Fusion proteins that facilitate 1,3-propanediol synthesis and uses thereof

Abstract

The application belongs to the field of biology and particularly relates to a fusion protein for promoting 1,3-propanediol synthesis and application thereof. The fusion protein is an NADPH-dependent alcohol dehydrogenase fusion protein or an NADH-dependent alcohol dehydrogenase fusion protein, which can be packaged into a diol dehydratase and promote 1,3-propanediol synthesis. It is verified by experiments that the fusion protein has an effect of improving the fermentation performance of a strain.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and particularly relates to a fusion protein for promoting synthesis of 1,3-propanediol and application thereof. BACKGROUND

[0002] 1,3-propanediol is an important diol, which can be used to synthesize polyester high molecular materials by polycondensation with dicarboxylic acid, and can also be used to produce plasticizers, surfactants, emulsifiers, demulsifiers, lubricating oils, dyes, inks, antifreezes, cosmetics and the like.

[0003] Propylene aldehyde method and ethylene oxide method are currently the main chemical synthesis methods for 1,3-propanediol. These two technologies are mature and reliable, but the production cost is high, and waste that pollutes the environment is generated, which limits large-scale application.

[0004] The biosynthesis method is more environmentally friendly than the chemical synthesis method, and there are a variety of natural high-yield 1,3-propanediol strains in nature. For example, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii and the like. These strains synthesize 1,3-propanediol by metabolizing glycerol under anaerobic or microaerobic conditions and simultaneously obtain substances and energy required for growth.

[0005] In the metabolic pathway for synthesizing 1,3-propanediol, glycerol dehydration is a very critical step. According to the dependence on cofactors, glycerol dehydratase can be divided into two types, one is vitamin B12-dependent glycerol dehydratase, and the other is vitamin B12-independent glycerol dehydratase. These two enzymes have low homology but similar catalytic principles, both of which use adenosine radicals to complete catalysis. The difference is that the adenosine radical required by the vitamin B12-dependent glycerol dehydratase comes from vitamin B12, and the adenosine radical required by the vitamin B12-independent glycerol dehydratase comes from S-adenosyl methionine. However, in the actual catalytic environment, the vitamin B12-dependent glycerol dehydratase can tolerate a certain concentration of oxygen, while the vitamin B12-independent glycerol dehydratase is extremely sensitive to oxygen, so the vitamin B12-dependent glycerol dehydratase is mainly used in engineering applications. For example, the E. coli engineering strain developed by DuPont Company for one-step fermentation to synthesize 1,3-propanediol from glucose (CN100471946C), and the Klebsiella pneumoniae engineering strain modified by Suman Lama to synthesize 1,3-propanediol from glucose as carbon source (https: / / doi.org / 10.1016 / j.ymben.2020.09.001).

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

[0007] The glycerol dehydratase operon is composed of multiple components and belongs to a relatively complex structure ( Figure 1 ). In comparison, the diol dehydratase structure is more complex, which is an operon sequence containing 19 genes in total ( Figure 2 ), and these proteins are tightly combined to form a cell microcompartment structure after expression. The bacterial microcompartment is a protein organelle widely existing in the bacterial kingdom, which is composed of a protein shell wrapping the enzyme core and active intermediates, and has functions such as accelerating catalysis, preventing side reactions, and wrapping toxic intermediates. The first identified bacterial microcompartment is carboxysome, which is used to enhance carbon dioxide fixation. More extensive studies have shown that bacterial microcompartment is involved in the catabolism of 1,2-propanediol, ethanolamine, choline, glycerol, rhamnose, caramel, and fucoidan.

[0008] In Klebsiella, diol dehydratase microcompartments are composed of multiple shell proteins, forming a 100-150 nanometer closed envelope, which contains diol dehydratase and propanediol dehydrogenase. The eight shell proteins are divided into three categories: hexamer, trimer and pentamer. They form a polymorphic pseudoicosahedral shell around the enzyme, which encloses the proteins related to propanediol metabolism in the microcompartments for reaction. The dense space increases the reaction efficiency and can also avoid the diffusion of intermediate aldehyde, reducing the cytotoxicity. From the perspective of spatial density and packaging of toxic substances, diol dehydratase microcompartments should have more advantages in the catalytic synthesis of 1,3-propanediol metabolic pathway. However, the native function of diol dehydratase microcompartments 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 3-hydroxypropionaldehyde, the product of glycerol dehydration, to synthesize 1,3-propanediol. 3-hydroxypropionaldehyde can only be reduced by the soluble alcohol dehydrogenase in the cytoplasm after being discharged from the microcompartments. Therefore, the synthesis process of 1,3-propanediol is not smooth, and the synthesis efficiency is low. In order to improve the defects of diol dehydratase microcompartments in the synthesis of 1,3-propanediol, it is necessary to supplement the alcohol dehydrogenase activity of the microcompartments through molecular biology modification, so that the synthesis of 1,3-propanediol is completed in the microcompartments, and the discharge of toxic product 3-hydroxypropionaldehyde is avoided. SUMMARY

[0009] The present application mainly develops NADPH-dependent alcohol dehydrogenase fusion protein (referred to as L-Yqhd) and NADH-dependent alcohol dehydrogenase fusion protein (referred to as L-Dhat) which can be packaged into diol dehydratase microcompartments and promote the synthesis of 1,3-propanediol, and verifies the improvement of fermentation performance of the strain. It is achieved by increasing the anchoring sequence in the catalytic microbody of the NADPH-dependent alcohol dehydrogenase fusion protein and the NADH-dependent alcohol dehydrogenase fusion protein (not containing the methionine encoded by the start codon).

[0010] Therefore, the present application provides a fusion protein in which the anchoring sequence in the catalytic microbody is connected in series with the NADPH-dependent alcohol dehydrogenase, or connected in series with the 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. Their common point is that they can catalyze the synthesis of 1,3-propanediol from 3-hydroxypropionaldehyde, and their difference is that they use different cofactors.

[0012] Preferably, the amino acid sequence of the anchoring 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 of the anchoring sequence in series with NADPH-dependent alcohol dehydrogenase is (SEQ ID NO: 3):

[0014] MNTSELETLIRTILSEQLNNFNLHTPTRILFGKGAIAGLREQIPHDARVLITYGGGSVKKTGVLDQVLDALKGMDVLEFGGIEPNPAYETLMNAVKLVREQKVTFLLAVGGGSVLDGTKFIAAAANYPENIDPWHILQTGGKEIKSAIPMGCVLTLPATGSESNAGAVISRKTTGDKQAFHSAHVQPVFAVLDPVYTYTLPPRQVANGVVDAFVHTVEQYVTKPVDAKIQDRFAEGILLTLIEDGPKALKEPENYDVRANVMWAATQALNGLIGAGVPQDWATHMLGHELTAMHGLDHAQTLAIVLPALWNEKRDTKRAKLLQYAERVWNITEGSDDERIDAAIAATRNFFEQLGVPTHLSDYGLDGSSIPALLKKLEEHGMTQLGENHDITLDVSRRIYEAAR.

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

[0016] MKTFSLQTRLYSGQGSLASYRMFDYLVPNVNFFGPNAISVVGERCQLLGGKKALLVTDKGLRAIKDGAVDKTLHYLREAGIEVAIFDGVEPNPKDTNVRDGLAVFRREQCDIIVTVGGGSPHDCGKGIGIAATHEGDLYQYAGIETLTNPLPPIVAVNTTAGTASEVTRHCVLTNTETKVKFVIVSWRNLPSVSINDPLLMIGKPAALTAATGMDALTHAVEAYISKDANPVTDAAAMQAIRLIARNLRQAVALGSNLQARENMAYASLLAGMAFNNANLGYVHAMAHQLGGLYDMPHGVANAVLLPHVARYNLIANPEKFADIAELMGENITGLSTLDAAEKAIAAITRLSMDIGIPQHLRDLGVKEADFPYMAEMALKDGNAFSNPRKGNEQEIAAIFRQAF.

[0017] The present application also provides a gene encoding the fusion protein as described above.

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

[0019]

[0020] Preferably, the anchor sequence is fused to 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 application provides a recombinant expression vector containing the coding gene. Specifically, it is a prokaryotic expression vector. The present application further provides a recombinant host bacterium 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 the pduQ site of Klebsiella michiganensis, and more specifically, the coding gene of the fusion protein L-Yqhd of the anchor sequence and 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 NADH-dependent alcohol dehydrogenase is integrated into the pduQ site.

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

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

[0025] Preferably, the recombinant host bacterium is a recombinant Klebsiella michiganensis, and the specific method is to inoculate the recombinant Klebsiella michiganensis into a fermentation medium, cultivate at 37°C, stir at a speed of 350 rpm, ventilate at a volume of 0.5vvm, control the pH to be 7.0, inoculate 150g of glycerol again after 24 hours of fermentation of the fermentation medium, and the total concentration of glycerol in the initial fermentation medium is 100g / L; end the fermentation after 60 hours of fermentation, and obtain 1,3-propanediol.

[0026] The present application is to perfect the catalytic function of the catalytic microbody, use the anchor sequence in the catalytic microbody to construct alcohol dehydrogenase Yqhd and Dhat into fusion proteins L-Yqhd and L-Dhat, so that they can be assembled into the catalytic microbody and perfect the function of the catalytic microbody, and further enhance the production capacity of the strain for 1,3-propanediol. The reason for carrying out the present application is that the diol dehydratase catalytic microbody has a dense structure, can increase the reaction concentration of local substances and encapsulate toxic intermediates from diffusion, but the original diol dehydratase catalytic microbody does not have the function of reducing 3-hydroxypropanal to 1,3-propanediol, resulting in its defects in the synthesis of 1,3-propanediol. In addition, most of the other 1,3-propanediol engineering bacteria use functionally corresponding soluble proteins to complete a series of catalysis, but there are defects such as loose catalytic environment and leakage of toxic intermediates. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of glycerol dehydratase operon.

[0028] Figure 2 Schematic diagram of diol dehydratase operon. Wherein: pocR, transcriptional repressor regulating expression of diol dehydratase module; pduF, propylene glycol diffusion promoting protein; pduA, pduB, pduJ, pduM, pduN, pduT, pduV, constituent proteins of bacterial microcompartment; pduC, pduD, pduE, three subunits of diol dehydratase; pduG, pduH, diol dehydratase reactivase; propionyl phosphate transferase, catalyzing conversion of propionyl-CoA and phosphate into propionyl phosphate and CoA; pduP, propionaldehyde dehydrogenase, catalyzing conversion of propionaldehyde into propionyl-CoA and releasing one molecule of NADH; PduQ, propanol dehydrogenase, catalyzing conversion of propionaldehyde into propanol; PduS, cobalamin reductase; PduO, adenosyltransferase of adenosylcobalamin triphosphate; propionate kinase, catalyzing conversion of propionyl phosphate into propionate and ATP. DETAILED DESCRIPTION

[0029] The present application is further described below through specific embodiments, so as to better understand the present application, but does not constitute limitation to the present application.

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

[0031] Table 1 Strains and plasmids used in the present study

[0032]

[0033]

[0034] Table 2 Primers used in the present study

[0035]

[0036]

[0037] Example 1: Construction and synthesis of the coding gene of the fusion protein L-Yqhd of the tandem of anchoring sequence and NADPH-dependent alcohol dehydrogenase Yqhd and the fusion protein L-Dhat of the tandem of anchoring sequence and NADH-dependent alcohol dehydrogenase Dhat

[0038] In order to anchor the fusion protein into the diol dehydratase catalytic microbody, the coding nucleotide sequence of the anchoring sequence (SEQ ID NO: 1) of the pduP gene site of Klebsiella michiganensis is concatenated with the coding sequence of the 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 catalytic microbody of diol dehydratase, the coding nucleotide sequence of the anchor sequence (SEQ ID NO: 2) of the pduQ gene site of Klebsiella michiganensis and the coding sequence of NADH-dependent alcohol dehydrogenase were connected in series to obtain the coding gene of L-Dhat with the nucleotide sequence of SEQ ID NO: 6.

[0040] The above sequence was synthesized by a DNA synthesis company to obtain the corresponding gene fragment for subsequent experiments.

[0041] Example 2: Integration and expression of the coding gene of L-Yqhd in Klebsiella michiganensis

[0042] Starting from Klebsiella michiganensis, the NADPH-dependent alcohol dehydrogenase fusion protein L-Yqhd was integrated and expressed by a two-step homologous recombination method, and the specific steps were as follows:

[0043] First, the basic DNA fragment of L-Yqhd fusion protein was inserted and expressed at the pduP gene site of Klebsiella michiganensis.

[0044] Using the genome of Klebsiella michiganensis as a template, primers pduP:L-Yqhd up arm up and pduP:L-Yqhd up arm down were used for PCR amplification to obtain DNA fragment pduP:L-Yqhd-1. The size of the DNA fragment is 436 bp, which is the upstream homologous arm of the pduP gene.

[0045] Using the genome of Klebsiella michiganensis as a template, primers pduP:L-Yqhd down arm up and pduP:L-Yqhd down arm down were used for PCR amplification to obtain DNA fragment pduP:L-Yqhd-2. The size of the DNA fragment is 485 bp, which is the downstream homologous 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 DNA fragment pduP:L-Yqhd-3. The size of the DNA fragment is 2665 bp, which contains a chloramphenicol resistance gene (cat), a levansucrase gene (sacB), and a 22 bp downstream of fragment pduP:L-Yqhd-1 and a 25 bp upstream of the coding sequence of gene L-Yqhd overlapping region.

[0047] The plasmid L-Yqhd was used as a template, and the 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 the DNA fragment is 1234, which contains the coding sequence of the fusion protein L-Yqhd and the 19bp upstream of the fragment pduP:L-Yqhd-2.

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

[0049] In the second step, the two-step homologous recombination fragment of L-Yqhd fusion protein was inserted and expressed at the pduP gene site of Klebsiella michiganii.

[0050] The molar mixture of the fragments pduP:L-Yqhd-1, pduP:L-Yqhd-2, pduP:L-Yqhd-3, and pduP:L-Yqhd-4 was used as a template, and the primers pduP:L-Yqhd up arm up and pduP:L-Yqhd down arm down were used for PCR amplification to obtain the DNA fragment pduP:L-Yqhd-I. The size of the DNA fragment is 4754bp, which is the splicing product of the four fragments pduP:L-Yqhd-1, pduP:L-Yqhd-2, pduP:L-Yqhd-3, and pduP:L-Yqhd-4. It is a DNA fragment containing the upstream homologous arm of pduP, the chloramphenicol resistance gene (cat), the levansucrose transferase gene (sacB), the coding sequence of L-Yqhd, and the downstream homologous arm of pduP.

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

[0052] In the third step, the construction of the 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 K. michiganensis by calcium chloride transformation method, and then the DNA fragment was electroporated into K. michiganensis with pKD46-km. The specific transformation steps are as follows:

[0054] First, prepare the K. michiganensis electroporation competent cells with the pKD46-km plasmid (the specific preparation steps of the 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”); 50 μl of competent cells were placed on ice, 50 ng of DNA fragment pduP:L-Yqhd-I was added, and the ice was placed for 2 minutes, and then transferred to a 0.2 cm Bio-Rad electric shock cup. Using a MicroPulser (Bio-Rad) electroporator, the electric shock parameters are voltage 2kv. After electric shock, 1 ml of LB medium was quickly transferred to the electric shock cup, blown 5 times and then transferred to a test tube, incubated at 75 rpm and 30°C for 2h. 200 μl of bacterial solution was spread on LB plates containing chloramphenicol (final concentration 34 μg / ml) and kanamycin (final concentration 50 μg / ml), and after overnight culture at 37°C, 8 single colonies were selected and verified by PCR using primers pduP:L-Yqhd up arm up and pduP:L-Yqhd down arm down. The correct colony amplified product was 4754 bp in size, and one correct single colony was selected and named YY-M001.

[0055] Fourth, 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 strain YY-M001. The specific transformation steps are as follows:

[0057] Firstly, prepare the electroporation competent cells of Michigan Klebsiella YY-M001 with pKD46-km plasmid; place 50 μl of the competent cells on ice, add 50 ng of DNA fragment pduP:L-Yqhd-II, and place on ice for 2 min, then transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporation instrument, and the voltage is 2 kv. After the electric shock, quickly transfer 1 ml of LB medium into the electric shock cup, shake 5 times, then transfer to a test tube, and incubate at 30°C at 75 rpm for 4 h. Transfer the bacterial solution to LB liquid medium containing 10% sucrose without sodium chloride (50 ml of medium in a 250 ml flask), and incubate for 24 h, then streak on LB solid medium containing 6% sucrose without sodium chloride. Select a single colony for PCR verification using primers pduP:L-Yqhd up arm up and pduP:L-Yqhd two-step down, and the correct colony has an amplified product of 983 bp. Select a correct single colony, and name it YY-M002.

[0058] Example 3: Integration and expression of the coding gene of L-Dhat in Michigan Klebsiella

[0059] Starting from Michigan Klebsiella, the NADH-dependent alcohol dehydrogenase fusion protein L-Dhat capable of anchoring in the diol dehydratase microcompartment is integrated and expressed by a two-step homologous recombination method, and the specific steps are as follows:

[0060] First, insert and express the basic DNA fragment of L-Dhat fusion protein at the pduQ gene site of Michigan Klebsiella.

[0061] Using the genome of Michigan Klebsiella as a template, use primers pduQ:L-Dhat up arm up and pduQ:L-Dhat up arm down to perform PCR amplification, and obtain DNA fragment pduQ:L-Dhat-1. The size of the DNA fragment is 595 bp, which is the upstream homologous arm of the pduQ gene.

[0062] Using the genome of Michigan Klebsiella as a template, use primers pduQ:L-Dhat down arm up and pduQ:L-Dhat down arm down to perform PCR amplification, and obtain DNA fragment pduQ:L-Dhat-2. The size of the DNA fragment is 600 bp, which is the downstream homologous arm of the pduQ gene.

[0063] The DNA fragment pduQ:L-Dhat-3 was obtained by PCR amplification using the primer pduQ:L-Dhat cat up and pduQ:L-Dhat sacb down with pXZ-CS as template. The size of the DNA fragment is 2661 bp, which contains chloramphenicol resistance gene (cat), levan sucrose transferase gene (sacB) and 18 bp downstream of the fragment pduQ:L-Dhat-1 and 25 bp upstream of the coding sequence of the gene L-Dhat.

[0064] The gene fragment pduQ:L-Dhat-4 was obtained by PCR amplification using the primer pduQ:L-Dhat Dhat up and pduQ:L-Dhat Dhat down with plasmid L-Dhat as template. The size of the DNA fragment is 1236, which contains the coding sequence of the fusion protein L-Dhat and 21 bp upstream of the fragment pduQ:L-Dhat-2.

[0065] The DNA fragment pduQ:L-Dhat-5 was obtained by PCR amplification using the primer pduQ:L-Dhat two-step up and pduQ:L-Dhat two-step down with plasmid L-Dhat as template. The size of the DNA fragment is 537 bp, which contains 18 bp downstream of the fragment pduQ:L-Dhat-1 and 519 bp upstream of the coding sequence of L-Dhat.

[0066] In the second step, the two-step homologous recombination fragment for inserting and expressing the L-Dhat fusion protein at the pduQ gene site of Klebsiella michiganii was constructed.

[0067] The DNA fragment pduQ:L-Dhat-I was obtained by PCR amplification using the primer pduQ:L-Dhat up arm up and pduQ:L-Dhat down arm down with the equimolar mixture of the fragments pduQ:L-Dhat-1, pduQ:L-Dhat-2, pduQ:L-Dhat-3, pduQ:L-Dhat-4 as template. The size of the DNA fragment is 5028 bp, which is the splicing product of the four fragments pduQ:L-Dhat-1, pduQ:L-Dhat-2, pduQ:L-Dhat-3, pduQ:L-Dhat-4 and is a DNA fragment containing the upstream homologous arm of pduQ, chloramphenicol resistance gene (cat), levan sucrose transferase gene (sacB), the coding sequence of L-Dhat and the downstream homologous arm of pduQ.

[0068] The DNA fragment pduQ:L-Dhat-II was obtained by PCR amplification using the primer pduQ:L-Dhat up arm up and pduQ:L-Dhat two-step down and the mixture of the fragments pduQ:L-Dhat-1 and pduQ:L-Dhat-5 as the template. The size of the DNA fragment pduQ:L-Dhat-II is 1114 bp, which is the splicing product of the two fragments pduQ:L-Dhat-1 and pduQ:L-Dhat-5 and contains the upstream homologous arm of pduQ and the 519 bp DNA fragment upstream of the coding region of L-Dhat gene.

[0069] Step 3, construction of the 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 K. michiganensis by the calcium chloride transformation method, and then the DNA fragment was electroporated into the K. michiganensis with the pKD46-km. The specific transformation steps are as follows:

[0071] First, the K. michiganensis with the pKD46-km plasmid was prepared for the electroporation of the competent cells (the specific preparation steps of the 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”); 50 μl of the competent cells were placed on ice, 50 ng of the DNA fragment pduQ:L-Dhat-I was added, and the mixture was placed on ice for 2 minutes, and then was transferred to a 0.2 cm Bio-Rad electroporation cup. The MicroPulser (Bio-Rad) electroporation instrument was used, and the voltage was 2 kv. After the electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, and then was transferred to a test tube after being blown 5 times, and was incubated at 75 rpm and 30°C for 2 h. 200 μl of the bacterial solution was spread on an LB plate containing chloramphenicol (the final concentration was 34 μg / ml) and kanamycin (the final concentration was 50 μg / ml), and was incubated at 37°C overnight. After the incubation, 8 single colonies were selected and subjected to PCR verification using the primers pduQ:L-Dhat up arm up and pduQ:L-Dhat down arm down. The size of the correct amplification product was 5028 bp, and one correct single colony was selected and named YY-M003.

[0072] Step 4, construction of the 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, the electroporation competent cells of K. michiganensis YY-M001 with pKD46-km plasmid were prepared; 50 μΐ of the competent cells were placed on ice, 50 ng of the DNA fragment pduQ:L-Dhat-II was added, and the mixture was placed on ice for 2 min, and then was transferred to a 0.2 cm Bio-Rad electroporation cuvette. The MicroPulser (Bio-Rad) electroporator was used, and the voltage was 2 kv. After the electroporation, 1 ml of LB medium was quickly transferred to the electroporation cuvette, and after 5 times of shaking, the mixture was transferred to a test tube, and was incubated at 30°C at 75 rpm for 4 h. The bacterial solution was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml medium in a 250 ml flask), and was incubated for 24 h. Then, the solution was streaked on LB solid 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 correct colony had an amplified product of 1114 bp, and a correct single colony was selected and was named YY-M004.

[0075] Example 4 Fermentation of wild-type K. michiganensis and recombinant strains YY-M002 and YY-M004 for producing 1,3-propanediol in a 5 L fermenter

[0076] The fermentation was performed in a 5 L fermenter (Shanghai Boxing, BIOTECH-5BG) under micro-aerobic conditions. The culture medium used is as follows:

[0077] The components of the seed culture medium were as follows: 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0078] The components of the fermentation culture medium were as follows: glycerol (50 g / L), yeast extract (1 g / L), NH4H2PO4(1 g / L), (NH4)2HPO4(3 g / L), MgSO4·7H2O (1 g / L), and KCl (74 mg / L), FeCl3·6H2O (2.4 mg / L), CoCl2·6H2O (0.3 mg / L), CuCl2·2H2O (0.15 mg / L), ZnCl2(0.3 mg / L), Na2MoO4·2H2O (0.3 mg / L), H3BO3(0.1 mg / L), and MnCl2·4H2O (0.5 mg / L). No vitamin B12 was added, and no antibiotic was added.

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

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

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

[0082] The amount of 1,3-propanediol is detected by the following method:

[0083] Sample preparation for detection: the fermentation liquid is centrifuged at 12000rpm in a 1.5ml centrifuge tube, and the supernatant is taken. After dilution by 10 times, it is filtered by a 0.22μm filter membrane. The detection conditions are: Agilent 1260 liquid chromatograph, differential refractive index detector, Borel HPX-87H chromatographic column, 5mM sulfuric acid mobile phase, column temperature 35°C, 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, YY-M004

[0086]

[0087] The fermentation experiment results show that the anchoring expression of NADPH or NADH dependent alcohol dehydrogenase in the diol dehydratase catalytic microbody can improve the synthesis ability, yield, conversion rate and production rate of 1,3-PDO by about 26%, which proves that the expression of L-Yqhd, L-Dhat fusion protein has a promoting effect on the synthesis of 1,3-propanediol.

Claims

1. A fusion protein, which is a fusion protein in which an anchoring sequence in a catalytic microbody is tandemly linked to an NADPH-dependent alcohol dehydrogenase, or a fusion protein in which an anchoring sequence in a tandem linked to an NADPH-dependent alcohol dehydrogenase is tandemly linked to an NADPH-dependent alcohol dehydrogenase, as shown in SEQ ID NO: 3; and the amino acid sequence of the fusion protein in which an anchoring sequence in a tandem linked to an NADPH-dependent alcohol dehydrogenase is tandemly linked to an NADPH-dependent alcohol dehydrogenase is shown in SEQ ID NO:

4.

2. The gene encoding the fusion protein as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

4. The recombinant expression vector encoding the gene as described in claim 2 or 3.

5. The recombinant expression vector as described in claim 4, characterized in that, It is a prokaryotic expression vector.

6. A recombinant host bacterium containing the encoding gene of claim 2 or 3.

7. The recombinant host bacterium as described in claim 6, characterized in that, It is Klebsiella Michigani.

8. The recombinant host bacterium as described in claim 7, characterized in that, The encoding gene was integrated into the genome of Klebsiella micranthae.

9. The recombinant host bacterium as described in claim 8, characterized in that, The encoding gene was integrated into the pduP or pduQ site of Klebsiella micrantha.

10. The encoding gene of claim 2 or 3, the recombinant expression vector of claim 4 or 5, or the recombinant host bacterium of any one of claims 6 to 9 in the preparation of 1,3-propanediol.

11. A method for preparing 1,3-propanediol, comprising the steps of: fermenting the recombinant host bacteria according to any one of claims 6 to 9 to produce 1,3-propanediol, further comprising the step of collecting the produced 1,3-propanediol.

12. The method as described in claim 11, characterized in that, The recombinant host bacterium is recombinant Klebsiella Michigani.

13. The method as described in claim 12, characterized in that, The recombinant Klebsiella Michigani was inoculated into a fermentation medium and cultured at 37°C with a stirring speed of 350 rpm, an aeration rate of 0.5 vvm, and a pH of 7.

0. After 20-30 hours of fermentation, glycerol was added again, with a total glycerol concentration of 100 g / L compared to the initial medium. Fermentation was stopped after 40-80 hours to obtain 1,3-propanediol.

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