Method for fermentation-production of pentanediamine comprising carbon dioxide stripping technique

Abstract

A method for fermentation-production of a pentanediamine, comprising: culturing a cell expressing a lysine decarboxylase to obtain a whole cell fermentation broth comprising a pentanediamine; and extracting the pentanediamine from the whole cell fermentation broth, and striping the whole cell fermentation broth of carbon dioxide contained therein before adding a strong base. The method greatly increases a production volume of the pentanediamine.

Claims

1. A method for preparing 1,5-pentanediamine by fermentation, consisting of the steps of: (1) culturing a cell overexpressing a lysine decarboxylase and adding the substrates lysine and pyridoxal phosphate to start whole-cell catalysis to obtain a fermentation broth comprising 1,5-pentanediamine, wherein the cell is a cell whose expression is enhanced by replacing the promoter of the lysine decarboxylase gene with T7 promoter, and the cell is E. coli BL21(DE3), and no acidic pH regulator is added to the fermentation broth, wherein the cells themselves generate CO.sub.2, to neutralize 1,5-pentanediamine; (2) removing the cells in the fermentation broth to obtain a clear liquid of fermentation broth; (3) removing carbon dioxide from the liquid obtained in step (2) by distillation under reduced pressure at about 0.095 MPa and at a temperature from 60 C. to 70 C., and then adding a base to obtain a treatment liquid; and (4) extracting a composition containing 1,5-pentanediamine from the treatment liquid obtained in the step (3) by distillation under the reduced pressure.

2. The method according to claim 1, wherein in step (2), the removing is centrifuging or filtering.

3. The method according to claim 1, wherein in step (3), the base includes sodium hydroxide, potassium hydroxide and/or calcium hydroxide.

4. The method according to claim 1, wherein the prepared 1,5-pentanediamine is greater than 30 g/L of the fermentation broth.

5. The method according to claim 4, wherein the prepared 1,5-pentanediamine is greater than 50 g/L of the fermentation broth.

6. The method according to claim 5, wherein the prepared 1,5-pentanediamine is greater than 80 g/L of the fermentation broth.

7. The method according to claim 6, wherein the prepared 1,5-pentanediamine is greater than 100 g/L of the fermentation broth.

8. The method according to claim 7, wherein the prepared 1,5-pentanediamine is greater than 120 g/L of the fermentation broth.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is plasmid profile of pET28a-cadA.

(2) FIG. 2 is the process curve of whole-cell catalysis for the production of pentanediamine.

(3) FIG. 3 is the change curve of pH value during the process of recycling carbon dioxide by alkaline solution.

(4) FIG. 4 is the process curve of the removal of carbon dioxide.

DETAILED DESCRIPTION OF EMBODIMENT

(5) The experimental methods used in the following embodiments are conventional methods unless with special description. Materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

Embodiment 1 Detection of Lysine and Pentanediamine by HPLC

(6) 1 mL whole-cell catalytic solution is centrifuged at 8000 g for 5 min, and the supernatant is collected to detect lysine content; 10 L supernatant is added into 2 mL centrifugal tube, and 200 L 0.5 mol/L NaHCO.sub.3 aqueous solution and 100 L 1% (volume ratio) dinitrofluorobenzene acetonitrile solution were added, and heated for 60 min in the dark area of the water bath at 6 C., then cooled to room temperature. 700 L 0.04 mol/L KH.sub.2PO.sub.4 aqueous solution (pH=7.20.05, pH value is adjusted with 40 g/L KOH aqueous solution) was added to dilute, and then shake well. The sample can be injected after keeping for 15 min and filtration, and the injection volume is 15 L;

(7) The chromatographic column used is C18 column (ZORBAX Eclipse XDB-C18, 4.6*150 mm, Agilent, USA); column temperature: 40 C.; ultraviolet detection wavelength: 360 nm; mobile phase A is 0.04 mol/L KH.sub.2PO.sub.4 (pH=7.2+0.05, adjusted by 40 g/100 mL KOH solution), mobile phase B is 55% (volume ratio) acetonitrile aqueous solution, the total flow rate of mobile phase is 1 mL/min. The elution process is as follows:

(8) At the beginning of elution (0 min), the volume fraction of mobile phase A in the total flow of the mobile phase is 86%, and volume fraction of mobile phase B in the total flow of the mobile phase is 14%; The elution process is divided into five stages, the volume fraction of mobile phase A and mobile phase D in the total flow of the mobile phase are linearly changed in each stage; the volume fraction of mobile phase A in the total flow of the mobile phase is 88%, and the volume fraction of mobile phase B in total flow of the mobile phase is 12% at the end of the first stage (conducting for 2 min from the beginning). The volume fraction of mobile phase A in the total flow of the mobile phase is 86%, and the volume fraction of mobile phase B in total flow of the mobile phase is 14% at the end of the second stage (conducting for 2 min from the end of the first stage). The volume fraction of mobile phase A in the total flow of the mobile phase is 70%, and the volume fraction of mobile phase B in total flow of the mobile phase is 30% at the end of the third stage (conducting for 6 min from the end of the second stage). The volume fraction of mobile phase A in the total flow of the mobile phase is 30%, and the volume fraction of mobile phase B in total flow of the mobile phase is 70% at the end of the fourth stage (conducting for 10 min from the end of the third stage). A standard curve is prepared using a commercially available L-lysine standard, then calculate the lysine concentration of the sample.

(9) In the detection of pentanediamine, 10 L sample supernatant is added into a 2 mL centrifugal tube containing 100 L 4.2 g/100 mL sodium bicarbonate aqueous solution. After blending, 200 L acetonitrile solution containing 2,4-dinitrofluorobenzene with volume percentage of 1% is added and mixed. And then keeping the derivatization reaction for 60 min at 60 C. (strictly timing, take out the centrifugal tube for slight oscillation and blending at 30 min, and then continue the derivatization reaction). Taking out the centrifugal tube and cooling to room temperature in the dark. Swirl shaking and mixing for 30 s after adding 1600 L acetonitrile. After filtration with organic filter membrane, the sample can be injected, and the injection volume is 15 L.

(10) The mobile phase A is 5.4 g/L potassium dihydrogen phosphate aqueous solution with pH 7.2 and the mobile phase B is acetonitrile aqueous solution with volume percentage of 80%. A and B are pumped into the mobile phase at a flow rate of 1 mL/min at a volume ratio of 5:95. The chromatographic column used is C18 (ZORBAX Eclipse XDB-C18, 4.6*150 mm, Agilent, USA); column temperature: 35 C.; detection wavelength: 360 nm.

(11) 1,5-pentanediamine hydrochloride (purchased from Sigma company) is used as standard, the concentration of 1,5-pentanediamine hydrochloride has a good linearity between 1-5 g/L. The standard curve is prepared by taking the concentration of the standard as the abscissa and the integral peak area of the standard as the ordinate.

(12) The concentration of 1,5-pentanediamine in the following embodiments is converted from the measured value of 1,5-pentanediamine hydrochloride calculated by the standard curve formula.

Embodiment 2 Construction of Engineered Bacteria for Whole-Cell Catalytic Production of Pentanediamine

(13) (I) Construction of Overexpression Plasmid Comprising Lysine Decarboxylase Gene cadA

(14) The ORF of the optimized lysine decarboxylase gene cadA was inserted after the T7 promoter and RBS of the pET28a(+) expression vector. The primers were designed using the optimized sequence of cadA, and the genomic DNA of the wild-type Escherichia coli K12 W3110 strain was used as a template. The cadA gene was amplified by PCR using the high-fidelity polymerase KAPA HiFi HotStar with P1 and P2 as primers. The nucleotide sequence of this gene is shown in SEQ ID NO. 1. The PCR procedure was performed for a total of 26 cycles, with each cycle including: denaturation at 98 C. for 30 seconds, annealing at 65 C. for 15 seconds, and extension at 72 C. for 150 seconds. A mutation site was introduced by primer P1 to obtain a fragment of the engineered lysine decarboxylase gene cadA*

(15) TABLE-US-00001 P1: (SEQIDNO.5) 5-CATGCCATGGCAGTTATTGCAATATTGAATCATATGGGAGT (Theunderlinedsequenceistherestriction recognitionsiteofNcoI) P2: (SEQIDNO.6) 5-ACGCGTCGACCTCCTTATGAGCAAAAAAGGGAAGTG-3 (Theunderlinedsequenceistherestriction recognitionsiteofSalI)

(16) The electrophoresis band of PCR products was recovered by gel extraction, and the DNA fragment of lysine decarboxylase gene cadA* and pET28a(+) plasmid were digested with restriction endonucleases Nco I and Sal I to obtain lysine decarboxylase gene cadA* gene fragment and large vector fragment after Enzyme digestion. The digested lysine decarboxylase gene cadA* gene fragment was ligated to the large vector fragment and transformed into competent cells of E. coli EC135 (Zhang et al, Plos Genetics, 2012, 8(9): e1002987). The LB plates containing 50 mg/L kanamycin were screened to obtain a transformant containing the recombinant plasmid, and the plasmid of which was extracted and sequenced. The plasmid with correct result was named pET28a-cadA* and shown in FIG. 1.

(17) (II) Replacement of the Promoter of Chromosomal cadB Gene.

(18) The genomic DNA of Escherichia coli BL21 (DE3) strain was used as a template, and PCR amplification was performed with primers P3 and P4, P5 and P6 respectively, and two DNA fragments of 510 bp and 610 bp in length were obtained, respectively, as shown in SEQ ID No. 2 and SEQ ID No. 3, respectively. Among them, the T7 promoter sequence and the lac regulatory sequence were introduced by primers P4 and P5. The PCR was performed as follows: denaturation at 94 C. for 30 s, annealing at 52 C. for 30 s, and extension at 72 C. for 30 s (30 cycles in total). As used herein, the primer sequence is as follows:

(19) TABLE-US-00002 P3: (SEQIDNO.7) 5-CGCGGATCCTGCGCCATTCTCAACATCCTT-3 (Theunderlinedsequenceistherestriction recognitionsiteofBamHI) P4: (SEQIDNO.8) 5-TCCGCTCACAATTCCCCTATAGTGAGTCGTATTATGCCGCAACATATT ATACCAACAG-3 P5: (SEQIDNO.9) 5-ACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCGAAATTAGGAG AAGAGCATGAG-3 P6: (SEQIDNO.10) 5-ATTGCGGCCGCTCCGCAGTATTCCAGTTAGCT-3 (Theunderlinedsequenceistherestriction recognitionsiteofNotI)

(20) A mixture of DNA molecules represented by SEQ ID No. 2 and SEQ ID No. 3 was used as a template, and P3 and P6 were used as primers, thereby an Overlap fragment of about 1.1 kb in length was amplified by Overlap PCR. The sequence of overlap fragment was shown in SEQ ID No. 4. The PCR procedure was performed for a total of 26 cycles, with each cycle including: denaturation at 94 C. for 30 seconds, annealing at 52 C. for 30 seconds, extension at 72 C. for 60 seconds.

(21) The sequence shown in nucleotides 477.sup.th to 495.sup.th from the 5 end in SEQ ID No. 4 is the T7 promoter sequence, and the sequence shown in nucleotides 496.sup.th to 520.sup.th from the 5 end in SEQ ID No. 4 is the lac regulatory sequence.

(22) Bam HI and Not I cleave the DNA molecule of SEQ ID No. 3 to obtain a gene fragment; while Bam HI and Not I cleave the pKOV plasmid to obtain a large fragment of the vector. The gene fragment was ligated to the large vector fragment to obtain a recombinant plasmid named pKOV-PT7-cadB. The recombinant plasmid was sequenced and verified the present of correct T7 promoter and lac regulatory gene sequences, and stored for later use.

(23) The constructed pKOV-PT7-cadB plasmid was electrotransformed into E. coli BL21 (DE3) strain and resuscitated in LB medium for 2 h at 30 C., 150 rpm. Monoclones positive for homologous recombination were selected according to the commercial guidelines for the pKOV plasmid of Addgene. It was confirmed by sequencing that its own promoter of the cadB gene on the chromosome was replaced by the T7 promoter, and the strain was named E. coli BL21 PcadB:: PT7.

(24) (3) Construction of Engineered Bacteria for Pentanediamine

(25) The plasmid pET28a-cadA* is transformed into competent cells of E. coli BL21 P.sub.cadB:: P.sub.T7. The engineered bacteria E. coli BL21 P.sub.cadB:: PT7/pET28a-cadA* is screened on LB plates containing 50 mg/L kanamycin for whole-cell catalysis of 1,5-pentanediamine.

Embodiment 3 Bacterial Culture and Catalytic Production of Pentanediamine

(26) Scraping engineered bacteria E. coli BL21 (DE3) P.sub.cadB:: P.sub.T7/pET28a-cadA* and inoculating into a 500 mL triangular flask containing 50 mL LB medium (containing 50 mg/L kanamycin (it can be 5-200 mg/L)), and culturing in shaker for 4 h at 37 C. and 220 rpm to obtain seed liquid, and OD.sub.600 is 4-5. The obtained seed liquid is inoculated into the 10 L fermentation tank containing 2 L inorganic salt medium at an inoculum concentration of 2%, and the culture temperature is 37 C., DO is more than 30%, and the tank pressure is 0.02-0.10 MPa. The concentration of glucose in the culture solution is maintained below 5 g/L by adding feeding liquid. When the OD.sub.600 of the bacteria in the culture medium is 30-40, adding 0.1 mM inducer IPTG. After 2 h, the OD.sub.600 of the bacteria in the culture medium is about 80, and the wet cells are obtained by centrifugation.

(27) Wherein, the compositions of inorganic salt medium and feeding liquid are as follows: inorganic salt medium: 2 g/L (NH.sub.4).sub.2HPO.sub.4, 4 g/L KH.sub.2PO.sub.4, 0.85 g/L citric acid, 0.7 g/L MgSO.sub.4.7H.sub.2O, 10 mg/L FeSO.sub.4.7H.sub.2O, 2.25 mg/L ZnSO.sub.4.7H.sub.2O, 0.2 mg/L CuSO.sub.4.5H.sub.2O, 0.5 mg/L MnSO.sub.4.5H.sub.2O, 0.23 mg/L NaB.sub.4O.sub.7.10H.sub.2O, 2.0 mg/L CaCl.sub.2.2H.sub.2O, 0.1 mg/L NH.sub.4Mo.sub.7O.sub.24, 0.15 mg/L CoCl.sub.2.6H.sub.2O, and water for the balance. The feeding liquid contains 700 g/L glucose and 20 g/L MgSO.sub.4.7H.sub.2O, and water for the balance.

(28) A catalytic liquid system containing 300 g/L lysine hydrochloride and 0.2 mmol/L PLP is prepared. After adjusting the temperature to 37 C., the stirring speed of fermentation tank is set to 500 rpm, and the whole-cell catalysis is started by adding 20 g/L wet bacteria (equivalent to cell dry weight of 4 g/L). The pH of the self-regulating catalytic system is adjusted by the by-product CO.sub.2 without adjusting the pH by adding acidic substances and without air supply. In the first 20 min of the start of the catalysis, the pH value rapidly increases to about 7.2, and then slowly increases to 7.8 in the next few hours (FIG. 2). The catalytic solution is taken out from the fermenter tank every 0.5 h, centrifuged at 12000g for 5 min, and the supernatant is decanted. The yield of 1,5-pentanediamine in the whole-cell catalysis process is detected according to the lysine and -pentanediamine method in embodiment 1. As shown in FIG. 2, the trend of lysine consumption and pentanediamine production is similar to that of pH value. The lysine is rapidly consumed in the first 0.5 h and the pentanediamine is rapidly accumulated, and then the change of the process is slow. The catalytic consumption rate of the substrate is 99.8% after catalyzing for 4 h, and the residual lysine hydrochloride is only 0.67 g/L.

Embodiment 4 Removal of Carbon Dioxide in Pentanediamine Catalytic Solution by Distillation Under Reduced Pressure

(29) The bacteria in the catalytic reaction solution are removed by centrifugation. Adding 500 mL of the centrifuged pentanediamine catalyst solution to the round bottom flask in the 20 L oil bath rotary evaporator, and carbon dioxide is removed by distillation under reduced pressure. In the process of carbon dioxide desorption, the vacuum meter reading of the circulating water vacuum pump reaches to about 0.095 Mpa, and the heating temperature of the water bath is set to 60 C., 70 C., 80 C., 90 C., 100 C., 120 C., 140 C. and 160 C., respectively, and then investigating the removal of carbon dioxide at different temperatures. After the pressure and temperature are stable, distillation under reduced pressure is carried out for 60 min. After the distillation, adding the ultra-pure water to the circular bottom flask, and fixing to the initial volume to determine the carbon dioxide content in the pentanediamine catalytic solution before and after the distillation. The total inorganic carbon (TIC) content of the catalytic solution is detected by the total organic carbon total nitrogen analyzer (Vario TOC) to calculate the carbon dioxide removal rate of the catalytic reaction solution. The calculation formula is as follows: the removal rate of carbon dioxide=(TIC content of the pentanediamine catalytic solution before distillation under reduced pressureTIC content in the pentanediamine catalytic solution after distillation under reduced pressure)/TIC content of the pentanediamine catalytic solution before distillation under reduced pressure100%. The experimental results show that the removal rate of carbon dioxide in the pentanediamine catalytic solution increases with the increase of heating temperature (Table 1).

(30) TABLE-US-00003 TABLE 1 Carbon dioxide removal rate of pentanediamine catalytic solution at different temperatures for distillation under reduced pressure Temperature ( C.) Removal rate of CO.sub.2 (%) 60 42.9 70 48.7 80 53.1 90 57.1 100 59.3 120 66.6 140 68.9 160 80.2

(31) During the removal of carbon dioxide, pentanediamine will also volatilize. In the distillation process under reduced pressure with different temperatures mentioned above, the temperature of the cooling circulating water is set to 20 C. Collecting the condensed fraction to detect the content of pentanediamine, and calculating the loss rate of pentanediamine in the carbon dioxide removal process. The calculation method is as follows: the loss rate of pentanediamine=(fraction pentanediamine contentfraction volume)/(pentanediamine content of the catalytic solution before distillationvolume of the catalytic solution added to the round bottom flask)100%. The results are shown in Table 2: the loss rate of pentanediamine increases with the increase of distillation temperature. When the heating temperature is set to 60 C., the loss rate of pentanediamine is less than 0.1%; and when the heating temperature is raised to 160 C., the loss rate of pentanediamine reaches to 8% or more. When the heating temperature is higher than 120 C., a large amount of pentanediamine volatilizes and escapes, and coagulates with the carbon dioxide gas into milky white pentaneamine carbonate on the surface of equipment pipe with lower temperature, which not only easily clogs the pipeline, but also increases the difficulty of equipment cleaning.

(32) TABLE-US-00004 TABLE 2 Loss rate of penanediamine at different temperatures for distillation under reduced pressure Temperature ( C.) Loss rate of penanediamine (%) 60 0.06 70 0.09 80 0.35 90 0.96 100 1.22 120 3.07 140 4.13 160 8.16

Embodiment 5 Recycling the Removed Carbon Dioxide by Alkaline Solution

(33) The bacteria in the catalytic reaction solution are removed by centrifugation. Adding 500 mL of the centrifuged pentanediamine catalyst solution to the round bottom flask of the 5 L rotary evaporator, and carbon dioxide is removed by distillation under reduced pressure. In the process of distillation, the vacuum meter reading of the circulating water vacuum pump is about 0.095 Mpa, and the heating temperature of the water bath is set to 40 C., 50 C., 60 C. and 70 C., respectively, and then investigating the removal of carbon dioxide at different temperatures. Adding 10 L sodium hydroxide solution with content of 6 g/L to the circulating water vacuum pump tank for absorbing carbon dioxide desorbed in the pentanediamine catalyst solution. The pH electrode is inserted into the tank to record the pH value of the alkaline solution of the circulating water vacuum pump tank in real time. As the removed carbon dioxide is chemically absorbed in the alkaline solution, the pH of the alkaline solution in the tank gradually decreases. The higher the heating temperature is, the faster the pH value of the alkaline solution in the tank decreases (FIG. 3).

(34) The total inorganic carbon (TIC) content of the alkaline solution of the circulating water vacuum pump tank is detected by the total organic carbon total nitrogen analyzer (type of Vario TOC) to calculate the absorption amount of carbon dioxide absorbed by alkaline solution. When the heating temperature of vacuum distillation is 50 C., the process curve of carbon dioxide absorption by the alkaline solution is shown in FIG. 4. The content of TIC of the alkaline solution in the circulating water vacuum pump tank increases gradually.

Embodiment 6 the Reduced Consumption of Strong Base to Replace Free Pentanediamine by Removal of Carbon Dioxide

(35) After the carbon dioxide is removed by the temperature and pressure conditions of embodiment 5 for 90 min, the pentanediamine catalyst solution is poured out from the round bottom flask and diluted with ultrapure water to 2 L, and then measuring the pH value of the diluted catalytic solution. When the heating temperature of the water bath is set to 40 C., 50 C., 60 C. and 70 C. for the removal of carbon dioxide, the pH value of the catalytic liquid rises from 7.66 to 8.45, 9.63, 9.75 and 9.90, respectively. Further, adjusting the pH value of the pentanediamine catalytic solution diluted to the equal volume with sodium hydroxide. After removing carbon dioxide, the pH of the catalytic solution of 100 mL is set to 11, 12 and 13, respectively, which need 42 mL, 72 mL and 106 mL of sodium hydroxide solution (100 g/L) respectively. While before the removal of carbon dioxide, diluting the catalyst solution with the same content of pentanediamine consumed 97 mL, 141 mL and 185 mL of sodium hydroxide solution, respectively. The results show that in the process of replacing free pentanediamine with strong base, the removal of carbon dioxide from the catalytic solution can significantly reduce the amount of strong base, and the amount of the formed solid residue after distillation of pentanediamine is much less.

Embodiment 7 the Removal of Carbon Dioxide Improves the Distillation Yield of Pentanediamine

(36) The bacteria in the catalytic reaction solution are removed by centrifugation. Adding 500 mL of the centrifuged pentanediamine catalyst solution to the round bottom flask in the 20 L rotary evaporator. The temperature of oil bath is set to 70 C. and the carbon dioxide is removed by distillation under reduced pressure. After 90 min, the sodium hydroxide with equivalent amount is added. The temperature of oil bath is set to 120 C., and the distillation under reduced pressure is continued for 120 min to collect the pentanediamine fraction. In the control experiment, the carbon dioxide is not removed, and the pentanediamine catalyst solution with the equivalent amount of sodium hydroxide is distilled directly under the same conditions to collect the pentanediamine fraction. After removal of carbon dioxide, 62.3 g of pentanediamine can be obtained by the distillation of the pentanediamine catalytic solution with the replacement of strong base, whereas only 11.8 g of pentanediamine could be obtained in the control experiment.