Fusion protein for promoting synthesis of 1, 3-propylene glycol and application thereof

Abstract

The invention belongs to the field of biology, and particularly relates to a fusion protein for promoting synthesis of 1, 3-propylene glycol and application of the fusion protein. The fusion protein NADPH (Nicotinamide Adenine Dinucleotide Phosphate) dependent alcohol dehydrogenase fusion protein or NADH (Nicotinamide Adenine Dinucleotide Hormone) dependent alcohol dehydrogenase fusion protein disclosed by the invention can be packaged into diol dehydratase and can promote the synthesis of 1, 3-propylene glycol. Experiments prove that the bacillus subtilis has an effect of improving the fermentation performance of the strain.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to a fusion protein for promoting the synthesis of 1,3-propanediol and an application thereof. Background Art

[0002] 1,3-Propanediol is an important diol that can be condensed with dicarboxylic acids to form polyester polymer materials. It can also be used to produce plasticizers, surfactants, emulsifiers, demulsifiers, lubricants, dyes, inks, antifreeze, cosmetics, etc.

[0003] The acrolein method and the ethylene oxide method are currently the main chemical synthesis methods used for 1,3-propylene glycol. These two technologies are mature and reliable, but the production costs are high and they produce waste that pollutes the environment, limiting their large-scale application.

[0004] Compared with chemical synthesis, biosynthesis is more environmentally friendly, and there are many strains in nature that naturally produce high 1,3-propanediol, such as Klebsiella pneumoniae, Klebsiella oxytoca, and Citrobacter freundii. These strains synthesize 1,3-propanediol by metabolizing glycerol under anaerobic or micro-anaerobic conditions and obtain the substances and energy required for growth.

[0005] In the metabolic pathway for the synthesis of 1,3-propanediol, glycerol dehydration is an extremely critical step. According to the cofactor dependence, glycerol dehydratase can be divided into two types, one is glycerol dehydratase that depends on vitamin B12, and the other is glycerol dehydratase that does not depend on vitamin B12. The homology of these two enzymes is low, but the catalytic principle is similar. Both use adenosine free radicals to complete the catalysis. The difference is that the adenosine free radicals required for the glycerol dehydratase that depends on vitamin B12 come from vitamin B12, and the adenosine free radicals required for the glycerol dehydratase that does not depend on vitamin B12 come from S-adenosylmethionine. However, in the actual catalytic environment, the glycerol dehydratase that depends on vitamin B12 can tolerate a certain concentration of oxygen, while the glycerol dehydratase that does not depend on vitamin B12 is extremely sensitive to oxygen. Therefore, in engineering applications, the glycerol dehydratase that depends on vitamin B12 is mainly used. For example, DuPont developed an engineered Escherichia coli strain (CN100471946C) that can synthesize 1,3-propanediol using glucose as a raw material through one-step fermentation, and Suman Lama modified an engineered Klebsiella pneumoniae strain that can synthesize 1,3-propanediol using glucose as a carbon source (https: / / doi.org / 10.1016 / j.ymben.2020.09.001).

[0006] According to the different organizational forms of the catalytic protein, the glycerol dehydratase that depends on vitamin B12 can be divided into two types: one is the water-soluble glycerol dehydratase dissolved in the cytoplasm, and the other is the diol dehydratase that is assembled with other auxiliary proteins to form a catalytic microbody. The functions and structures of glycerol dehydratase and diol dehydratase are very similar. Both can catalyze the dehydration of glycerol, but the function of diol dehydratase is more inclined to the utilization of 1,2-propanediol. Among different types of Klebsiella, some have both glycerol dehydratase operons ( Figure 1 ) and the diol dehydratase operon ( Figure 2 ) such as Klebsiella pneumoniae; some only have a glycerol dehydratase operon such as Klebsiella terrestris; some only have a diol dehydratase operon such as Klebsiella michella.

[0007] The glycerol dehydratase operon is composed of multiple components and is a relatively complex structure ( Figure 1 ). In comparison, the structure of diol dehydratase is more complex, consisting of an operator sequence containing a total of 19 genes ( Figure 2 ), these proteins will be tightly bound to form a cell microcompartment structure after expression. Bacterial microcompartments are protein organelles widely present in bacteria. They are composed of a protein shell that encapsulates the enzyme core and active intermediates. They have functions such as accelerating catalysis, preventing side reactions, and encapsulating toxic intermediates. The first bacterial microcompartment identified was the carboxysome, which was used to enhance carbon dioxide fixation. More extensive studies have shown that bacterial microcompartments are involved in the decomposition and metabolism of 1,2-propanediol, ethanolamine, choline, glycerol, rhamnose, caramel, and fucoidan.

[0008] In Klebsiella, the diol dehydratase microcompartment is composed of multiple shell proteins, forming a closed envelope of 100-150 nanometers, which contains diol dehydratase and propanediol dehydrogenase. These eight shell proteins are divided into three categories: hexamers, trimers and pentamers. They form a polymorphic pseudo-icosahedral shell around the enzyme, encapsulating proteins related to propanediol metabolism in the microchamber for reaction. The dense space will increase the reaction efficiency and avoid the diffusion of intermediate aldehydes, reducing cytotoxicity. From the perspective of spatial density and encapsulation of toxic substances, the diol dehydratase microcompartment should have more advantages in the metabolic pathway of catalyzing the synthesis of 1,3-propanediol. However, the original function of the natural diol dehydratase microcompartment is to metabolize 1,2-propanediol in the environment into propionic acid for cell growth. It lacks alcohol dehydrogenase that can catalyze the reduction of glycerol dehydration product 3-hydroxypropanal to synthesize 1,3-propanediol. 3-hydroxypropanal can only be discharged from the microcompartment into the cytoplasm to be reduced by soluble alcohol dehydrogenase in the cytoplasm, which leads to the unsmooth synthesis of 1,3-propanediol and low synthesis efficiency. In order to improve the defects of the diol dehydratase microcompartment in the synthesis of 1,3-propanediol, it is necessary to supplement the alcohol dehydrogenase activity of the microcompartment through molecular biological modification, so that the synthesis of 1,3-propanediol can be completed in the microcompartment and avoid the excretion of the toxic product 3-hydroxypropanal. Summary of the invention

[0009] The present invention mainly develops NADPH-dependent alcohol dehydrogenase fusion protein (abbreviated as L-Yqhd) and NADH-dependent alcohol dehydrogenase fusion protein (abbreviated as L-Dhat) that can be packaged into diol dehydratase microcompartments and promote 1,3-propanediol synthesis, and verifies its improvement on the fermentation performance of the strain. It is achieved by increasing the anchor sequence in the aforementioned catalytic microbody of the NADPH-dependent alcohol dehydrogenase fusion protein and the NADH-dependent alcohol dehydrogenase fusion protein (excluding methionine encoded by the start codon).

[0010] The present invention thus provides a fusion protein in tandem with an NADPH-dependent alcohol dehydrogenase due to an anchor sequence in the catalytic microbody, or a fusion protein in tandem with an NADH-dependent alcohol dehydrogenase.

[0011] The NADPH-dependent alcohol dehydrogenase Yqhd is derived from Escherichia coli and is an NADPH-type alcohol dehydrogenase; the NADH-dependent alcohol dehydrogenase Dhat is derived from Klebsiella pneumoniae and is an NADH-type alcohol dehydrogenase. The similarity between the two is that they can catalyze 3-hydroxypropanal to synthesize 1,3-propanediol, and the similarities and differences are that they use different cofactors.

[0012] Preferably, the amino acid sequence of the anchor sequence is MNTSELETLIRTILSEQL (SEQ ID NO: 1) or MKTFSLQTRLYSGQGSLA (SEQ ID NO: 2).

[0013] More preferably, the amino acid sequence of the fusion protein L-Yqhd in which the anchor sequence is connected in series with the NADPH-dependent alcohol dehydrogenase is (SEQ ID NO: 3):

[0014] MNTSELETLIRTILSEQLNNFNLHTPTRILFGKGAIAGLREQIPHDARVLITYGGGSVKKTGVLDQVLDALKGMDVLEFGGIEPNPAYETLMNAVKLVREQKVTFLLAVGGGSVLDGTKFIAAAANYPENIDPWHILQTGGKEIKSAIPMGCVLTLPATGSESNAGAVISRKTTGDKQAFHSAHVQPVFAVLDPVYTYTLPP RQVANGVVDAFVHTVEQYVTKPVDAKIQDRFAEGILLTLIEDGPKALKEPENYDVRANVMWAATQALNGLIGAGVPQDWATHMLGHELTAMHGLDHAQTLAIVLPALWNEKRDTKRAKLLQYAERVWNITEGSDDERIDAAIAATRNFFEQLGVPTHLSDYGLDGSSIPALLKKLEEHGMTQLGENHDITLDVSRRIYEAAR.

[0015] Preferably, the amino acid sequence of the fusion protein L-Dhat in which the anchor sequence is connected in series with the NADH-dependent alcohol dehydrogenase is (SEQ ID NO: 4):

[0016] MKTFSLQTRLYSGQGSLASYRMFDYLVPNVNFFGPNAISVVGERCQLLGGKKALLVTDKGLRAIKDGAVDKTLHYLREAGIEVAIFDGVEPNPKDTNVRDGLAVFRREQCDIIVTVGGGSPHDCGKGIGIAATHEGDLYQYAGIETLTNPLPPIVAVNTTAGTASEVTRHCVLTNTETKVKFVIVSWRNLPSVSINDPLLMI GKPAALTAATGMDALTHAVEAYISKDANPPVTDAAAMQAIRLIARNLRQAVALGSNLQARENMAYASSLLAGMAFNNANLGYVHAMAHQLGGLYDMPHGVANAVLLPHVARYNLIANPEKFADIAELMGENITGLSTLDAAEKAIAAITRLSMDIGIPQHLRDLGVKEADFPYMAEMALKDGNAFSNPRKGNEQEIAAIFRQAF.

[0017] The present invention also provides a gene encoding the fusion protein.

[0018] Preferably, the nucleotide sequence of the gene encoding the fusion protein L-Yqhd in which the anchor sequence is connected in series with the NADPH-dependent alcohol dehydrogenase is (SEQ ID NO: 5):

[0019]

[0020] Preferably, the anchor sequence and the nucleic acid sequence of the gene encoding the NADH-dependent alcohol dehydrogenase fusion protein L-Dhat (SEQ ID NO: 6):

[0021]

[0022] The present invention provides a recombinant expression vector containing the coding gene. Specifically, it is a prokaryotic expression vector. The present invention further provides a recombinant host bacteria containing the coding gene. Preferably, it is Klebsiella michiganensis. More preferably, the coding gene is integrated into the genome of Klebsiella michiganensis. More preferably, it is integrated into the pduP gene site or pduQ site of Klebsiella michiganensis. More specifically, in the embodiment, the coding gene of the fusion protein L-Yqhd in which the anchor sequence is connected in series with the NADH-dependent alcohol dehydrogenase is integrated into the pduP site, and the coding gene of the fusion protein L-Dhat of the anchor sequence and the NADH-dependent alcohol dehydrogenase is integrated into the pduQ site.

[0023] The present invention further provides the use of the encoding gene or its expression vector or its recombinant host bacteria in the preparation of 1,3-propanediol.

[0024] The present invention also provides a method for preparing 1,3-propylene glycol, which comprises the following steps: fermenting the recombinant host bacteria to produce 1,3-propylene glycol, and further comprising the step of collecting the produced 1,3-propylene glycol.

[0025] Preferably, the recombinant host bacteria is a recombinant Klebsiella michiganensis. The specific method is to inoculate the recombinant Klebsiella michiganensis into a fermentation medium, culture at 37° C., stir at 350 rpm, aerate at 0.5 vvm, and control the pH to 7.0. After 24 hours of fermentation, 150 g of glycerol is added again to make a total of 100 g / L with the glycerol concentration in the initial culture medium. After 60 hours of fermentation, the fermentation is terminated to obtain 1,3-propanediol.

[0026] In order to improve the catalytic function of the catalytic microbody, the present invention uses the anchor sequence in the catalytic microbody to construct the alcohol dehydrogenase Yqhd and Dhat into fusion proteins L-Yqhd and L-Dhat, so that they can be assembled into the catalytic microbody and improve the function of the catalytic microbody, thereby enhancing the strain's production capacity for 1,3-propylene glycol. The reason for carrying out the present invention is that the diol dehydratase catalytic microbody can increase the reaction concentration of local substances and wrap the toxic intermediates to prevent diffusion because of its dense structure, but the original diol dehydratase catalytic microbody does not have the function of reducing 3-hydroxypropanal to 1,3-propylene glycol, resulting in defects in the synthesis of 1,3-propylene glycol. In addition, most other 1,3-propylene glycol engineering bacteria use soluble proteins corresponding to the function to complete a series of catalysis, but there are defects such as loose catalytic environment and leakage of toxic intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the glycerol dehydratase operon.

[0028] Figure 2 Schematic diagram of the diol dehydratase operon. Among them: pocR, a transcriptional repressor that regulates the expression of the diol dehydratase module; pduF, a propanediol diffusion-promoting protein; pduA, pduB, pduJ, pduM, pduN, pduT, and pduV, constituent proteins of bacterial microcompartments; pduC, pduD, and pduE, three subunits of diol dehydratase; pduG and pduH, diol dehydratase resurrection enzymes; propionyl phosphotransferase, catalyzing the conversion of propionyl-CoA and phosphate into propionyl phosphate and CoA; pduP, propionaldehyde dehydrogenase, catalyzing propionaldehyde into propionyl-CoA and releasing one molecule of NADH; PduQ, propanol dehydrogenase, catalyzing propionaldehyde into propanol; PduS, cobalamin reductase; PduO, adenosine triphosphate cobalamin adenosyltransferase; and propionate kinase, catalyzing propionyl phosphate into propionate and ATP. DETAILED DESCRIPTION

[0029] The present invention is further described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0030] The bacterial species and plasmids used in the examples are shown in Table 1, and the primer information used is shown in Table 2.

[0031] Table 1 Bacterial strains and plasmids used in this study

[0032]

[0033]

[0034] Table 2 Primers used in this study

[0035]

[0036]

[0037] Example 1: Construction and synthesis of genes encoding the fusion protein L-Yqhd of the anchor sequence and the NADPH-dependent alcohol dehydrogenase Yqhd in series and the fusion protein L-Dhat of the anchor sequence and the NADH-dependent alcohol dehydrogenase Dhat in series

[0038] In order to anchor the fusion protein into the diol dehydratase catalytic microbody, the coding nucleotide sequence of the anchor sequence (SEQ ID NO: 1) of the pduP gene site of Klebsiella michiganensis was concatenated with the coding sequence of NADPH-dependent alcohol dehydrogenase Yqhd to obtain the coding gene of L-Yqhd with the nucleotide sequence shown in SEQ ID NO: 5.

[0039] To anchor the fusion protein into the diol dehydratase catalytic microbody, the nucleotide sequence encoding the anchor sequence (SEQ ID NO: 2) of the pduQ gene site of Klebsiella michiganensis was concatenated with the coding sequence of NADH-dependent alcohol dehydrogenase to obtain the coding gene of L-Dhat with the nucleotide sequence shown in SEQ ID NO: 6.

[0040] The above sequence was handed over to a DNA synthesis company to synthesize the corresponding gene fragment for use in subsequent experiments.

[0041] Example 2: Integrating the gene encoding L-Yqhd into Klebsiella michiganensis for expression

[0042] Starting from Klebsiella michiganensis, a two-step homologous recombination method was used to integrate and express the NADPH-dependent alcohol dehydrogenase fusion protein L-Yqhd. The specific steps are as follows:

[0043] In the first step, a basic DNA fragment was amplified to insert and express the L-Yqhd fusion protein at the pduP gene site of Klebsiella michiganensis.

[0044] Using the genome of Klebsiella michiganensis as a template, PCR amplification was performed using primers pduP:L-Yqhd up arm up and pduP:L-Yqhd up arm down to obtain a DNA fragment pduP:L-Yqhd-1. The DNA fragment is 436 bp in size and is the upstream homology arm of the pduP gene.

[0045] Using the genome of Klebsiella michiganensis as a template, PCR amplification was performed using primers pduP:L-Yqhd down arm up and pduP:L-Yqhd down arm down to obtain a DNA fragment pduP:L-Yqhd-2. The DNA fragment is 485 bp in size and is the downstream homology arm of the pduP gene.

[0046] Using pXZ-CS as a template, primers pduP:L-Yqhd cat up and pduP:L-Yqhd sacb down were used for PCR amplification to obtain a DNA fragment pduP:L-Yqhd-3. The DNA fragment is 2665 bp in size and contains a chloramphenicol resistance gene (cat), a fructan sacrose transferase gene (sacB), and a fragment of the overlapping region of 22 bp downstream of the pduP:L-Yqhd-1 fragment and 25 bp upstream of the gene L-Yqhd coding sequence.

[0047] Plasmid L-Yqhd was used as a template and primers pduP:L-Yqhd Yqhd up and pduP:L-Yqhd Yqhd down were used for PCR amplification to obtain the gene fragment pduP:L-Yqhd-4. The size of this DNA fragment is 1234, which contains the fusion protein L-Yqhd coding sequence and the upstream 19bp of the fragment pduP:L-Yqhd-2.

[0048] Using plasmid L-Yqhd as a template, primers pduP:L-Yqhd two-step up and pduP:L-Yqhd two-step down were used for PCR amplification to obtain a DNA fragment pduP:L-Yqhd-5. The DNA fragment is 569 bp in size, including 22 bp downstream of the fragment pduP:L-Yqhd-1 and 547 bp upstream of L-Yqhd.

[0049] In the second step, a two-step homologous recombination fragment was constructed to insert and express the L-Yqhd fusion protein at the pduP gene site of Klebsiella michiganensis.

[0050] Using an equimolar mixture of fragments pduP:L-Yqhd-1, pduP:L-Yqhd-2, pduP:L-Yqhd-3, and pduP:L-Yqhd-4 as a template, PCR amplification was performed using primers pduP:L-Yqhd up arm up and pduP:L-Yqhd down arm down to obtain a DNA fragment pduP:L-Yqhd-I with a size of 4754 bp. It is the splicing product of four fragments, pduP:L-Yqhd-1, pduP:L-Yqhd-2, pduP:L-Yqhd-3, and pduP:L-Yqhd-4, and is a DNA fragment containing the pduP upstream homologous arm, chloramphenicol resistance gene (cat), fructan sucrose transferase gene (sacB), L-Yqhd coding sequence, and pduP downstream homologous arm.

[0051] Using a molar mixture of fragments pduP:L-Yqhd-1 and pduP:L-Yqhd-5 as a template, PCR amplification was performed using primers pduP:L-Yqhd up arm up and pduP:L-Yqhd two-step down to obtain a DNA fragment pduP:L-Yqhd-II. The DNA fragment is 983 bp in size and is the splicing product of the two fragments pduP:L-Yqhd-1 and pduP:L-Yqhd-5. It is a DNA fragment containing both the upstream homology arm of pduP and 547 bp upstream of the coding region of the L-Yqhd gene.

[0052] Step 3: Construction of recombinant bacteria YY-M001

[0053] The fragment pduP:L-Yqhd-I was used for the first homologous recombination: first, the pKD46-km plasmid (nucleotide sequence as shown in SEQ ID NO: 8) was transformed into Klebsiella michiganensis by calcium chloride transformation method, and then the DNA fragment was electroporated into Klebsiella michiganensis carrying pKD46-km. The specific transformation steps are as follows:

[0054] First, prepare Klebsiella michiganensis electroporation competent cells with pKD46-km plasmid (for the specific preparation steps of competent cells, refer to the method in the literature "Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation [J]. Nucleic Acids Res, 1998, 16 (13): 6127-6145"); place 50 μl of competent cells on ice, add 50 ng of DNA fragment pduP:L-Yqhd-I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of bacterial solution and spread it on LB plate containing chloramphenicol (final concentration of 34 μg / ml) and kanamycin (final concentration of 50 μg / ml). After culturing at 37°C overnight, select 8 single colonies and use primers pduP:L-Yqhd uparm up and pduP:L-Yqhd down arm down for PCR verification. The size of the correct colony amplification product is 4754 bp. Select a correct single colony and name it YY-M001.

[0055] Step 4: Construction of recombinant bacteria YY-M002

[0056] The fragment pduP:L-Yqhd-II was used for the second homologous recombination: the DNA fragment pduP:L-Yqhd-II was electroporated into the strain YY-M001. The specific transformation steps are as follows:

[0057] First, prepare the electroporation competent cells of Michigan Klebsiella YY-M001 with pKD46-km plasmid; place 50 μl competent cells on ice, add 50 ng DNA fragment pduP:L-Yqhd-II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameter is 2 kv voltage. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, blow 5 times and transfer to a test tube, incubate at 75 rpm and 30 ° C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml conical flask), culture for 24 hours, and then streak culture on LB solid culture medium without sodium chloride containing 6% sucrose. A single colony was selected and PCR verification was performed using primers pduP:L-Yqhd uparm up and pduP:L-Yqhd two-step down. The size of the correct colony amplification product was 983bp. A correct single colony was selected and named YY-M002.

[0058] Example 3: Integrating the gene encoding L-Dhat into Klebsiella michiganensis for expression

[0059] Starting from Klebsiella michiganensis, a two-step homologous recombination method was used to integrate and express the NADH-dependent alcohol dehydrogenase fusion protein L-Dhat that can be anchored in the diol dehydratase microcompartment. The specific steps are as follows:

[0060] In the first step, a basic DNA fragment was amplified to insert and express the L-Dhat fusion protein at the pduQ gene site of Klebsiella michiganensis.

[0061] Using the genome of Klebsiella michiganensis as a template, PCR amplification was performed using primers pduQ:L-Dhat up arm up and pduQ:L-Dhat up arm down to obtain a DNA fragment pduQ:L-Dhat-1. The DNA fragment is 595 bp in size and is the upstream homology arm of the pduQ gene.

[0062] Using the genome of Klebsiella michiganensis as a template, PCR amplification was performed using primers pduQ:L-Dhat down arm up and pduQ:L-Dhat down arm down to obtain a DNA fragment pduQ:L-Dhat-2. The DNA fragment is 600 bp in size and is the downstream homology arm of the pduQ gene.

[0063] Using pXZ-CS as a template, PCR amplification was performed using primers pduQ:L-Dhat cat up and pduQ:L-Dhat sacb down to obtain a DNA fragment pduQ:L-Dhat-3. The DNA fragment is 2661 bp in size and contains a chloramphenicol resistance gene (cat), a fructan sacose transferase gene (sacB), and a fragment of the 18 bp downstream of the pduQ:L-Dhat-1 fragment and a 25 bp overlapping region upstream of the gene L-Dhat coding sequence.

[0064] Using plasmid L-Dhat as a template, primers pduQ:L-Dhat Dhat up and pduQ:L-Dhat Dhat down were used for PCR amplification to obtain gene fragment pduQ:L-Dhat-4. The size of this DNA fragment is 1236, containing the fusion protein L-Dhat coding sequence and 21bp upstream of the fragment pduQ:L-Dhat-2.

[0065] Using plasmid L-Dhat as a template, primers pduQ:L-Dhat two-step up and pduQ:L-Dhat two-step down were used for PCR amplification to obtain a DNA fragment pduQ:L-Dhat-5. The DNA fragment is 537 bp in size, including 18 bp downstream of the fragment pduQ:L-Dhat-1 and 519 bp upstream of the L-Dhat coding sequence.

[0066] In the second step, a two-step homologous recombination fragment was constructed to insert and express the L-Dhat fusion protein at the pduQ gene site of Klebsiella michiganensis.

[0067] Using an equimolar mixture of fragments pduQ:L-Dhat-1, pduQ:L-Dhat-2, pduQ:L-Dhat-3, and pduQ:L-Dhat-4 as a template, PCR amplification was performed using primers pduQ:L-Dhat uparm up and pduQ:L-Dhat downarm down to obtain a DNA fragment pduQ:L-Dhat-I. The DNA fragment was 5028 bp in size and was the splicing product of four fragments, pduQ:L-Dhat-1, pduQ:L-Dhat-2, pduQ:L-Dhat-3, and pduQ:L-Dhat-4. It was a DNA fragment containing a pduQ upstream homologous arm, a chloramphenicol resistance gene (cat), a fructan sucrose transferase gene (sacB), an L-Dhat coding sequence, and a pduQ downstream homologous arm.

[0068] Using an equimolar mixture of fragments pduQ:L-Dhat-1 and pduQ:L-Dhat-5 as a template, PCR amplification was performed using primers pduQ:L-Dhat uparm up and pduQ:L-Dhat two-step down to obtain a DNA fragment pduQ:L-Dhat-II. The DNA fragment is 1114 bp in size, which is the splicing product of the two fragments pduQ:L-Dhat-1 and pduQ:L-Dhat-5, and is a DNA fragment containing both the upstream homology arm of pduQ and 519 bp upstream of the coding region of the L-Dhat gene.

[0069] Step 3: Construction of recombinant strain YY-M003

[0070] The fragment pduQ:L-Dhat-I was used for the first homologous recombination: first, the pKD46-km plasmid was transformed into Klebsiella michiganensis by calcium chloride transformation method, and then the DNA fragment was electroporated into Klebsiella michiganensis carrying pKD46-km. The specific transformation steps are as follows:

[0071] First, prepare Klebsiella michiganensis electroporation competent cells with pKD46-km plasmid (for the specific preparation steps of competent cells, refer to the method in the literature "Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation [J]. Nucleic Acids Res, 1998, 16 (13): 6127-6145"); place 50 μl of competent cells on ice, add 50 ng of DNA fragment pduQ:L-Dhat-I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. 200 μl of bacterial solution was spread on an LB plate containing chloramphenicol (final concentration of 34 μg / ml) and kanamycin (final concentration of 50 μg / ml). After overnight culture at 37°C, 8 single colonies were selected and PCR verification was performed using primers pduQ:L-Dhat up arm up and pduQ:L-Dhat down arm down. The size of the correct colony amplification product was 5028 bp. A correct single colony was selected and named YY-M003.

[0072] Step 4: Construction of recombinant strain YY-M004

[0073] The fragment pduQ:L-Dhat-II was used for the second homologous recombination: the DNA fragment pduQ:L-Dhat-II was electroporated into strain YY-M003. The specific transformation steps are as follows:

[0074] First, prepare the electroporation competent cells of Michigan Klebsiella YY-M001 with pKD46-km plasmid; place 50 μl competent cells on ice, add 50 ng DNA fragment pduQ:L-Dhat-II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameter is 2 kv voltage. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, blow 5 times and transfer to a test tube, incubate at 75 rpm and 30 ° C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml conical flask), culture for 24 hours, and then streak culture on LB solid culture medium without sodium chloride containing 6% sucrose. A single colony was selected and PCR verification was performed using primers pduQ:L-Dhat up arm up and pduQ:L-Dhat two-step down. The size of the correct colony amplification product was 1114 bp. A correct single colony was selected and named YY-M004.

[0075] Example 4 Fermentation of wild-type Klebsiella michiganensis and recombinant strains YY-M002 and YY-M004 in a 5L fermenter to produce 1,3-propylene glycol

[0076] The fermentation was carried out microaerobically in a 5L fermenter (BIOTECH-5BG, Shanghai Baoxing). The culture medium used was as follows:

[0077] Seed culture medium components: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0078] Fermentation medium components: glycerol (50g / L), yeast extract (1g / L), NH4H2PO4 (1g / L), (NH4)2HPO4 (3g / L), MgSO4·7H2O (1g / L), KCl (74mg / L), FeCl3·6H2O (2.4mg / L), CoCl2·6H2O (0.3mg / L), CuCl2·2H2O (0.15mg / )L, ZnCl2 (0.3mg / L), Na2MoO4·2H2O (0.3mg / L), H3BO3 (0.1mg / L) and MnCl2·4H2O (0.5mg / L). No need to add vitamin B12 or any antibiotics.

[0079] The experiment includes the following steps: (1) Primary seed culture: 20 ml of seed culture medium is placed in a 100 ml Erlenmeyer flask and sterilized at 115°C for 15 min. After cooling, a single clone is picked and inoculated into the culture medium and cultured at 37°C and 250 rpm for 6 hours to obtain the primary seed solution.

[0080] (1) Secondary seed culture: 150 ml of seed culture medium was placed in a 500 ml Erlenmeyer flask and sterilized at 115° C. for 15 min. After cooling, the primary seed solution was inoculated into the seed culture medium at an inoculum rate of 1% (V / V), and cultured at 37° C. and 250 rpm for 12 hours to obtain the secondary seed solution, which was used to inoculate the fermentation medium.

[0081] (2) Fermentation culture: The volume of the fermentation medium in 5 L was 3 L, and sterilized at 115°C for 25 min. The seed solution was diluted to a final concentration of OD 550 =0.1 inoculum was inoculated into the fermentation medium, cultured at 37°C, with a stirring speed of 350 rpm, aeration of 0.5 vvm, and pH controlled at 7.0. After 24 hours of fermentation, 150 g of glycerol was added again, and the total glycerol concentration in the initial medium was 100 g / L. Fermentation was terminated after 60 hours of fermentation.

[0082] The amount of 1,3-propylene glycol was determined as follows:

[0083] Sample preparation: Take the fermentation broth, centrifuge at 12000rpm in a 1.5ml centrifuge tube to take the supernatant, dilute it 10 times and filter it with a 0.22μm filter membrane. Detection conditions: Agilent 1260 liquid chromatograph, differential refractive index detector, Bio-Rad HPX-87H column, 5mM sulfuric acid mobile phase, column temperature 35℃, flow rate 0.5ml / min.

[0084] The experimental results are shown in Table 3.

[0085] Table 3 Fermentation results of wild-type Klebsiella michiganensis, recombinant bacteria YY-M002 and YY-M004

[0086]

[0087] The results of fermentation experiments showed that anchoring the expression of NADPH or NADH-dependent alcohol dehydrogenase in the diol dehydratase catalytic microbody could increase the synthesis capacity, yield, conversion rate and production rate of 1,3-PDO by about 26%, proving that the expression of L-Yqhd and L-Dhat fusion proteins has a promoting effect on the synthesis of 1,3-propanediol.

Claims

1. A fusion protein, which is a fusion protein in which an anchor sequence in a catalytic microbody is connected in series with an NADPH-dependent alcohol dehydrogenase, or a fusion protein in which an anchor sequence in a catalytic microbody is connected in series with an NADH-dependent alcohol dehydrogenase.

2. The fusion protein according to claim 1, characterized in that The NADPH-dependent alcohol dehydrogenase Yqhd is derived from Escherichia coli; the NADH-dependent alcohol dehydrogenase Dhat is derived from Klebsiella pneumoniae; the anchor sequences are the anchor sequences of the pduP site and the pduQ site, respectively, and more specifically, their amino acid sequences are shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein L-Yqhd of the anchor sequence and NADPH-dependent alcohol dehydrogenase in series is shown in SEQ ID NO: 3; the amino acid sequence of the fusion protein L-Dhat of the anchor sequence and NADH-dependent alcohol dehydrogenase in series is shown in SEQ ID NO:

4. The gene encoding the fusion protein according to any one of claims 1 to 3.

5. The coding gene according to claim 4, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:5 or SEQ ID NO:

6.

6. The recombinant expression vector encoding a gene as claimed in claim 4 or 5, preferably it is a prokaryotic expression vector.

7. A recombinant host bacterium containing the encoding gene of claim 4 or 5; preferably, it is Klebsiella michiganensis; more preferably, the encoding gene is integrated into the genome of Klebsiella michiganensis; more preferably, it is integrated into the pduP gene site or pduQ site of Klebsiella michiganensis.

8. Use of the coding gene or its expression vector or its recombinant host bacteria according to claim 4 or 5 in the preparation of 1,3-propylene glycol.

9. A method for preparing 1,3-propylene glycol, comprising the steps of: fermenting the recombinant host bacteria according to claim 7 to produce 1,3-propylene glycol, and further comprising the step of collecting the produced 1,3-propylene glycol.

10. The method according to claim 9, characterized in that The recombinant host bacteria is a recombinant Klebsiella Michigan. A more specific method is to inoculate the recombinant Klebsiella Michigan into a fermentation medium, culture at 37°C, with a stirring speed of 350 rpm, a ventilation volume of 0.5 vvm, and a pH control of 7.

0. After 20-30 hours of fermentation culture, glycerol is again added to a total of 100 g / L with the glycerol concentration in the initial culture medium; and the fermentation is terminated after 40 hours to 80 hours to obtain 1,3-propanediol.

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