Recombinant vector, engineered bacterium for synthesizing 1,5-pentanediol and its application

Abstract

The present invention relates to the field of genetic engineering technology, and particularly to a recombinant vector, an engineered bacterium for synthesizing 1,5-pentanediol, and their applications. The recombinant vector provided by the present invention can express 5-aminovaleramidase, lysine 2-monooxygenase, carboxylic acid reductase, and phosphopantetheinyl transferase in bacteria. Using the recombinant vector provided by the present invention to construct a recombinant bacterium, a complete 1,5-pentanediol metabolic pathway can be constructed only through three key enzymes, namely 5-aminovaleramidase, lysine 2-monooxygenase, and carboxylic acid reductase. The metabolic pathway is extremely short, and the metabolic efficiency is higher. It can biosynthesize 1.28 g / L of 1,5-pentanediol under the condition of consuming 19.43 g / L of glucose. The strain has strong stability and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to recombinant vectors, engineering bacteria for synthesizing 1,5-pentanediol, and their applications. Background Art

[0002] 1,5-Pentanediol (1,5-PDO) is a diol with important industrial application value and is widely used in the manufacture of polyesters, polyurethanes, plasticizers, inks, and coatings, etc. Biosynthesis of 1,5-pentanediol generally uses glucose as a substrate and finally obtains 1,5-pentanediol through lysine, 5-aminovaleric acid, 5-hydroxypentanoic acid, and 5-hydroxypentanal. However, in the process from lysine to 1,5-PDO, there are limiting factors such as a long metabolic pathway and low enzyme activity, and at the same time, there is also the problem of low 1,5-PDO production, which seriously restricts its application and development.

[0003] For example, Chinese patent document CN112708588A uses recombinant microorganisms overexpressing genes of module A, module B, and module C to complete the steps. Module A includes 4-hydroxybutyryl-CoA transferase abfT gene and aldehyde dehydrogenase bld gene; module B includes amidase davA gene, lysine monooxygenase davB gene, 4-aminobutyrate aminotransferase gabT gene, and alcohol dehydrogenase yqhD gene; module C includes aspartokinase lysC gene, dihydropicolinate synthase dapA gene, and diaminopimelic acid ddh gene; the finally constructed Escherichia coli produces 1,5-pentanediol with a yield of 1.1 g / L.

[0004] And Chinese patent document CN117025497A discloses another genetically engineered bacterium for catalytic preparation of 1,5-pentanediol. The host is Escherichia coli BL21(DE3), and lysine decarboxylase CadA, 4-aminobutyrate transaminase GabT, and aldehyde-ketone reductase YahK are overexpressed, but the yield of 1,5-pentanediol biosynthesis by this genetically engineered bacterium is only 0.36 g / L. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides recombinant vectors, engineering bacteria for synthesizing 1,5-pentanediol, and their applications. The recombinant bacteria constructed using the recombinant vectors provided by the present invention can achieve the synthesis of 1,5-pentanediol, have an extremely short metabolic pathway, higher metabolic efficiency, strong strain stability, and are suitable for industrial production.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides recombinant vectors for constructing engineering bacteria, including a first recombinant vector and a second recombinant vector;

[0008] The first recombinant vector includes a first starting vector and a first coding gene inserted into the first starting vector; the first coding gene includes a 5-aminovaleramidase coding gene and a lysine 2-monooxygenase coding gene; the amino acid sequence of the 5-aminovaleramidase is as shown in SEQ ID NO.28; the amino acid sequence of the lysine 2-monooxygenase is as shown in SEQ ID NO.29;

[0009] The second recombinant vector includes a second starting vector and a second coding gene inserted into the second starting vector; the second coding gene includes a carboxylic acid reductase coding gene and a phosphopantetheine transferase coding gene.

[0010] Preferably, the nucleotide sequence of the 5-aminovaleramidase coding gene is as shown in SEQ ID NO.2; the nucleotide sequence of the lysine 2-monooxygenase coding gene is as shown in SEQ ID NO.1.

[0011] Preferably, the second recombinant vector is the recombinant vector pET28a- caR - Sfp .

[0012] Preferably, the first starting vector is the pETDuet-1 vector.

[0013] Preferably, the promoter of the first coding gene is the trc promoter.

[0014] The present invention provides the application of the recombinant vector described in the above technical solution in constructing an engineering bacterium for synthesizing 1,5-pentanediol.

[0015] The present invention provides an engineering bacterium for synthesizing 1,5-pentanediol, which includes a primitive strain and a recombinant vector transferred into the primitive strain; the recombinant vector is the recombinant vector described in the above technical solution; the primitive strain includes Escherichia coli ( Escherichia coli ).

[0016] The present invention provides the application of the recombinant vector described in the above technical solution or the engineering bacterium described in the above technical solution in synthesizing 1,5-pentanediol.

[0017] The present invention provides a method for synthesizing 1,5-pentanediol, including the following steps:

[0018] Ferment the engineering bacterium described in the above technical solution in a fermentation broth containing glucose to obtain a fermentation broth containing 1,5-pentanediol.

[0019] Preferably, the fermentation broth further contains pyruvic acid and isopropyl-β-D-thiogalactopyranoside.

[0020] Beneficial effects:

[0021] The recombinant vector provided by the present invention can express and synthesize 5-aminovaleramidase, lysine 2-monooxygenase, carboxylic acid reductase, and phosphopantetheine transferase in the bacterial cell. The recombinant bacterium constructed by using the recombinant vector provided by the present invention can construct a complete 1,5-pentanediol metabolic pathway only through three key enzymes, namely 5-aminovaleramidase, lysine 2-monooxygenase, and carboxylic acid reductase. The metabolic pathway is extremely short and the metabolic efficiency is higher. It can biosynthesize 1.28 g / L of 1,5-pentanediol by consuming 19.43 g / L of glucose. The strain has strong stability and is suitable for industrial production. Brief description of the drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0023] Figure 1 For DavA gene and DavB electrophoretogram of the gene;

[0024] Figure 2 For DavA gene, caR gene and SfP electrophoretogram of the gene;

[0025] Figure 3 For the yields of 1,5-pentanediol, 5-hydroxypentanoic acid, and 5-hydroxypentanal in the fermentation broth of recombinant bacterium QDE01 at different times;

[0026] Figure 4 For the yields of 1,5-pentanediol in the fermentation broth of recombinant bacteria QDE01, QDE02, and QDE03 at different times;

[0027] Figure 5 For the cell concentrations of recombinant bacteria QDE01, QDE02, and QDE03 in the fermentation broth at different times. Detailed implementation manners

[0028] The present invention provides a recombinant vector for constructing an engineered bacterium, including a first recombinant vector and a second recombinant vector;

[0029] The first recombinant vector includes a first starting vector and a first coding gene inserted into the first starting vector; the first coding gene includes a 5-aminovaleramidase coding gene ( DavA gene) and a lysine 2-monooxygenase coding gene ( DavBgene); the amino acid sequence of the 5-aminovaleramidase is shown in SEQ ID NO.28; the amino acid sequence of the lysine 2-monooxygenase is shown in SEQ ID NO.29;

[0030] The second recombinant vector includes a second starting vector and a second coding gene inserted into the second starting vector; the second coding gene includes a carboxylic acid reductase coding gene ( caR gene) and a phosphopantetheinyl transferase coding gene ( SfP gene). Phosphopantetheinyl transferase can phosphopantetheinylate carboxylic acid reductase, thereby increasing the specific activity of carboxylic acid reductase.

[0031] SEQ ID NO.28:

[0032] MRIALYQGAPKPLDVPGNLQRLRHQAQLAAERGAQLLVCPEMFLTGYNIGLAQVERLAEAADGPAAMTVVEIAQAHRIAIVYGYPERGDDGAIYNSVQLIDAHGRSLSNYRKTHLFGELDRSMFSPGADHFPVVELEGWKVGLLICYDIEFPENARRLALDGAELILVPTANMTPYDFTCQVTVRARAQENQCYLVYANYCGAEDEIEYCGQSSIIGPDGSLLAMAGRDECQLLAELEHERVVQGRTAFPYLTDLRQELHLRKG*;

[0033] SEQ ID NO.29:

[0034] MNKKNRHPADGKKPITIFGPDFPFAFDDWLEHPAGLGSIPAERHGEEVAIVGAGIAGLVAAYELMKLGLKPVVYEASKLGGRLRSQAFNGTDGIVAELGGMRFPVSSTAFYHYVDKLGLETKPFPNPLTPASGSTVIDLEGQTYYAEKPTDLPQLFHEVADAWADALESGAQFADIQQAIRDRDVPRLKELWNKLVPLWDDRTFYDFVATSRSFAKLSFQHREVFGQVGFGTGGWDSDFPNSMLEIFRVVMTNCDDHQHLVVGGVEQVPQGIWRHVPERCVHWPEGTSLSTLHGGAPRTGVKRIARASDGRLAVTDNWGDTRHYSAVLATCQTWLLTTQIDCEESLFSQKMWMALDRTRYMQSSKTFVMVDRPFWKDKDPETGRDLLSMTLTDRLTRGTYLFDNGNDKPGVICLSYSWMSDALKMLPHPVEKRVQLALDALKKIYPKTDIAGHIIGDPITVSWEADPYFLGAFKGALPGHYRYNQRMYAHFMQQDMPAEQRGIFIAGDDVSWTPAWVEGAVQTSLNAVWGIMNHFGGHTHPDNPGPGDVFNEIGPIALAD*。

[0035] As an embodiment, the nucleotide sequence of the 5-aminovaleramidase encoding gene is as shown in SEQ ID NO.2; the nucleotide sequence of the lysine 2-monooxygenase encoding gene is as shown in SEQ ID NO.1.

[0036] As an embodiment, the second recombinant vector is the recombinant vector pGCS. The recombinant vector pGCS in the present invention is obtained by replacing the T7 promoter in the pET28a plasmid with the trc promoter and inserting caR gene and sfPConstructed by gene, disclosed in the literature [Chen Y, Song W, Wang G, et al. Metabolic engineering of high L-lysine-producing Escherichia coli for de novo production of L-lysine-derived compounds[J]. ACS Synthetic Biology, 2024, 13(9): 2948-2959.].

[0037] As one embodiment, the first starting vector is the pETDuet-1 vector. As another embodiment, the T7 promoter in the pETDuet-1 vector is replaced with the trc promoter as the promoter of the first coding gene. As one embodiment, the DavB gene is located between the SalⅠ and AflⅡ restriction enzyme cleavage sites of the pETDuet-1 vector, and the DavA gene is located between the SacⅠ and BamHⅠ restriction enzyme cleavage sites of the pETDuet-1 vector. The trc promoter can enable better expression of the first coding gene and the second coding gene in the host bacterium.

[0038] The recombinant vector provided by the present invention can express and synthesize 5-aminovaleramidase, lysine 2-monooxygenase, carboxylic acid reductase, and phosphopantetheine transferase in the bacterium. A new metabolic pathway for producing 1,5-pentanediol can be constructed only by three key enzymes, namely 5-aminovaleramidase, lysine 2-monooxygenase, and carboxylic acid reductase. The recombinant bacterium constructed by using the recombinant vector provided by the present invention can achieve the synthesis of 1,5-pentanediol. The metabolic pathway is extremely short, the metabolic efficiency is higher, the strain stability is strong, and it is suitable for industrial production.

[0039] Based on the above advantages, the present invention provides the application of the recombinant vector described in the above technical solution in constructing an engineering bacterium for synthesizing 1,5-pentanediol.

[0040] Based on the above advantages, the present invention provides an engineering bacterium for synthesizing 1,5-pentanediol. The engineering bacterium includes a primitive strain and a recombinant vector transferred into the primitive strain; the recombinant vector is the recombinant vector described in the above technical solution; the primitive strain includes Escherichia coli ( Escherichia coli ).

[0041] As one embodiment, the primitive strain can be Escherichia coli. As another embodiment, the Escherichia coli is E.coli QDE. The E.coliQDE is preserved in the laboratory of Qilu University of Technology and is disclosed in the literature

Li N, Xue L, Wang Z, et al. Improving the synthesis efficiency of amino acids such as L-lysine by assembling artificial cellulosome elements Dockerin protein in vivo[J]. Fermentation, 2022, 8(11): 578.

[0042] Based on the above advantages, the present invention provides the use of the recombinant vector described in the above technical solution or the engineered bacterium described in the above technical solution in the synthesis of 1,5-pentanediol.

[0043] Based on the above advantages, the present invention provides a method for synthesizing 1,5-pentanediol, comprising the following steps:

[0044] Ferment the engineered bacterium described in the above technical solution in a fermentation broth containing glucose to obtain a fermentation broth containing 1,5-pentanediol.

[0045] As an implementation manner, the fermentation broth further contains pyruvic acid and isopropyl-β-D-thiogalactopyranoside.

[0046] To further illustrate the present invention, the recombinant vector, the engineered bacterium for synthesizing 1,5-pentanediol and their applications provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1 Construction of Recombinant Plasmid

[0048] 1. Obtain from NCBI DavA The nucleotide sequences of the genes (GenBank: CP061335.1) and DavB (GenBank: CP101700.1) genes were optimized according to the usage frequency of rare codons in Escherichia coli and were entrusted to GenScript Biotech Corporation for synthesis to obtain the optimized DavB gene sequence (SEQ ID NO.1) and DavA gene sequence (SEQ ID NO.2), specifically as follows:

[0049] DavB Gene (SEQ ID NO.1):

[0050]

[0051] DavA Gene (SEQ ID NO.2):

[0052] 5'-atgcgtatcgcgctgtaccagggcgcgccgaaaccgctggatgttccgggtaacctgcagcgtctgcgtcaccaggcgcagctggcggctgaacgtggtgcgcagctgctggtgtgcccggaaatgttcctgaccggctacaacatcggcctggcgcaggttgaacgtctggcggaagcggcggacggtccggcggcgatgaccgttgttgaaatcgcgcaggcgcaccgtatcgcgatcgtttacggttacccggaacgtggtgatgatggtgcgatctacaacagcgttcagctgatcgatgcgcacggtcgttctctgtctaactaccgtaaaacccacctgttcggtgaactggatcgtagcatgttctctccgggcgcggatcacttcccggttgttgaactggaaggttggaaagttggcctgctgatctgctacgacatcgaatttccggaaaacgcgcgtcgtctggcgctggatggtgcggaactgatcctggttccgaccgcgaacatgactccgtacgatttcacctgccaggttaccgttcgtgctcgtgcgcaggaaaaccagtgctacctggtttacgcgaactactgcggtgcggaagatgaaatcgaatactgcggccagagcagcatcatcggtccggatggtagcctgctggcgatggcgggccgtgatgaatgccagctgctggcggaactggaacacgaacgtgttgttcagggccgtaccgcgttcccgtacctgaccgatctgcgtcaggaactgcacctgcgtaaaggctaa-3'.

[0053] 2. Using the optimized DavB gene sequence as a template, perform PCR amplification using primers DavB-F and DavB-R to obtain the DavB gene sequence containing the restriction site sequence; using the optimizedDavA Using the gene sequence as a template, PCR amplification was carried out with primers DavA-F and DavA-R to obtain a gene sequence containing a restriction enzyme site sequence. DavA The primer sequences are as follows:

[0054] DavB-F (SEQ ID NO.3):

[0055] 5'-ggatcttccagagatggatccATGAATAAAAAGAACAGGCACCCC-3';

[0056] DavB-R (SEQ ID NO.4):

[0057] 5'-ctgccgttcgacgatgagctcTTAGTCGGCTAAGGCAATCGG-3';

[0058] DavA-F (SEQ ID NO.5):

[0059] 5'-ggatcttccagagatgtcgacAAGGAGATATACCATGCGTATCGCGCTGTACCAG-3';

[0060] DavA-R (SEQ ID NO.6):

[0061] 5'-ctgccgttcgacgatcttaagTTAGCCTTTACGCAGGTGCAGTTC-3';

[0062] Among them, 5'-ggatcttccagagatggatcc-3' (SEQ ID NO.7) is the BamHⅠ restriction enzyme site sequence and vector homologous arm sequence;

[0063] 5'-ctgccgttcgacgatgagctc-3' (SEQ ID NO.8) is the SacⅠ restriction enzyme site sequence and vector homologous arm sequence;

[0064] 5'-ggatcttccagagatgtcgac-3' (SEQ ID NO.9) is the SalⅠ restriction enzyme site sequence and vector homologous arm sequence;

[0065] 5'-ctgccgttcgacgatcttaag-3' (SEQ ID NO.10) is the AflⅡ restriction enzyme site sequence and vector homologous arm sequence;

[0066] 5'-AAGGAGATATACC-3' (SEQ ID NO.35) is the ribosome binding site (RBS) sequence.

[0067] The reaction system for PCR amplification was as follows: 2 μL of template, 2 μL of upstream primer, 2 μL of downstream primer, 25 μL of 2×Phanta Ultra-Fidelity Enzyme, and 19 μL of ddH2O. The reaction program for PCR amplification was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 45 s, for 30 cycles; final extension at 72°C for 10 min.

[0068] 3. Entrust Sangon Biotech to synthesize the trc promoter sequence (shown in SEQ ID NO.11), specifically as follows:

[0069] 5'-gcatgcCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAACGCTCGTCATCAAAATCACTCCGCGAAATttgacaattaatcatccggctcgtataatgGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATttgacaattaatcatccggctcgtataatgGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATAcatatg-3';

[0070] The trc promoter sequence was ligated between the SphI and NdeI restriction sites of the pETDuet-1 vector, replacing the T7 promoter on the pETDuet-1 vector, to obtain a new pETDuet-1 vector (denoted as the pETDuet vector).

[0071] 4. After subjecting the pETDuet vector obtained in Step 3 and the DavB gene sequence amplified in Step 2 to double digestion with SalⅠ and AflⅡ respectively, ligate overnight at 37°C to obtain the recombinant plasmid pETDuet-d avB .

[0072] 5. After subjecting the recombinant plasmid pETDuet- davB obtained in Step 4 and the DavB gene sequence amplified in Step 2 to double digestion with SacⅠ and BamHⅠ respectively, ligate overnight at 37°C to obtain the recombinant plasmid pETDuet- davB - davA .

[0073] Example 2 Construction of Escherichia coli for Biosynthesizing 1,5-Pentanediol

[0074] Transform the plasmid pET28a- caR - sfP and the recombinant plasmid pETDuet- davB - davA constructed in Example 1 into the same strain of Escherichia coli E.coli QDE, and culture overnight at 37°C on an LB medium containing double resistance of 50 μg / mL kanamycin and 100 μg / mL ampicillin.

[0075] Pick single colonies for PCR identification (primers are shown in Table 1), and the results are as shown in Figure 1 and Figure 2 . Among them, Figure 1 M is Marker DL2000, 1 is the DavA gene, and 2 is the DavB gene; Figure 2 M is Marker DL5000, 1 is the DavA gene, 2 is the caR gene, and 3 is the SfP gene. Determine that the DavB gene, DavA gene, caR gene, Sfp gene have been successfully transferred into Escherichia coli to obtain the recombinant strain QDE01.

[0076] Table 1 Primer Sequences Used for PCR Identification

[0077]

[0078] Example 3 Biosynthesis of 1,5-Pentanediol by Recombinant Strain QDE01

[0079] Activate the recombinant strain QDE01 constructed in Example 2 in a shaker at 37°C to OD 600It was 2 to obtain the seed solution; the seed solution was inoculated into the LB medium containing 0.5 g / L pyruvate at an inoculation amount of 1% (v / v), and cultured in a shaker until the OD 600 value reached 1, then isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.02 mmol / L and 20 g / L glucose were added, and fermentation was carried out in a shaker at 30 °C and samples were taken regularly.

[0080] A control group was set up: the recombinant bacterium QDE01 was replaced with a control strain for fermentation; the construction method of the control strain was similar to that of the recombinant bacterium QDE01, except that the plasmid pET28a- caR - sfP was replaced with the plasmid pET28a (the T7 promoter was replaced by the trc promoter), and the recombinant plasmid pETDuet- davB - davA was replaced with the pETDuet vector constructed in Example 1.

[0081] The ultraviolet high performance liquid chromatography determination method was used to detect the yields of 1,5-pentanediol, 5-hydroxypentanoic acid and 5-hydroxypentanal in the fermentation broth every 1 d, and the results were as Figure 3 shown.

[0082] From Figure 3 it can be seen that the DavB gene 、DavA gene and caR gene after expressing the optimized codons of the present invention, using three key enzymes of 5-aminovaleramidase, lysine 2-monooxygenase and carboxylic acid reductase can construct a complete 1,5-pentanediol metabolic pathway, and 1.28 g / L of 1,5-pentanediol can be biosynthesized under the condition of consuming 19.43 g / L of glucose. A complete and efficient 1,5-pentanediol biosynthesis pathway was constructed. The control E.coli QDE did not produce 1,5-pentanediol.

[0083] Comparative Example 1

[0084] The construction method of the recombinant bacterium QDE02 was similar to that of the recombinant bacterium QDE01, except that the recombinant plasmid pETDuet- davB - davA was replaced with the recombinant plasmid pETDuet- davB - davA -g abT - yahK ;

[0085] The construction method of the recombinant bacterium QDE03 was similar to that of the recombinant bacterium QDE01, except that the recombinant plasmid pETDuet- davB - davA was replaced with the recombinant plasmid pETDuet- davB - davA - davT - eyqhD .

[0086] The recombinant plasmid pETDuet- davB - davA -gabT - yahK The construction method is as follows:

[0087] 1) Using the plasmid pGGY containing the 4-aminobutyrate aminotransferase encoding gene ( gabT gene) as a template, PCR amplification was performed using primers gabT-F and gabT-R to obtain gabT the gene sequence; using the plasmid pGGY containing yahK gene as a template, PCR amplification was performed using primers yahK-F and yahK-R to obtain yahK the gene sequence;

[0088] 2) Insert the gabT gene sequence between the NdeⅠ and MunⅠ restriction enzyme cleavage sites of the recombinant plasmid pETDuet- davB - davA constructed in Example 1 to obtain the recombinant plasmid pETDuet- davB - davA -g abT ;

[0089] 3) Insert the yahK gene sequence between the SgfⅠ and KpnⅠ restriction enzyme cleavage sites of the recombinant plasmid pETDuet- davB - davA -g abT to obtain the recombinant plasmid pETDuet- davB - davA -g abT - yahK .

[0090] The construction method of the recombinant plasmid pETDuet- davB - davA - davT - eyqhD is as follows:

[0091] 1) Using the plasmid pGDE containing davT gene as a template, PCR amplification was performed using primers davT-F and davT-R to obtain davT the gene sequence; using the plasmid pGDE containing eyqhD gene as a template, PCR amplification was performed using primers eyqhD-F and eyqhD-R to obtain eyqhD the gene sequence;

[0092] 2) Insert the davT gene sequence between the NdeⅠ and MunⅠ restriction enzyme cleavage sites of the recombinant plasmid pETDuet- davB - davA constructed in Example 1 to obtain the recombinant plasmid pETDuet- davB - davA - davT ;

[0093] 3) Insert the eyqhD gene sequence into the recombinant plasmid pETDuet- davB - davA - davTBetween the SgfⅠ and KpnⅠ restriction sites, the recombinant plasmid pETDuet- davB - davA - davT - eyqhD .

[0094] Both the plasmid pGGY and the plasmid pGDE are disclosed in the literature [Chen Y, Song W, Wang G, et al. Metabolic engineering of high L-lysine-producing Escherichia coli for de novo production of L-lysine-derived compounds[J]. ACS Synthetic Biology, 2024, 13(9): 2948-2959.]. The primer sequences are shown in Table 2.

[0095] Table 2 Sequences of different primers

[0096]

[0097] Note: 5'-taagaaggagatatacatatg-3' (SEQ ID NO.30) is the NdeⅠ restriction site sequence and the vector homologous arm sequence; 5'-tggccggccgatatccaattg-3' (SEQ ID NO.31) is the MunⅠ restriction site sequence and the vector homologous arm sequence; 5'-atcggccggccacgcgatcgcaaggag-3' (SEQ ID NO.32) is the SgfⅠ restriction site sequence and the vector homologous arm sequence; 5'-ctgccgttcgacgatggtacc-3' (SEQ ID NO.33) and 5'-tttrccagactcgagggtrcc-3' (SEQ ID NO.34) are both the KpnⅠ restriction site sequence and the vector homologous arm sequence.

[0098] Comparative Example 2

[0099] After verifying the constructed recombinant bacteria QDE01, QDE02, and QDE03 without errors, fermentation was carried out by the method of Example 3 and samples were taken regularly. The yields of 1,5-pentanediol and the cell concentrations in the fermentation broths of different recombinant bacteria were detected, and the results are shown in Figure 4 and Figure 5 .

[0100] The results show that the 1,5-PDO production of the recombinant strain QDE01 reached 1.3 g / L at 6 d. The 1,5-PDO production of strains QDE02 and QDE03 was only 1.13 g / L and 0.87 g / L at 6 d. In addition, the cell concentrations of QDE02 and QDE03 were significantly lower than that of the recombinant strain QDE01. It can be seen that excessive insertion of genes on the plasmid is not conducive to the growth of recombinant bacteria, thereby affecting the 1,5-pentanediol production. The recombinant strain QDE01 constructed in the present invention has a shorter metabolic pathway, a high 1,5-pentanediol production, and good cell growth, which is more conducive to the later industrial production.

[0101] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Construct a recombinant vector of an engineering bacterium, characterized in that: The first recombinant vector and the recombinant vector pET28a- R - Sf composition; The first recombinant vector consists of a first starting vector and a first coding gene inserted into the first starting vector; the first coding gene consists of a 5-aminovaleramidase coding gene and a lysine 2-monooxygenase coding gene; the amino acid sequence of the 5-aminovaleramidase is shown in SEQ ID NO.28; the amino acid sequence of the lysine 2-monooxygenase is shown in SEQ ID NO.29; the nucleotide sequence of the 5-aminovaleramidase coding gene is shown in SEQ ID NO.2; and the nucleotide sequence of the lysine 2-monooxygenase coding gene is shown in SEQ ID NO.

1.

2. The recombinant vector according to claim 1, characterized in that The first starting vector is a pETDuet-1 vector.

3. The recombinant vector according to claim 1, characterized in that The promoter of the first coding gene is trc promoter.

4. Use of the recombinant vector according to any one of claims 1 to 3 in constructing an engineered bacterium for synthesizing 1,5-pentanediol.

5. An engineered bacterium for synthesizing 1,5-pentanediol, characterized in that: The engineered bacteria include an original strain and a recombinant vector transferred into the original strain; the recombinant vector is the recombinant vector according to any one of claims 1 to 3; the original strain includes Escherichia coli ( Escherichia coli ).

6. Use of the recombinant vector according to any one of claims 1 to 3 or the engineered bacteria according to claim 5 in the synthesis of 1,5-pentanediol.

7. A method for synthesizing 1,5-pentanediol, characterized in that: The following steps are involved: The engineered bacteria according to claim 5 are fermented in a fermentation liquid containing glucose to obtain a fermentation liquid containing 1,5-pentanediol.

8. The method according to claim 7, characterized in that The fermentation broth also contains pyruvic acid and isopropyl-β-D-thiogalactopyranoside.

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