Method for producing pentamethylene diamine through liquid-solid two-phase fermentation

Abstract

The invention relates to a method for producing pentamethylene diamine through liquid-solid two-phase fermentation. The method comprises the following steps: inoculating a seed culture medium with an activated recombinant escherichia coli strain, and culturing to obtain a seed solution; according to the recombinant escherichia coli, pET28a (+) is taken as a carrier, and a lysine decarboxylase gene CadA is subjected to heterologous expression; inoculating the obtained seed solution into a fermentation conical flask containing a solid culture medium for fermentation culture and induction, and centrifuging and resuspending the obtained bacterial solution to obtain a bacterial suspension; and mixing the obtained bacterial suspension with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, carrying out a catalytic reaction, and centrifuging to obtain 1, 5-pentanediamine. Compared with common fermentation, the method has the advantages that the growth density of E.coli is obviously improved by 2.41 times, and the yield of pentamethylene diamine reaches 524.71 mM in the catalysis process. The two-phase fermentation system is beneficial to improving the growth density and the production efficiency of E.coli by providing a more suitable growth environment and improving oxygen transfer, so that the accurate control on the expression process is realized.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, in particular to a method for producing pentamethylenediamine by liquid-solid biphase fermentation. Background Art

[0002] Lysine decarboxylase (LDC, EC 4.1.1.18) can catalyze L-lysine to produce pentamethylenediamine, a process that requires the participation of the coenzyme factor pyridoxal phosphate (PLP). Pentamethylenediamine is a five-carbon diamine that can undergo polycondensation with dicarboxylic acids to produce polyamide, and the global annual market volume of polyamide can reach 3.5 million tons.

[0003] L-lysine can be catalyzed by lysine decarboxylase to produce pentamethylenediamine, releasing carbon dioxide. The enzyme is specific to L-lysine and has no catalytic activity for other L-amino acids and L-lysine derivatives, D-amino acids and other amines. It is widely distributed. Currently, the two L-lysine decarboxylases derived from Escherichia coli are mainly studied, namely the inducible type (CadA) and the constitutive type (IdcC). Compared with decarboxylases from other sources, they have the best catalytic effect because they use L-lysine as a substrate and have high activity. The above two lysine decarboxylases are effective biocatalysts for producing pentamethylenediamine using industrial-grade L-lysine. The inducible CadA has been widely used in the process of whole-cell biocatalysis and microbial fermentation to produce pentamethylenediamine.

[0004] However, the traditional lysine decarboxylase production method has problems such as low yield, low enzyme activity, and poor stability. Liquid-solid biphasic fermentation technology can significantly improve the growth rate and metabolic activity of bacteria, thereby increasing the expression level of exogenous genes, increasing the number of effective viable bacteria and enriching products at the same time, providing a reference for establishing a large-scale fermentation process. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a method for producing pentamethylenediamine by liquid-solid biphasic fermentation. In the E. coli expression system, the expression of exogenous genes is affected by many factors, among which the optimization of fermentation conditions is a key regulatory means. Therefore, the present invention adopts a biphasic fermentation system to provide E. coli with a more stable attachment growth surface, which is more conducive to the high-density growth of E. coli, thereby achieving precise control of the target protein.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a method for producing 1,5-pentanediamine by liquid-solid biphase fermentation, comprising the following steps:

[0008] S1, inoculating the activated recombinant Escherichia coli strain into a seed culture medium for cultivation to obtain a seed solution; the seed culture medium is an LB liquid culture medium containing kanamycin; the recombinant Escherichia coli uses pET28a(+) as a vector to heterologously express the lysine decarboxylase gene CadA;

[0009] S2, inoculating the seed solution obtained in step S1 into a fermentation conical flask containing a solid culture medium for fermentation and induction, and centrifuging and resuspending the obtained bacterial solution to obtain a bacterial suspension;

[0010] S3, mixing the bacterial suspension obtained in step S2 with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, performing a catalytic reaction, and centrifuging to obtain 1,5-pentanediamine. The buffer solution is a PBS solution (10×, pH=5.6)

[0011] In one embodiment of the present invention, in step S1, the lysine decarboxylase gene CadA is derived from E. coli MG1655; the nucleotide sequence of the lysine decarboxylase is shown as SEQ ID NO 1.

[0012] In one embodiment of the present invention, in step S1, the recombinant Escherichia coli uses E. coli BL21 (DE3) as an expression host.

[0013] In one embodiment of the present invention, in step S2, the inoculation amount of the seed solution is 2%-4%.

[0014] In one embodiment of the present invention, in step S2, the components of the solid culture medium are 15g / L-18g / L agar, 10g / L-15g / L beef powder, 10g / L-15g / L peptone, 5g / L-6g / L sodium chloride and 2g / L-3g / L dipotassium hydrogen phosphate; and the pH value is 7.4-7.6.

[0015] Preferably, the solid culture medium comprises 18 g / L agar, 10 g / L beef powder, 10 g / L peptone, 5 g / L sodium chloride and 2 g / L dipotassium hydrogen phosphate.

[0016] In one embodiment of the present invention, in step S2, the fermentation conical flask is a three-thorn conical flask or a baffle conical flask.

[0017] In one embodiment of the present invention, in step S2, the fermentation culture conditions are: 34°C-37°C, 200-220rpm, 20h-22h.

[0018] In one embodiment of the present invention, in step S3, the amount of the bacterial suspension added is 2 gDCW / L-3 gDCW / L.

[0019] The method according to claim 1, characterized in that in step S3, the molar concentration of lysine hydrochloride is 500mM-1000mM;

[0020] And / or, the molar concentration of the pyridoxal phosphate is 0.1 mM-1 mM.

[0021] In one embodiment of the present invention, in step S3, the catalytic reaction conditions are: 34° C.-37° C., 200 rpm-220 rpm for 6 h-10 h.

[0022] In one embodiment of the present invention, in step S3, the centrifugation condition is: 5000 rpm-8000 rpm for 10 min-15 min.

[0023] The above technical solution of the present invention has the following advantages compared with the prior art:

[0024] The present invention provides a method for producing pentamethylenediamine by liquid-solid biphasic fermentation. The present invention constructs a recombinant strain that overexpresses lysine decarboxylase and optimizes the fermentation process. The present invention adopts a biphasic fermentation system, introduces a solid culture medium below the liquid culture medium, optimizes the composition of the biphasic fermentation culture medium and selects a suitable expression container, which can significantly improve the growth rate and metabolic activity of bacteria, thereby improving the expression level of exogenous genes. The group using biphasic fermentation can significantly increase the E. coli growth density by 2.41 times compared to the group using ordinary fermentation. At the same time, in the subsequent whole-cell catalytic process, the group using biphasic fermentation can achieve a pentamethylenediamine production of 524.71mM, which is significantly higher than other groups, including conventional ordinary fermentation. The biphasic fermentation system helps to improve the growth density and production efficiency of E. coli by providing a more suitable growth environment and improving oxygen transfer, thereby achieving precise control of the expression process. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 is the OD at different times in different groups in the embodiment of the present invention 600 ;

[0027] Figure 2 It is the yield of pentamethylenediamine under different groups in the examples of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0029] A method for optimizing the conditions for preparing lysine decarboxylase by dual-phase fermentation, the specific steps of the method are as follows:

[0030] The present invention first solves the problem of constructing a recombinant Escherichia coli strain expressing lysine decarboxylase. The lysine decarboxylase gene CadA is copied from Escherichia coli and is heterologously expressed using pET28a(+) as a vector.

[0031] Furthermore, the Escherichia coli is E. coli BL21 (DE3), and the gene encoding lysine decarboxylase CadA comes from the genome of E. coli MG1655 (GenBank: NC_000913.3).

[0032] Secondly, the present invention optimizes the fermentation conditions of the recombinant bacteria, specifically considering the selection of the conical flask, the fermentation method and the specific composition of the bottom solid culture medium for optimization and determination.

[0033] The optimization of fermentation method includes the following steps:

[0034] (1) Cultivation of the recombinant bacterial seed solution: a single clone of the recombinant bacteria was inoculated into a 2 mL LB shaking tube containing kanamycin medium and cultured in a shaking incubator at 37° C. with a shaking speed of 220 rpm for overnight culture.

[0035] (2) Two-phase fermentation: The primary seed solution cultured in step (1) was inoculated into a fermentation conical flask at an inoculation amount of 4% and cultured at 34° C. for 10 h.

[0036] (3) The fermentation in step (2) was continued for 10 h, and IPTG inducer was added. The fermentation was terminated by culturing at 220 rpm and 37°C for 12 h.

[0037] (4) Ordinary liquid fermentation method: The primary seed liquid cultured in step (2) is inoculated into a liquid fermentation medium, and the conical flask is placed in a shaking incubator at 34° C. and 220 rpm for culture.

[0038] (5) After culturing for 10 h according to step (4), IPTG inducer was added for induction for 12 h, and the culture conditions were adjusted to 37°C and 220 rpm.

[0039] The choice of conical flask style mainly included ordinary conical flask A1 and three-thorn conical flask A2.

[0040] The bottom solid culture medium of the two-phase fermentation was optimized. The specific culture medium components are as follows:

[0041] A3 nutrient agar (Nutrient agarmedium, Wokai, 69002765): Weigh 33 g of this product, add it to 1 L of distilled water, heat and boil to dissolve, and sterilize it by high pressure at 115°C for 30 min. The specific ingredients are shown in Table 1.

[0042] A4 broth agar medium (Broth agar medium, Wokai, 69026066): Weigh 45g of this product, add it to 1L of distilled water, heat and boil to dissolve, and sterilize it by high pressure at 121℃ for 30min. The specific ingredients are shown in Table 1.

[0043] Table 1 Culture medium involved in the experiment

[0044]

[0045] During the fermentation process, samples were taken at 3 h, 6 h, 10 h, and 22 h to determine the bacterial concentration, and the pentamethylenediamine production was determined at 22 h.

[0046] The bacterial cell concentration and pentamethylenediamine production under different conditions were analyzed and compared to obtain the optimized fermentation conditions for preparing lysine decarboxylase by liquid-solid biphasic fermentation.

[0047] Materials and Methods

[0048] LB medium: peptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water to 1000 mL, autoclave at 121 °C for 20 min.

[0049] Fermentation medium: peptone 12 g, yeast extract 24 g, glycerol 5 g, K2HPO4·3H2O 16.4 g, KH2PO4 2.31 g, distilled water to 1000 mL, autoclave at 121°C for 20 min.

[0050] The specific fermentation conditions optimization groups involved in this experiment are shown in Table 2.

[0051] Table 2 Groups under different conditions

[0052]

[0053] DEEMM derivatization of 1,5-pentanediamine: The solution after whole cell catalysis was centrifuged at 8000 r / min, the supernatant was diluted, and then derivatized with DEEMM. After adding the sample according to the DEEMM pentamethylenediamine derivatization system shown in Table 3, the derivatization reaction was carried out at 70°C for 2 hours in a water bath. The reaction solution was filtered through a filter membrane, and the filtrate was subjected to liquid chromatography analysis.

[0054] Table 3 DEEMM pentamethylenediamine derivatization system

[0055]

[0056] Detection method of 1,5-pentanediamine: HPLC was used to qualitatively and quantitatively analyze 1,5-pentanediamine in the sample. Liquid chromatography separation was performed on a C18 column and maintained at 35°C. Mobile phase A was 100% acetonitrile, B was 25 mM sodium acetate buffer (pH = 4.8), flow rate was 1 mL / min, and detection wave was performed at 284 nm. Gradient program: 2 min, 25% A, 30 min, 60% A, 31 min, 20% A, 40 min, 20% A.

[0057] Example 1: Construction of lysine decarboxylase recombinant vector

[0058] Acquisition of lysine decarboxylase CadA gene: Using the E. coli MG1655 (bio-81704, China Culture Collection Center) genome (GenBank: NC_000913.3) as a template, primers k12-CadA-F (5'-CTTTAAGAAGGAGATATACCATGAACGTTATTGCAATATTGAATCA-3', SEQ ID NO 2) and k12-CadA-R (5'-CAGTGGTGGTGGTGGTGGTGTTTT TTGCTTTCTTCTTTCAATACCT-3', SEQ ID NO 3) were used to amplify the lysine decarboxylase gene CadA. Using plasmid pET28a (+) as a template, primers pET28a-F (5'-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3', SEQ ID NO 4) and pET28a-R (5'-CACCA CCACCACCACCACTG-3', SEQID NO 5) were used to linearize the plasmid and introduce homology arms. The two PCR products were homologously recombined and connected using the ClonExpressⅡOne Step Cloning Kit (Novozyme C113) to construct plasmid pET28a-CadA. After sequencing verification, the plasmid was transformed into E. coli BL21 (DE3) to obtain BL21-CadA, which was stored in a -80°C refrigerator.

[0059] Example 2: Optimization of external fermentation conditions

[0060] Activate BL21-CadA stored in a -80°C refrigerator by streaking on a plate, pick a suitable single colony and inoculate it into LB liquid medium containing kanamycin for overnight culture for 12 hours at 220rpm. Under sterile operation, inoculate the fermentation conical flask with an inoculation amount of 4% of the overnight culture seed liquid. The fermentation conical flask conditions refer to groups A1, A2, A3 and A4. Place the conical flasks of groups A1, A2, A3 and A4 in a shaker at 34°C and 220rpm for culture. After 10 hours of culture, add 0.5mM IPTG to induce for 12 hours, and the culture conditions are adjusted to 37°C and 220rpm. Measure OD at 3h, 6h, 10h and 22h respectively. 600 .

[0061] Example 3: Whole-cell catalytic synthesis of 1,5-pentanediamine

[0062] The fermented cells in Example 2 were collected, centrifuged at 5000 rpm for 10 min, the supernatant was removed, the cells were resuspended in PBS buffer, 800 mM L-lysine hydrochloride and 0.1 mM pyridoxal phosphate solution were added, and the reaction was catalyzed at 37°C and 220 rpm for 6 h. The reaction was terminated by boiling at 100°C for 5 min. After the reaction was completed, the cells were centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was diluted and the content was determined.

[0063] Optimizing external fermentation conditions is a key step to improve microbial production efficiency. The present invention carefully adjusts the selection of conical flasks, whether to use a two-phase fermentation system, and the specific composition of the bottom solid culture medium. According to the growth curve, pentamethylenediamine production ( Figure 1 and Figure 2 ) results are shown.

[0064] By comparing groups A1 and A2, it was found that the growth enhancement effect of microorganisms in the early stage of fermentation was not obvious, but in the late stage of fermentation and after the use of inducers, the three-thorn conical flask had a significant promoting effect on the growth of E. coli compared with the ordinary conical flask. The three-thorn conical flask can provide favorable conditions for the growth of aerobic microorganisms such as E. coli by improving the dissolved oxygen efficiency and improving the cell suspension state, further improving the growth rate and accumulation of microorganisms.

[0065] As the culture time increased, the OD 600 All of them showed different degrees of growth. The growth density of E. coli using two-phase fermentation was significantly better than that of ordinary liquid fermentation without bottom solid culture medium, indicating that two-phase fermentation has a significant promoting effect on the growth of E. coli. When the culture time was 22h, the growth density of E. coli in group A4 using two-phase fermentation was significantly increased by 2.41 times compared with A1 using ordinary fermentation.

[0066] By comparing the cell density in A3 and A4 culture media, it was found that the bottom solid culture medium components of the dual-phase fermentation also affect the growth of E. coli. The broth agar culture medium has the best effect on promoting the growth density of E. coli. When the culture time is 22h, the cell density of A4 is significantly increased by 1.5 times compared with group A3. In the subsequent whole-cell catalytic process, the yield of pentamethylenediamine in A4 reached the highest level compared with other groups, which was 524.71mM.

[0067] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for producing 1,5-pentanediamine by liquid-solid biphase fermentation, characterized in that: The following steps are involved: S1. Inoculate the activated recombinant Escherichia coli strain into a seed culture medium for cultivation to obtain a seed solution; the recombinant Escherichia coli uses pET28a(+) as a vector to heterologously express the lysine decarboxylase gene CadA; S2, inoculating the seed solution obtained in step S1 into a fermentation conical flask containing a solid culture medium for fermentation and induction, and centrifuging and resuspending the obtained bacterial solution to obtain a bacterial suspension; S3, mixing the bacterial suspension obtained in step S2 with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, performing a catalytic reaction, and centrifuging to obtain 1,5-pentanediamine.

2. The method according to claim 1, characterized in that In step S1, the lysine decarboxylase gene CadA is derived from E. coli MG1655; the nucleotide sequence of the lysine decarboxylase is shown in SEQ ID NO 1.

3. The method according to claim 1, characterized in that In step S1, the recombinant Escherichia coli uses E. coli BL21 (DE3) as the expression host.

4. The method according to claim 1, characterized in that: In step S2, the inoculation amount of the seed solution is 2%-4%.

5. The method according to claim 1, characterized in that In step S2, the components of the solid culture medium are 15g / L-18g / L agar, 10g / L-15g / L beef powder, 10g / L-15g / L peptone, 5g / L-6g / L sodium chloride and 2g / L-3g / L dipotassium hydrogen phosphate; and the pH value is 7.4-7.

6.

6. The method according to claim 1, characterized in that In step S2, the fermentation conical flask is a three-thorn conical flask or a baffle conical flask.

7. The method according to claim 1, characterized in that In step S2, the fermentation culture conditions are: 34°C-37°C, 200rpm-220rpm, 20h-22h.

8. The method according to claim 1, characterized in that: In step S3, the amount of the bacterial suspension added is 2 gDCW / L-3 gDCW / L.

9. The method according to claim 1, characterized in that: In step S3, the molar concentration of lysine hydrochloride is 500mM-1000mM; And / or, the molar concentration of the pyridoxal phosphate is 0.1 mM-1 mM.

10. The method according to claim 1, characterized in that In step S3, the catalytic reaction conditions are: 34°C-37°C, 200rpm-220rpm for 6h-10h.

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