Method for preparing pentamethylene diamine through continuous conversion and production strain thereof

Abstract

The invention relates to a method for preparing pentamethylene diamine through continuous conversion and a production strain thereof. The invention belongs to the technical field of microorganisms, and particularly relates to a genetically engineered bacterium capable of continuously producing pentamethylene diamine and application of the genetically engineered bacterium. According to the pentamethylene diamine biotransformation genetically engineered bacterium disclosed by the invention, kdsD, lpxL, pagP, lpxP, eptA, lpxM, OmpF and OmpC are deleted, so that the thallus can be self-settled after the transformation of the strain is completed, convenience is provided for subsequent continuous transformation and extraction and purification of pentamethylene diamine, the accumulation of pentamethylene diamine reaches a relatively high level after multiple continuous transformation processes, and the cost is saved. The invention also provides a thallus sedimentation mode, which is characterized in that 10-20 mM of Ca < 2 + >, Mg < 2 + > and 5-40 g / L of LMat01 are added on the basis of the genetically engineered bacterium disclosed by the invention, so that thalli can be completely settled within 15 minutes, and can still be completely settled within 15 minutes after multiple times of transformation. According to the invention, by establishing a thallus sedimentation process, a strain required by synthesis of pentamethylene diamine and an efficient preparation method thereof are constructed, so that a continuous preparation process of pentamethylene diamine is realized.

Description

Technical field: The present invention belongs to the technical field of microorganisms, and particularly relates to a genetically engineered bacterium capable of continuously producing pentamethylenediamine and an application thereof. Background technology: 1,5-Diaminopentane, also known as cadaverine, has a molecular formula of C7H 14 N2, in the cell, pentamethylenediamine is produced by decarboxylation of L-lysine by lysine decarboxylase (EC4.1.1.18) (e.g. Figure 1 Pentamethylenediamine is widely used in agriculture, food, medicine, and industry. In industry, 1,5-pentamethylenediamine is a highly valuable platform compound that can be polymerized with dibasic acids to produce the bio-based polyamide PA5X. For example, PA54, synthesized by condensation with succinic acid, exhibits excellent oil swelling resistance, impact resistance, and anti-permeability properties; PA56, synthesized by condensation with adipic acid, exhibits good moisture absorption and breathability, as well as high tensile strength; and PA510, synthesized by condensation with sebacic acid, exhibits strong mechanical properties, a high melting point, and low density. Bio-based polyamide PA5X can be widely used in automotive parts, packaging materials, electronic equipment, and aviation technology. Currently, the global demand for polyamide materials is increasing, making the large-scale industrial production of pentamethylenediamine, a precursor to bio-based polyamide materials, extremely important.

[0001] The microbial production of pentamethylenediamine is a novel and potentially competitive production route, while existing technologies focus more on the expression of lysine decarboxylase and the construction of recombinant bacteria.

[0002] For example, the Chinese invention patent application with publication number CN118755652A, "A recombinant Escherichia coli and a method for synthesizing 1,5-pentanediamine," discloses a method for constructing recombinant Escherichia coli to synthesize 1,5-pentanediamine. The method uses pET28a(+) as a vector to heterologously express the lysine decarboxylase gene cadA, thereby improving the enzymatic activity of the lysine decarboxylase.

[0003] Chinese invention patent application publication number CN118516387A, "A recombinant Escherichia coli for producing pentamethylenediamine, its construction method, and fermentation-scaled application," discloses a method for producing cadaverine by constructing a recombinant microorganism and culturing the microorganism in a 50 L fermentation tank, supplemented with glucose and ammonium sulfate in a fixed ratio, and fermenting.

[0004] The present invention realizes a continuous preparation process of pentamethylenediamine by establishing a bacterial cell sedimentation process, constructing a strain required for pentamethylenediamine synthesis and an efficient preparation method thereof. Summary of the invention: In order to solve the above technical problems, the present invention provides a genetically engineered bacterium that can efficiently produce pentamethylenediamine and a process for efficiently and continuously preparing pentamethylenediamine using the same.

[0005] One of the technical solutions provided by the present invention is a genetically engineered bacterium that can efficiently produce pentamethylenediamine. The genetically engineered bacterium is derived from a starting strain of Escherichia coli. lldD LPS transporter mutant gene msbA 148, and on this basis, the core polysaccharide core synthesis-related gene kdsD, lipid A fatty acid chain modification-related genes pagP, lpxM, phosphate group modification-related genes lpxL, lpxP, eptA and outer membrane protein-related genes OmpF, OmpC were superimposed and deleted, and the lysine decarboxylase promoter P cadA Replaced with an environmental / nutrient factor-controlled promoter; Furthermore, the starting strain of the genetically engineered bacteria may be Escherichia coli K12, DH5α, W3110, BL21, MG1655, but is not limited to the above strains; Furthermore, the starting strain is Escherichia coli K12; Furthermore, the gene msbA 148, the nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing; Furthermore, the gene lldD , encoding L-lactate dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO: 4 in the sequence listing; Furthermore, the gene kdsD encodes D-arabinose 5-phosphate isomerase, and the nucleotide sequence is shown in SEQ ID NO: 5 in the sequence listing; Furthermore, the gene lpxL encodes lauroyl acyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 6 in the sequence listing; Furthermore, the gene pagP encodes lipid A palmitoyltransferase, and the nucleotide sequence is shown as SEQ ID NO: 7 in the sequence listing; Furthermore, the gene lpxP encodes palmitoyl acyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing; Furthermore, the gene eptA encodes phosphoethanolamine transferase, and the nucleotide sequence is shown in SEQ ID NO: 9 in the sequence listing; Furthermore, the gene lpxM encodes lipid A myristoyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 10 in the sequence listing; Furthermore, the gene OmpF encodes outer membrane protein F, and the nucleotide sequence is shown in SEQ ID NO: 11 in the sequence listing; Furthermore, the gene OmpC encodes outer membrane protein C, and the nucleotide sequence is shown in SEQ ID NO: 12 in the sequence listing; Furthermore, the environmental / nutrient factor controlled promoter can be a promoter controlled by pH, temperature, dissolved oxygen, etc., or a promoter controlled by multiple nutrient factors such as lactose, xylose, arabinose, etc.; Furthermore, the controllable promoter is a temperature-regulated promoter pR-pL promoter; Furthermore, the genetically engineered bacteria expresses a signal peptide while replacing the promoter, so that the recombinant strain can express lysine decarboxylase in large quantities in the periplasmic space of the cells; Furthermore, the signal peptide is encoded by the gene pelBs.

[0006] The second technical solution provided by the present invention is a bacterial sedimentation agent, wherein the sedimentation agent is LMat01, and the structure is: 3-aminopropyl-3-mercaptopropyl-N-n-butylaminopropyl silica, such as Figure 11 As shown; Furthermore, the bacterial sedimentation agent is a composition comprising Ca 2+ Mg 2+ and LMat01; Furthermore, the Ca 2+ The sources of Mg include but are not limited to: CaCl2, Ca(NO3)2, calcium acetate; the Mg 2+ Sources include but are not limited to: MgCl2, MgSO4, Mg(NO3)2; Preferably, the bacterial sedimentation agent comprises: CaCl2, MgCl2 and LMatO1; Furthermore, the concentration of the bacterial sedimentation agent used in the bacterial sedimentation system is 10-20 mM CaCl2, 10-20 mM MgCl2, and 5-40 g / L LMatO1; Preferably, the concentration of the bacterial sedimentation agent used in the bacterial sedimentation system is 10 mM CaCl2, 10 mMMgCl2, and 5 g / L LMatO1; Furthermore, the method for using the bacterial sedimentation agent is to add the bacterial sedimentation agent to the fermentation liquid and let it stand for 15 to 120 minutes to obtain precipitated bacteria.

[0007] The third technical solution provided by the present invention is the use of the bacterial sedimentation agent described in the second technical solution in the continuous conversion and preparation of pentamethylenediamine, particularly the use of the genetically engineered bacteria described in the first technical solution as the production strain in the continuous conversion and preparation of pentamethylenediamine. Furthermore, the continuous conversion is performed after the conversion of a single batch of pentamethylenediamine is completed, and the sedimentation agent described in the second technical solution is added to the fermentation broth to precipitate the bacteria, and then the bacteria are recovered (the supernatant is used for product extraction), and the substrate lysine is added to the bacteria again to carry out the conversion process of the next batch, and the above conversion, bacteria sedimentation and feeding process are repeated to complete multiple batches of continuous conversion; Furthermore, the feed concentration of the substrate lysine is 100-210 g / L, preferably 210 g / L; Furthermore, the above transformation, bacterial cell sedimentation and feeding process were repeated to complete 50 batches of transformation process continuously; Furthermore, the method for preparing pentamethylenediamine by continuous conversion is as follows: (1) Bacterial growth stage: Use fully synthetic culture medium with an inoculation volume of 10%. During the initial 6 to 12 hours of fermentation, control the culture temperature at 25 to 36°C, the rotation speed at 200 to 900 r / min, the ventilation volume at 200 to 500 L / h, and the dissolved oxygen at more than 50% to achieve rapid bacterial growth. (2) Induction enzyme production stage: bacterial volume (OD 600 When the cells grow to 13-16, the temperature is controlled at 37-50°C, the rotation speed is 900 r / min, the ventilation volume is 500 L / h, and the enzyme production is induced for 1-5 hours; (3) Conversion stage: After the induction of enzyme production, the temperature is controlled at 37-50°C, the rotation speed is 200 r / min, the ventilation volume is 0 L / h, and 100-210 g / L of lysine substrate is added for bioconversion of pentamethylenediamine until the lysine content drops below 0.6 g / L. This is the end of the first batch conversion stage. (4) Continuous transformation stage after bacterial sedimentation: After the first batch of transformation stage, a final concentration of 10-20 mM Ca was added to the fermentation broth. 2+ 、10~20 mM Mg 2+ and 5-40 g / L LMat01, let it stand for 15-120 min (sedimentation rate reaches more than 80%), then recover the lower layer of bacteria; add 100-210 g / L lysine substrate to the lower layer of bacteria again, control the temperature at 37-50°C, rotate at 200 r / min, and ventilate at 0 L / h, until the lysine content is below 0.6 g / L, end the second batch conversion stage, and repeat this step to enter the continuous conversion stage.

[0008] The process involved four stages: fermentation culture of the bacteria, efficient enzyme production at variable temperature, rapid conversion of the target product, and continuous conversion after sedimentation. The conversion was repeated 50 times, producing pentamethylenediamine at a level of 62.76-129.90 g / L, and the lysine conversion rate reached 97%-98% of the theoretical conversion rate.

[0009] Furthermore, the culture medium used in the fermentation process was a fully synthetic culture medium (g / L): diammonium hydrogen phosphate 25, potassium dihydrogen phosphate 5, citric acid 25, ammonium sulfate 5, MgSO4 0.5, FeSO4•7H2O 1, CaCl2•2H2O 1, ZnSO4•7H2O 1, CuSO4•5H2O 1, MnSO4•H2O 1, Na2B4O7•10H2O 1, (NH4)6Mo7O 24 •4H2O 1, glycerol 30, the rest is water; Furthermore, the efficient key enzyme preparation process and continuous conversion process established by the present invention are not limited to the preparation of pentamethylenediamine, but also include other chemicals with similar reaction processes, such as pyruvate, alanine, lactic acid, α-ketoglutaric acid, succinic acid, itaconic acid and various functional sugars, etc.

[0010] Beneficial effects: 1. The genetically engineered bacteria provided by the present invention have a significant and efficient ability to convert lysine into pentamethylenediamine. The bacteria are cultured at 25-50°C for 6-12 hours, induced to produce enzymes for 1-5 hours, converted to produce pentamethylenediamine for 2-14 hours, allowed to settle for 0.5-2 hours, and continuously converted to produce pentamethylenediamine for 3-141 hours. The time from cell culture to complete pentamethylenediamine conversion is 13-154 hours. After 50 consecutive conversions, the pentamethylenediamine production level is 62.76-129.90 g / L, and the lysine conversion rate reaches 97%-98% of the theoretical conversion rate.

[0011] 2. The efficient conversion process of pentamethylenediamine of the present invention: the bacteria are rapidly grown using glycerol at 25-36°C for 6-12 hours to form bacteria; after rapid induction of enzyme production for 1-5 hours, pentamethylenediamine is converted and synthesized at 37-50°C, and a sedimentation aid Ca is added. 2+ Mg 2+ And LMat01 is then continuously converted to synthesize pentamethylenediamine. That is, using the recombinant bacteria of the present invention and its continuous conversion process for preparing pentamethylenediamine, the production process of pentamethylenediamine only requires heating to induce enzyme production, and rapid sedimentation under the action of an auxiliary agent, thereby achieving continuous conversion of lysine as a raw material to produce pentamethylenediamine.

[0012] 3. The starting strain of the genetically engineered bacteria of the present invention is Escherichia coli. The lysine decarboxylase induced by temperature increase is located between the cell membrane and the cell wall, and is not released into the fermentation system. The cofactor PLP (pyridoxal phosphate) is produced during bacterial growth. In the process of converting lysine to pentamethylenediamine, intact cells can be used to directly convert the substrate to the product, providing a basis for a continuous conversion process.

[0013] 4. The genetically engineered bacteria for pentamethylenediamine bioconversion of the present invention utilize a fully synthetic culture medium during bacterial culture, resulting in a clear culture fluid, which facilitates subsequent product separation and extraction. The raw materials used in the conversion process of the present invention can be either lysine fermentation liquor that has not undergone post-extraction treatment, or lysine hydrochloride and lysine sulfate. The efficiency of the conversion process of the present invention is not affected by residual lysine fermentation liquor. The pentamethylenediamine salt formed after the conversion process of the present invention is easily extracted and purified.

[0014] 5. The genetically engineered bacteria for the bioconversion of pentamethylenediamine of the present invention delete kdsD, lpxL, pagP, lpxP, eptA, lpxM, OmpF, and OmpC, so that the bacteria can self-sediment after the transformation is completed, which provides convenience for subsequent continuous transformation and extraction and purification of pentamethylenediamine. After multiple continuous transformation processes, pentamethylenediamine accumulates to a high level, saving costs.

[0015] 6. The present invention provides a bacterial sedimentation method, which is based on the genetically engineered bacteria of the present invention by adding 10-20 mM Ca 2+ Mg 2+ With 5-40 g / L LMat01, the bacteria can be completely precipitated in 15 min, and even after multiple transformations, they can still be completely precipitated in 15 min. Description of the drawings:

[0016] Figure 1 Lysine decarboxylation reaction; Figure 2 Strain construction process; Figure 3 Physical map of recombinant plasmid pT-cadA; Figure 4 Results of functional identification of lysine decarboxylase promoter; Figure 5 Results of functional identification of lysine decarboxylase signal peptide; Figure 6 Comparison of sedimentation results between strains GLK08, GLK09 and the starting strain K12; Figure 7 Comparison of sedimentation rates of strain GLK09 and starting strain K12 under the action of adjuvant; Figure 8 Pentamethylenediamine HPLC detection spectrum; Figure 9 Pentamethylenediamine conversion results at the bench-scale level; Figure 10 Flow chart of the continuous conversion preparation of pentamethylenediamine.

[0017] Figure 11 is the structural formula of LMat01. Specific implementation method: In order to make the purpose, technical solutions and advantages of this patent more clear, the following is a further detailed description of this patent in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0018] The new material LMat01 used in the present invention is a porous silica composite material, the full name of which is: 3-aminopropyl-3-mercaptopropyl-N-n-butylaminopropyl silica, with the structural formula as follows: Figure 11 shown.

[0019] The construction flow chart of the engineering bacteria in the present invention is as follows Figure 2 shown.

[0020] The present invention is further explained below through specific examples.

[0021] Example 1: Site-directed mutagenesis and expression of lipopolysaccharide transporter 1. msbA 148 site-directed mutagenesis (CT) Using the Escherichia coli K12 genome as a template, the upstream and downstream fragments were amplified by PCR using primers Msb-1F / Msb-2R and Msb-2F / Msb-1R, respectively. The upstream fragment was ~169 bp and the downstream fragment was ~1616 bp after gel recovery. The upstream and downstream fragments were mixed as templates for overlapping PCR to obtain msbA The mutation in which the C base at position 148 is replaced by a T base msbA 148 gene fragment ~ 1750 bp. Primer sequences are shown in Table 1 below.

[0022] Table 1 Primer sequence list

[0023] 2. Construction of expression plasmid will pass Bam HI and Sma I double-digested plasmid pET28a(+) and Bam HI-digested mutant gene fragment msbA 148 enzyme inactivation and ligation to obtain plasmid pET28a-msbA 148 (~5083bp).

[0024] 3. Plasmid pSK- lldD ::difEry's Construction by E.coli K12 genome was used as template and primers were used to lldD -F / lldD -R amplification lldD , and plasmid vector pSKsym( Sma I) Ligation to construct plasmid pSKsym- lldD ; Using the enzyme cutting site information carried by the amplified sequence itself, the two ends of dif Repeat sequence encoding erythromycin resistance selection marker gene sequence ( dif Ery) and pSKsym- lldD Ligation to construct the recombinant plasmid pSK- lldD :: dif Ery.

[0025] described dif The nucleotide sequence of the Ery resistance gene is shown in SEQ ID NO: 13 in the sequence listing.

[0026] 4. Gene expression pET28a- msbA 148 was used as template and amplified with primers PUBB-I / II to obtain a 2.9 kb fragment containing the promoter PamyL (SEQ ID NO: 1) and the mutated target gene ( msb A148, (SEQ ID NO: 3)) and terminator TamyL (SEQ ID NO: 2) were cloned into plasmid pSK- s ::difEry lldD I site to obtain pSK- Stu ::difEry- lldD 148, and then plasmid pSK- msbA ::difEry- lldD 148 as template, primer msbA -F / lldD -R amplified the mutation cassette and electrotransformed it into Escherichia coli K12 to obtain lldD Integration at the gene locus lldD strain GLKM with 148 genes.

[0027] Example 2: Deletion of genes related to lipopolysaccharide synthesis in strain GLKM 1. Obtaining kdsD, lpxL, pagP, lpxP, eptA, and lpxM genes by msbA K12 genomic DNA was used as a template, and primers kdsD-up1 / kdsD-up2 and kdsD-dn1 / kdsD-dn2 were used to amplify the upstream fragment (393 bp) and the downstream fragment (459 bp). The upstream and downstream fragments of the kdsD gene were overlapped by PCR to obtain the target gene kdsD, which was 833 bp in size. Using lpxL-up1 / lpxL-up2 and lpxL-dn1 / lpxL-dn2 as primers, PCR amplification obtained the upstream fragment (481 bp) and the downstream fragment (395 bp). The upstream and downstream fragments of the lpxL gene were overlapped by PCR to obtain the target gene lpxL, with a fragment size of 833 bp. Using pagP-up1 / pagP-up2 and pagP-dn1 / pagP-dn2 as primers, PCR amplification obtained the upstream fragment (268 bp) and downstream fragment (646 bp), and the upstream and downstream fragments of the pagP gene were overlapped by PCR to obtain the target gene pagP, which was 895 bp in size. Using lpxP-1F / lpxP-2R as primers, PCR amplification yielded a gene fragment of lpxP with a size of 1296 bp; Using eptA-1F / eptA-2R as primers, PCR amplification yielded a gene fragment of eptA with a size of 1119 bp; The lpxM gene fragment of 850 bp was amplified by PCR using lpxM-F / lpxM-R primers; 2. Plasmid construction (1) Construction of recombinant plasmid with pSKsym The target gene fragments kdsD, lpxL, pagP, lpxP, lpxM and eptA obtained in step 1 were respectively E.coli The plasmid pSKsym digested with enzyme I was ligated to obtain the recombinant plasmids pSKsym-kdsD, pSKsym-lpxL, pSKsym-pagP, pSKsym-lpxP, pSKsym-lpxM and pSKsym-eptA.

[0028] The constructed plasmids pSKsym-kdsD, pSKsym-lpxL, pSKsym-pagP, pSKsym-lpxP and pSKsym-eptA were SmaThe full length of the plasmid was verified by RV enzyme digestion, and the band sizes were 3790 bp, 3811 bp, 3852 bp, 4253 bp and 3813 bp respectively; the plasmid pSKsym-lpxM was Eco The full length was verified by enzyme digestion with I, and the band size was 3807 bp.

[0029] (2) Construction of resistance gene recombinant fragments Will Kpn Ery were cloned into dif I digested the plasmids pSKsym-kdsD, pSKsym-lpxL, and pSKsym-pagP to obtain pSKsym-kdsD::difEry, pSKsym-lpxL::difEry, and pSKsym-pagP::difEry; Plasmid pSKsym-lpxP Sma RV, Eco After BI enzyme digestion to remove the middle fragment of 435 bp, the plasmid with the middle sequence removed was separated from the resistance fragment. Sna Ery ligation to obtain plasmid pSKsym-lpxP::difEry; Plasmid pSKsym-eptA dif RV, Eco I enzyme digestion, remove the middle fragment of 353 bp, remove the middle sequence of the plasmid and the resistance fragment Sal Ery ligation to obtain plasmid pSKsym-eptA::difEry; Plasmid pSKsym-lpxM was reversely amplified using primers lpxM-inF / lpxM-inR, and the middle fragment of 271 bp was removed and combined with the resistance fragment. dif Ery was ligated to obtain the plasmid pSKsym-lpxM::difEry.

[0030] 3. RED recombination technology to delete genes The mutant cassette kdsD::difEry was amplified from pSKsym-kdsD::difEry using primers kdsD-up1 / kdsD-up2 and electroporated into the GLKM strain. Transformants with the correct mutant size and containing the resistance selection marker were confirmed by colony PCR. The erythromycin resistance selection marker was removed by subculture, and the recombinant strain GLK01 with the kdsD gene deleted was obtained. The mutant cassette lpxL::difEry was amplified from pSKsym-lpxL::difEry using primers lpxL-up1 / lpxL-up2 and electroporated into strain GLK01. Transformants of the correct mutant size containing the resistance selection marker were confirmed by colony PCR. After subculture, the erythromycin resistance selection marker was removed, and recombinant strain GLK02 with stacked deletions of the kdsD and lpxL genes was obtained. The mutation cassette pagP::difEry was amplified from pSKsym-pagP::difEry using primers pagP-up1 / pagP-dn2, electroporated into the strain GLK02, and screened for erythromycin resistance. Colony PCR confirmed that transformants with the correct mutant size containing the resistance selection marker were obtained. After subculture, the erythromycin resistance selection marker was removed, and the recombinant strain GLK03 with stacked deletions of the kdsD, lpxL and pagP genes was screened.

[0031] The mutant cassette lpxP::difEry was amplified from pSKsym-lpxP::difEry using primers lpxP-1F / lpxP-2R and electroporated into the strain GLK03. Transformants of the correct mutant size containing the resistance selection marker were confirmed by colony PCR. After subculture and removal of the erythromycin resistance selection marker, the recombinant strain GLK04 with stacked deletions of the kdsD, lpxL, pagP, and lpxP genes was obtained. The mutant cassette eptA::difEry was amplified from pSKsym-eptA::difEry using primers eptA-1F / eptA-2R and electroporated into the strain GLK04. Transformants of the correct mutant size containing the resistance selection marker were obtained by colony PCR verification. After subculture, the erythromycin resistance selection marker was removed, and the recombinant strain GLK05 with stacked deletions of the kdsD, lpxL, pagP, lpxP and eptA genes was obtained. The mutant cassette lpxM::difEry was amplified from pSKsym-lpxM::difEry using primers lpxM-F / lpxM-R and electroporated into the strain GLK05. Transformants of the correct mutant size containing the erythromycin resistance marker were confirmed by colony PCR. After subculture and removal of the erythromycin resistance selection marker, the recombinant strain GLK06 with stacked deletions of the kdsD, lpxL, pagP, lpxP, eptA, and lpxM genes was obtained.

[0032] Example 3: Deletion of outer membrane protein ompF and ompC genes 1. ompF gene deletion by dif The K12 genome was used as a template and primers ompF-upF / ompF-upR and ompF-dnF / ompF-dnR were used to amplify the upstream (340 bp) and downstream (307+26=333 bp) fragments, respectively. The mutation cassette (653 bp) was amplified by overlapping PCR using the above and downstream fragments as templates and cloned into pSKsym ( E.coli I site), constructing plasmid pSKsym-ompF (3610 bp), which can be Sma EV single cut; Will Eco Ery was cloned into pSKsym-ompF dif RV site, complete plasmid pSKsym-ompF- Eco Construction of Ery (4781 bp).

[0033] The primers ompF-upF / ompF-upR were used to convert the pSKsym-ompF- dif The mutation cassette ompF- dif Ery was electroporated into the strain GLK06, screened under erythromycin resistance, and colony PCR was used to verify that transformants of the correct mutant size containing the resistance screening marker were obtained. After subculture, the erythromycin resistance screening marker was removed, and the recombinant strain GLK07 with the ompF gene deleted was screened.

[0034] 2. ompC gene deletion by dif K12 genome was used as template, and ompC was amplified with primers ompC-F / ompC-R, with a size of 1261 bp. It was then ligated with plasmid pSKsym to obtain pSKsym-ompC (3953 bp). The recombinant plasmid can be E.coli RI or Eco I single cut; The plasmid pSKsym-ompC was Pst RI and Eco I double enzyme digestion, remove the middle sequence 503 bp, fill the sticky ends, and then Pst Ery ligation to construct plasmid pSKsym-ompC:: dif Ery (4621 bp) was electroporated into strain GLK07.

[0035] After screening under erythromycin resistance, colony PCR was used to verify that transformants of the correct mutant size containing the resistance selection marker were obtained. After subculture, the erythromycin resistance selection marker was removed and the recombinant strain GLK08 with the ompC gene deleted was obtained.

[0036] Example 4: Acquisition of Escherichia coli chromosomal lysine decarboxylase function 1. Obtaining the plasmid pPL-pelBs by dif The K12 genome was used as a template to PCR amplify the pR-pL promoter (primers pL-F and pL-R). The PCR product was approximately 1.37 kb in size. E.coli HI and Bam After digestion with enzyme I, it was ligated with plasmid pET-20b containing the signal peptide sequence pelBs to obtain plasmid pPL-pelBs, with a size of 5.0 kb. The nucleotide sequence of the pPL-pelBs sequence is shown in SEQ ID NO: 14 in the sequence listing.

[0037] 2. Acquisition of the cadA gene by Spe The K12 genome was used as a template and cadA-F / cadA-R as primers to amplify the cadA gene fragment with a size of 2148 bp. 3. Ligation of cadA gene with plasmid pPL-pelBs Use restriction enzymes E.coli I and Nco HI was used for enzyme digestion, and the cadA gene and plasmid pPL-pelBs were connected to obtain the recombinant plasmid pR-pL-pelBs-cadA, abbreviated as pT-cadA. Its physical map is shown in Bam As shown, the recombinant plasmid contains a temperature-controlled promoter, a signal peptide, and the complete cadA gene, and has the function of temperature-controlled secretory expression of cadA. The recombinant plasmid pT-cadA was electroporated into the GLK08 strain to obtain the recombinant strain GLK09.

[0038] Example 5: Determination of lysine decarboxylase activity in strain GLK09 strain GLK09, strain GLK08 and starting strain Figure 3 K12 cells were cultured in a shaking incubator at 30°C and 40°C, at 200 rpm for 9 hours. The culture medium contained (g / L): 5g of yeast extract, 10g of peptone, and 10g of sodium chloride. Lactose (5g / L) was also added as an inducer. Lysine decarboxylase (cadA) specific activity was measured in cell lysates.

[0039] The enzyme activity assay was performed as follows: 50 μL of 5 g / L L-lysine stock solution was added to 50 μL of cell disruption solution (control group: 50 μL of water) and the mixture was reacted for 15 min. 400 μL of 10 g / L TNBS solution was then added to the mixture, and the mixture was incubated at 42°C for 6 min. 400 μL of the reaction solution was removed and 2 mL of toluene was added, followed by shaking for 90 s. After standing for 5 min, TNP-pentanediamine was in the upper toluene phase, while TNP-lysine was still in the aqueous phase. Approximately 1.8 mL of the toluene layer was transferred to a quartz cuvette and the reading was taken at 340 nm on a UV spectrophotometer.

[0040] The cell disruption solution was prepared as follows: 30 mL of fermentation broth was placed in a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded, 10 mL of ddH₂O was added, vortexed, and the volume was filled up to 30 mL with ddH₂O. The cell suspension was centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded, 10 mL of PBS was added, vortexed, and the volume was filled up to 30 mL with PBS. The cell suspension was centrifuged at 12,000 rpm for 5 minutes. The cells were washed once. The cells were resuspended in 10 mL of PBS and disrupted by sonication (sonication conditions: 3 s on, 2 s off, temperature 25°C, disruption time 30 minutes, power 40%). The cell debris was removed by centrifugation at 8,000 rpm for 15 minutes at 4°C.

[0041] The results are as follows E.coli As shown, without the addition of an inducer, strain GLK09 produced only minimal lysine decarboxylase activity at 30°C. However, when cultured at 40°C, the LDC activity of strain GLK09 was 20 times higher than that at 30°C, sufficient for the rapid formation of pentamethylenediamine. When the inducer was added, the LDC activity of strain GLK09 at 40°C was calibrated to 100%. In comparison, the LDC activity of strain GLK09 at 30°C was significantly lower, indicating that increasing the temperature effectively enhanced the activity of lysine decarboxylase.

[0042] Example 6: Identification of GLK09 cell activity and secretory expression of lysine decarboxylase strain GLK09, strain GLK08 and starting strain Figure 4 K12 was cultured at 30°C in a shaking incubator at 200 r / min for 9 h. The culture medium consisted of (g / L): diammonium hydrogen phosphate 25, potassium dihydrogen phosphate 5, citric acid 25, ammonium sulfate 5, MgSO4 0.5, FeSO4•7H2O 1, CaCl2•2H2O 1, ZnSO4•7H2O 1, CuSO4•5H2O 1, MnSO4•H2O 1, Na2B4O7•10H2O 1, (NH4)6Mo7O 24•4H2O 1, glycerol 10.

[0043] After 9 hours of incubation at 30°C, the temperature was raised to 40°C and the temperature was raised for 2 hours to induce enzyme production. The fermentation broth was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was directly measured as the enzyme activity in the fermentation broth; the enzyme activity measured after the cells collected by centrifugation were resuspended in culture medium to the starting volume was used as the enzyme activity in the periplasmic space; the enzyme activity measured in the cell lysis solution was used as the total enzyme activity in the periplasmic space and intracellular space. Typical measurement results are shown below. E.coli shown. After culturing and inducing enzyme production in GLK09, the specific activity of lysine decarboxylase in the cell lysate was calibrated as 100%. The enzyme activity in the fermentation broth was extremely low, while the activity in the periplasm was close to that in the cell lysate. This indicates that the signal peptide effectively expresses lysine decarboxylase in the periplasm.

[0044] Example 7: Determination of strain sedimentation properties strain GLK09, strain GLK08 and starting strain Figure 5 K12 was inoculated into a 250 mL shake flask (50 mL of liquid) and cultured in a shaking incubator at 37°C and 200 r / min for 9 h. The culture medium consisted of (g / L): diammonium hydrogen phosphate 25, potassium dihydrogen phosphate 5, citric acid 25, ammonium sulfate 5, MgSO4 0.5, FeSO4•7H2O 1, CaCl2•2H2O 1, ZnSO4•7H2O 1, CuSO4•5H2O1, MnSO4•H2O 1, Na2B4O7•10H2O 1, (NH4)6Mo7O 24 •4H2O 1, glycerol 10. After 9 h, raise the temperature to 40℃ and continue culturing for 4 h, then add lysine salt with a final concentration of 80 g / L. The conversion is completed when the lysine content is below 0.6 g / L. After completion, add 40 mL of bacterial solution to a 50 mL centrifuge tube, let it stand in a 4℃ refrigerator, and observe the sedimentation of the bacteria.

[0045]

[0046] The results are as follows E.coli As shown in the figure, after standing for 5 hours, the sedimentation rate of the starting strain K12 was about 50%, the sedimentation rate of the strain GLK08 was about 75.54%, and the sedimentation rate of the strain GLK09 was about 80.98%. After the starting strain was modified, the sedimentation rate increased by 20-30%, which shows that the genetic modification of the present invention has an effect on the sedimentation rate of the starting strain. Figure 6 K12 enhanced sedimentation. Furthermore, the sedimentation rate of GLK09 was higher than that of GLK08, indicating that the ligation of the promoter and signal peptide pPL-pelBs further improved the sedimentation rate.

[0047] Example 8: Effect of Induced Enzyme Production and Additives on Sedimentation in a 5 L Fermenter The strain GLK09 and the starting strain K12 were used in a 5 L fermenter to convert lysine to pentamethylenediamine to verify the effect of temperature-induced lysine decarboxylase production and the effect of additives on the sedimentation of the strain GLK09. The process is as follows: Bacterial growth stage: Use fully synthetic culture medium with an inoculation of 10%, culture at 37°C for 9 h, rotation speed of 200-900 r / min, ventilation volume of 200-500 L / h, and dissolved oxygen controlled at above 50% for rapid bacterial growth; culture medium composition (g / L): diammonium hydrogen phosphate 25, potassium dihydrogen phosphate 5, citric acid 25, ammonium sulfate 5, MgSO4 0.5, FeSO4•7H2O 1, CaCl2•2H2O 1, ZnSO4•7H2O 1, CuSO4•5H2O 1, MnSO4•H2O 1, Na2B4O7•10H2O 1, (NH4)6Mo7O 24 •4H2O 1, glycerol 30.

[0048] Induction enzyme production stage: bacterial volume (OD 600 ) When the culture medium was grown to 15 hours, the temperature was raised to 40°C, the rotation speed was 900 r / min, the ventilation volume was 500 L / h, and the enzyme production was induced for 4 hours; Conversion stage: After the induction of enzyme production is completed, the temperature is controlled at 40°C, the rotation speed is 200 r / min, the ventilation volume is 0 L / h, and a lysine substrate with a final concentration of 100 g / L is added. The conversion is continued for 6 h until the lysine content is below 0.6 g / L, at which point the conversion stage is terminated.

[0049] After completing the transformation, conduct group experiments: (1) Add 40 mL of K12 bacterial solution to a 50 mL centrifuge tube, place it in a 4°C refrigerator, and observe the sedimentation of the bacteria; (2) Add 40 mL of GLK09 bacterial solution to a 50 mL centrifuge tube, place it in a 4°C refrigerator, and observe the sedimentation of the bacteria; (3) Add 40 mL of GLK09 bacterial solution to a 50 mL centrifuge tube, add 10 mM CaCl2, and place it in a 4°C refrigerator to observe the sedimentation of the bacteria; (4) Add 40 mL of GLK09 bacterial solution to a 50 mL centrifuge tube, add 10 mM MgCl2, and place it in a 4°C refrigerator to observe the sedimentation of the bacteria; (5) Add 40 mL of GLK09 bacterial solution and 5 g / L of LMat01 to a 50 mL centrifuge tube, place it in a 4°C refrigerator, and observe the sedimentation of the bacteria; (6) Add 40 mL of GLK09 bacterial solution to a 50 mL centrifuge tube, and at the same time add 10 mM CaCl2, 10 mM MgCl2, and 5 g / L LMat01. Place the tube in a 4°C refrigerator and observe the sedimentation of the bacteria.

[0050] (7) Add 40 mL of K12 bacterial solution to a 50 mL centrifuge tube, add 10 mM CaCl2, and place it in a 4°C refrigerator to observe the sedimentation of the bacteria; (8) Add 40 mL of K12 bacterial solution and 10 mM MgCl2 to a 50 mL centrifuge tube, place it in a 4°C refrigerator, and observe the sedimentation of the bacteria; (9) Add 40 mL of K12 bacterial solution and 5 g / L of LMat01 to a 50 mL centrifuge tube, place it in a 4°C refrigerator, and observe the sedimentation of the bacteria; (10) Add 40 mL of K12 bacterial solution to a 50 mL centrifuge tube, and add 10 mM CaCl2, 10 mM MgCl2, and 5 g / L LMat01 at the same time. Place the tube in a refrigerator at 4°C and observe the sedimentation of the bacteria.

[0051] The settlement results are as follows E.coli As shown in the figure, complete sedimentation is defined as a sedimentation rate exceeding 80%. As can be seen from the figure, after adding CaCl2, MgCl2, LMat01, and their combination to the bacterial broth, none of these three sedimentants, nor their combination, helped K12 achieve complete sedimentation within 5 hours. However, they showed a significant sedimentation effect on strain GLK09, increasing the sedimentation rate from 18.5% to 45.7%, 37.0%, 57.3%, and 81.0%, respectively, at 15 minutes. LMat01 and the combination of the three sedimentants were particularly effective, as shown in Table 2 below. However, according to prior art disclosures, LMat01 has never been used for bacterial sedimentation.

[0052] Table 2 Effects of adding three additives on the sedimentation rate of GLK09 and K12 (settling for 15 minutes)

[0053] Pentamethylenediamine HPLC test results are as follows Figure 7 As shown in the figure. During the fermentation process, lysine, pentamethylenediamine, bacterial concentration, and lysine decarboxylase activity were Figure 8 shown.

[0054] The fermentation results of strain GLK09 in a 5 L fermenter showed that glycerol was successfully used to accumulate bacterial cells in the aerobic stage, and lysine was quickly converted into pentamethylenediamine in the conversion stage.

[0055] Example 9: Fermentation and conversion of 50 batches of bacterial strains in a 30-ton fermenter to produce pentamethylenediamine The fermentation process in Example 8 was scaled up to a 30-ton scale (with an initial liquid charge of 10 tons). To ensure continuous addition of glycerol and lysine, the fermentor and feeding tank were selected, and pre-operation fermentor preparation was completed according to standard factory procedures. One main fermentor, one glycerol feed tank, one lysine feed tank, and one seed tank were used. 50% glycerol was added and sterilized. The concentrated lysine extract solution was sterilized and stirred for later use. The process was as follows: Bacteria growth stage: inoculate the seed tank (inoculation amount 10%), start fermentation, 37℃, ventilation 180-340 L / h, rotation speed 200-600 r / min (control the dissolved oxygen above 50% by gradually increasing ventilation and rotation speed).

[0056] Induction enzyme production stage: After 12 hours, the fermentation temperature was raised to 40°C and the glycerol content was measured every 2 hours. When the glycerol was exhausted, ventilation was stopped and the stirring speed was reduced to 180 r / min.

[0057] Conversion stage: 10 tons of lysine solution with a concentration of 210 g / L was added, and the conversion temperature was 40°C. After 6 hours, the lysine consumption dropped below 0.6 g / L, and the first batch of conversion process was completed. The product was then post-extracted and crystals were prepared.

[0058] Continuous transformation stage after sedimentation: After the first batch transformation stage, add 10 mM CaCl2, 10 mMM MgCl2 and 5 g / L LMat01 to a final concentration, let it stand for 15 min (sedimentation rate reaches more than 80%) to recover the bacteria, and the upper fermentation liquid enters the post-extraction stage of pentamethylenediamine. The steps are as follows: Figure 9 As shown; a liquid solution containing 210 g / L lysine (10 tons) was added to the lower layer of bacteria again, the temperature was controlled at 40°C, the rotation speed was 200 r / min, and the ventilation volume was 0 L / h. When the lysine content was below 0.6 g / L, the second batch conversion stage was ended, and this step was repeated to enter the continuous conversion stage.

[0059] After a single batch of conversion is completed, the cells are recovered after sedimentation and re-fed. A liquid containing 210 g / L lysine is added to the recovered cells in the lower layer to start the next batch of conversion. 50 batches of conversion are completed continuously (sedimentation time is 15 minutes each). The continuous conversion process of pentamethylenediamine is shown in the figure below. Figure 10 Figure 10 The yields of pentamethylenediamine in different batches during the fermentation process are shown in Table 3.

[0060] Table 3 Continuous conversion production results of 50 batches of 30-ton tanks

[0061] In summary, the present invention achieves a simple preparation process for the efficient production of pentamethylenediamine from lysine by recombinant bacteria after knocking out the genes of the starting bacteria through genetic engineering technology and dynamically regulating the expression of the lysine decarboxylase encoding gene on the chromosome of the starting bacteria under simple conditions. After simple modifications, the technology of the present invention can also be used for other industrially important microbial metabolites, but not limited to, such as L-lactic acid, acetic acid, pyruvic acid, succinic acid, malic acid and other organic acids; proline, alanine, lysine, methionine, glutamic acid, arginine and other amino acids; thiamine, vitamin B 12 or, the construction of bacterial strains, fermentation production, and establishment and application of new process technologies for short-chain alcohols such as ethanol and propanol.

Claims

1. A genetically engineered bacterium for producing pentamethylenediamine, characterized in that: The genetically engineered bacteria are derived from Escherichia coli starting strains lldD LPS transporter mutant gene msbA 148, deleted the core polysaccharide core synthesis related gene kdsD, lipid A fatty acid chain modification related genes pagP, lpxM, phosphate group modification related genes lpxL, lpxP, eptA and outer membrane protein related genes OmpF, OmpC, and the lysine decarboxylase promoter P cadA Obtained by replacing it with an environmental / nutritional factor-controlled promoter.

2. A genetically engineered bacterium for producing pentamethylenediamine according to claim 1, characterized in that: The starting strains of the genetically engineered bacteria include but are not limited to Escherichia coli K12, DH5α, W3110, BL21, and MG1655.

3. The genetically engineered bacterium for producing pentamethylenediamine according to claim 1, wherein: The gene msbA 148, the nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing; The gene lldD , encoding L-lactate dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO: 4 in the sequence listing; The gene kdsD encodes D-arabinose 5-phosphate isomerase, and the nucleotide sequence is shown in SEQ ID NO: 5 in the sequence listing; The gene lpxL encodes lauroyl acyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 6 in the sequence listing; The gene pagP encodes lipid A palmitoyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing; The gene lpxP encodes palmitoyl acyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing; The gene eptA encodes phosphoethanolamine transferase, and the nucleotide sequence is shown in SEQ ID NO: 9 in the sequence listing; The gene lpxM encodes lipid A myristoyltransferase, and the nucleotide sequence is shown in SEQ ID NO: 10 in the sequence listing; The gene OmpF encodes outer membrane protein F, and the nucleotide sequence is shown in SEQ ID NO: 11 in the sequence listing; The gene OmpC encodes outer membrane protein C, and the nucleotide sequence is shown in SEQ ID NO: 12 in the sequence listing.

4. The genetically engineered bacterium for producing pentamethylenediamine according to claim 1, wherein: The controllable promoter is a temperature-regulated promoter pR-pL promoter; The genetically engineered bacteria expresses a signal peptide while replacing the lysine decarboxylase promoter.

5. A bacterial sedimentation agent, characterized in that The sedimentation agent is LMat01; the structure of the sedimentation agent LMat01 is: 3-aminopropyl-3-mercaptopropyl-N-n-butylaminopropyl silica.

6. A bacterial sedimentation agent according to claim 5, characterized in that: The sedimentation agent also contains Ca 2+ Mg 2+ ; The Ca 2+ The sources of Mg include but are not limited to: CaCl2, Ca(NO3)2, calcium acetate; the Mg 2+ Sources include but are not limited to: MgCl2, MgSO4, Mg(NO3)2; The concentration of the bacterial sedimentation agent in the bacterial sedimentation system is 10-20 mM Ca 2+ 、10-20mM Mg 2+ , 5-40 g / L LMatO1; preferably 10-20 mM CaCl2, 10-20 mM MgCl2, 5-40 g / L LMatO1.

7. A method for bacterial sedimentation, characterized in that: The bacterial cell sedimentation agent according to claim 5 or 6 is added to a system containing bacterial cells and allowed to stand for 15 to 120 minutes to obtain precipitated bacterial cells.

8. Use of the genetically engineered bacteria according to claim 1 in the continuous conversion preparation of pentamethylenediamine, characterized in that: The genetically engineered bacteria according to claim 1 are used to bioconvert lysine to prepare pentamethylenediamine. After a single batch of conversion is completed, the sedimentation agent according to claim 5 or 6 is added to the fermentation broth to precipitate the bacteria, and then the bacteria are recovered. The substrate lysine is re-added to the bacteria to carry out the conversion process of the next batch, and the above-mentioned conversion, bacteria sedimentation and feeding process are repeated to complete multiple batches of continuous conversion.

9. The use according to claim 8, wherein The genetically engineered bacteria of claim 1 are used to biotransform lysine to prepare pentamethylenediamine. After the first transformation is completed, 10-20 mM Ca is added to the transformation system. 2+ 、10~20 mM Mg 2+ and 5-40 g / L LMat01, and after standing for 15-120 min, recover the lower layer of bacteria; add 100-210 g / L lysine substrate to the lower layer of bacteria again, control the temperature at 37-50°C, and continue until the lysine content is below 0.6 g / L, ending the second batch conversion stage, and repeating this step to enter the continuous conversion stage.

10. The use according to claim 9, characterized in that The continuous conversion is not less than 50 times.

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