The use of the microbial preparation of claim 1 in the preparation of products containing butanediamine.

3. A method for producing butanediamine by fermentation, characterized in that, The method involves first inoculating the seed culture of microbial preparation A as described in claim 1 into a co-fermentation medium, fermenting and culturing it, and then inoculating it into a co-fermentation medium containing the seed culture of microbial preparation B as described in claim 1, and continuing fermentation and culturing to produce butanediamine.

4. The method according to claim 3, characterized in that, The specific steps of the method are as follows: (1) The microbial preparation A according to claim 1 is inoculated into seed culture medium A and fermented to obtain seed liquid A; (2) The microbial preparation B according to claim 1 is inoculated into seed culture medium B and fermented for 10-30 h to obtain seed liquid B; (3) Inoculate the seed liquid A obtained in step (1) into the co-fermentation medium at an inoculation amount of 4-10% by volume, and ferment for 72-120 h to obtain fermentation liquid A; (4) Inoculate the fermentation broth A from step (3) into a co-fermentation medium containing seed liquid B, and ferment for 2 to 5 days to obtain a fermentation broth containing butanediamine.

5. The method according to claim 4, characterized in that, In step (4), seed liquid B is inoculated into the co-fermentation medium at a volume ratio of 1.0 to 1.5%, and fermentation liquid A is inoculated into the co-fermentation medium at a volume ratio of 2 to 3%.

6. The method according to claim 3 or 4, characterized in that, The co-fermentation medium consists of the following components (g / L): ammonium sulfate 25-35, sucrose 55-65, MgSO4·7H2O 3-7, KH2PO4 25-35, MnSO4·H2O 0.02, FeSO4·7H2O 0.02, VB1 0.00045, biotin 0.00005, calcium carbonate 25-35, yeast extract 3-7, peptone 15-25, NaCl 0.5, MgCl2 0.95, KCl 0.186, with the remainder being water.

7. The use of the method according to any one of claims 3 to 6 in the preparation of products containing butanediamine.

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