Recombinant microorganism for producing 3-aminopropanol by fermentation and construction method and application thereof

Abstract

The present application relates to the recombinant microorganism for producing 3-amino propanol by fermentation method and its construction method and application. Specifically, the present application provides a construction method of the recombinant microorganism for producing 3-amino propanol, which is introducing the coding gene of omega-transaminase into the microorganism producing 3-hydroxy propionaldehyde, wherein the omega-transaminase can catalyze the conversion of 3-hydroxy propionaldehyde into 3-amino propanol in the presence of amino donor. The present application introduces the omega-transaminase capable of catalyzing the synthesis of 3-amino propanol with 3-hydroxy propionaldehyde as the substrate into the microorganism producing 3-hydroxy propionaldehyde based on gene editing, molecular cloning and other technologies, which first creates a brand new full-path biosynthesis process route of 3-amino propanol.

Description

Technical Field

[0001] This invention relates to the preparation of 3-aminopropanol, specifically to the preparation and synthesis of 3-aminopropanol via a microbial route. Background Technology

[0002] 3-Aminopropanol (also known as 3-AP) is an important fine chemical intermediate widely used in pharmaceuticals, pesticides, and dyes, playing a crucial role in the synthesis of drugs such as cyclophosphamide and cyclophosphamide, as well as key raw materials such as vitamin B5 (panthenol). With the improvement of people's living standards, the application of panthenol in daily chemical products is increasing, especially in hair care products and specialized cosmetics, thus significantly boosting the market demand for 3-aminopropanol. Currently, the mainstream preparation process for 3-aminopropanol uses 3-hydroxypropionitrile as a raw material, employing a catalyst for hydrogenation reduction to obtain 3-aminopropanol. In addition, several other feasible processes have been reported: ① Synthesizing 3-aminopropanol from ethyl 3-aminopropionate and 2-cyanoethanol; ② Synthesizing ketoximes from methyl isobutyl ketone or cyclohexanone, followed by condensation with acrylonitrile and catalytic hydrogenation cracking to prepare 3-aminopropanol; ③ Synthesizing 3-aminopropanol from dimethyl sulfoxide (DMSO) and dichloromethane; ④ Using 1,4-butyrolactone as a starting material, sequentially adding hydrazine hydrate and sodium nitrite aqueous solution to generate acyl azide, which is then rearranged to generate 3-aminopropanol. However, the above-mentioned process routes generally suffer from problems such as poor catalyst activity, low yield of 3-aminopropanol, high selectivity of by-products, difficulty in product separation, harsh reaction conditions, low safety, and high equipment requirements.

[0003] Patent CN115819253A discloses a process route for the catalytic synthesis of 3-aminopropanol from β-alanine. Compared with the aforementioned production processes, this route is simpler, more environmentally friendly, and sustainable, with its main advantage lying in the acquisition of the raw material (β-alanine). The mainstream preparation process uses β-hydroxypropionitrile, obtained through a series of complex chemical processes from the petroleum derivative acrylic acid. In contrast, the β-alanine used in CN115819253A can be obtained through bioconversion of renewable biomass feedstocks (such as glucose and glycerol). The reaction process is mild and environmentally friendly, and the biosynthesis of β-alanine has already achieved commercial production. Although this "bioconversion + chemical catalysis" process achieves partial green synthesis of 3-aminopropanol, it still does not fundamentally solve the problems of high energy consumption, high cost, and difficulty in guaranteeing product quality in 3-aminopropanol production. Therefore, the development of a fully biological production process for 3-aminopropanol remains a challenge for the industry.

[0004] With the rapid development of synthetic biology, it has attracted much attention due to its green, environmentally friendly, safe, and efficient advantages. Developing methods using synthetic biology to directly produce 3-aminopropanol from renewable biomass feedstocks such as glycerol and glucose is the fundamental way to achieve its truly green production. Unfortunately, a natural biosynthetic pathway for 3-aminopropanol does not exist in nature; therefore, constructing an artificial biosynthetic pathway for 3-aminopropanol is key to realizing its green biomanufacturing. Summary of the Invention

[0005] The purpose of this invention is to provide a biosynthetic pathway for 3-aminopropanol (also known as 3-AP). Through structural studies, 3-aminopropanol has two active functional groups: an amino group (-NH₂) and a hydroxyl group (-OH). Based on the principle of retrosynthesis, the inventors hypothesized that, while keeping the hydroxyl group unchanged, an amino group could be added to the other end of a substrate with an existing hydroxyl group to obtain the target product 3-aminopropanol. After in-depth analysis, 3-hydroxypropanal (also known as 3-HPA) emerged as a candidate substrate. The next question is how to attach an amino group to 3-HPA biologically. Therefore, the screening of specific enzymes is crucial.

[0006] Existing research often uses ω-transaminases to achieve amino group transfer. However, the optimal substrates for ω-transaminases are usually α-keto acids or methyl ketones. Regarding their catalytic activity against aldol substrates, current techniques show that ω-transaminases have a catalytic preference for cyclic or long-chain aliphatic aldols, with catalytic efficiency positively correlated with the carbon chain number of the substrate. The catalytic efficiency for short-chain aldols decreases significantly. Furthermore, there are no reports in the current technology on the use of ω-transaminases to catalyze 3-hydroxypropionaldehyde. Through extensive screening, the applicant has successfully obtained several ω-transaminases (i.e., ω-TA2, ω-TA12, and ω-TA13) with high catalytic activity for 3-HPA, which can catalyze the conversion of 3-HPA to 3-AP with high conversion rates. In this application, ω-TA13 (whose amino acid sequence is shown in SEQ ID NO: 10) was selected as the most effective one by example. However, those skilled in the art will understand that ω-transaminases such as ω-TA2 (whose amino acid sequence is shown in SEQ ID NO: 11) and ω-TA12 (whose amino acid sequence is shown in SEQ ID NO: 12), which can catalyze the synthesis of 3-aminopropanol from 3-hydroxypropanal, can achieve similar technical effects. Therefore, the source of the substrate 3-HPA needs to be addressed next.

[0007] To achieve a fully biosynthetic pathway, the applicant has studied microorganisms that naturally produce 3-HPA. Currently, the main bacterial species used to produce 3-HPA from glycerol include: *Bacillus*, *Citrobacter*, *Enterobacter*, *Clostridium*, *Klebsiella*, and *Lactobacillus*. *Klebsiella pneumoniae* and *Lactobacillus reuteri* have been extensively studied, both of which can naturally synthesize 3-HPA without the need for exogenous addition of expensive vitamin B12. In the preferred embodiment of this application, *Klebsiella pneumoniae*, which has a high 3-HPA production capacity, is selected as the substrate bacteria. However, those skilled in the art will understand that microorganisms capable of naturally producing 3-HPA or genetically engineered to produce 3-hydroxypropionaldehyde can have the same or similar effects. In the 3-HPA synthesis process of this application, if ω-transaminase catalytic conversion is carried out, problems such as substrate transport to the active site, complex operation process, large enzyme usage, and high production cost must be considered. Therefore, this invention proposes for the first time to introduce the coding sequence of ω-transaminase into microorganisms that can produce 3-HPA.

[0008] Therefore, in one aspect of the present invention, a recombinant microorganism for preparing 3-aminopropanol by fermentation and a method thereof are provided, wherein the coding sequence of an ω-transaminase, such as ω-TA13 transaminase, is introduced into the microorganism that produces 3-hydroxypropanol.

[0009] In a specific embodiment of the present invention, the "microorganism producing 3-hydroxypropanal" includes microorganisms that naturally produce 3-hydroxypropanal or recombinant microorganisms that can produce 3-hydroxypropanal after genetic engineering modification.

[0010] In a specific embodiment of the present invention, wild-type Klebsiella pneumoniae, which naturally produces high levels of 3-HPA, was selected as the substrate bacterium, and the encoding gene of ω-TA13 was introduced into it (i.e., the TA enzyme transamination pathway was introduced, for example, the encoding gene of ω-TA13 was introduced into the kana-resistant pUC57 vector and the recombinant vector was transduced into wild-type Klebsiella pneumoniae) to express the ω-TA13 enzyme, thereby opening up the biological pathway for the synthesis of 3-AP in Klebsiella pneumoniae. The product 3-HPA of Klebsiella pneumoniae can be used as a substrate, and under the catalysis of the ω-TA13 enzyme, 3-AP (i.e., 3-aminopropanol) can be obtained through a transamination reaction, realizing the full-pathway biosynthesis of 3-aminopropanol.

[0011] To achieve the accumulation of 3-HPA, in a preferred embodiment of the present invention, the method further includes blocking or weakening the 3-hydroxypropionic acid degradation pathway in the 3-hydroxypropionic acid-producing microorganism. Specifically, when the 3-hydroxypropionic acid-producing microorganism is Klebsiella pneumoniae, there are two downstream degradation pathways of 3-HPA in wild-type Klebsiella pneumoniae. One pathway involves the degradation of 3-HPA to 3-HP by the action of NAD-dependent aldehyde dehydrogenase puuc, and the other pathway involves the degradation of 3-HPA to 1,3-PDO by the action of NADH-dependent alcohol dehydrogenase dhaT or NADPH-dependent alcohol dehydrogenase yqhD (see the literature Production of 3-Hydroxypropionic Acid From Glycerol by Recombinant Klebsiella pneumoniae ΔdhaT ΔyqhD Which Can Produce Vitamin B12 Naturally). Therefore, in order to achieve the accumulation of 3-HPA after fermentation and provide substrate accumulation for the subsequent transaminase transamination reaction, in the preferred embodiment of this application, the puuc, dhaT, and yqhD genes in the wild-type Klebsiella pneumoniae substrate were sequentially knocked out using gene editing technology, and a recombinant Klebsiella strain 3HPAL028 was successfully constructed. This strain lacks the key genes puuc (its nucleotide sequence is shown in SEQ ID NO: 1), dhaT (its nucleotide sequence is shown in SEQ ID NO: 2), and yqhD (its nucleotide sequence is shown in SEQ ID NO: 3) in the two degradation pathways of 3-HPA. Therefore, during fermentation, 3-HPA can be accumulated, providing substrate accumulation for the subsequent transaminase transamination reaction.

[0012] In a more specific embodiment of the present invention, the gene encoding the transaminase ω-TA13 (SEQ ID NO: 4) is ligated into a plasmid (e.g., pUC57) to construct an expression plasmid for ω-TA13 (e.g., pUC57-ω-TA13). This expression plasmid is then introduced into strain 3HPAL028 to obtain the recombinant strain 3HPAL403, which can produce and accumulate 3-HPA while also expressing the transaminase ω-TA13. Thus, ω-TA13 can undergo a transamination reaction using 3-HPA as a substrate to generate 3-AP. Preferably, an amino donor such as L-alanine, a pyridoxal 5'-phosphate cofactor (PLP), and an IPTG inducer are also added to the transamination reaction. Results show that the recombinant strain 3HPAL403 can ferment and produce 3-AP at a yield of 1.8 g / L. The in vivo metabolic pathway for the production of 3-AP by the recombinant strain 3HPAL403 is as follows: Figure 1As shown.

[0013] Those skilled in the art will understand that the innovation of this invention lies in introducing the gene encoding an ω-transaminase that can catalyze the conversion of 3-hydroxypropanal to 3-aminopropanol into Klebsiella pneumoniae, thereby enabling the expression of ω-transaminase. The above-mentioned knockout of the puuc, dhaT, and yqhD genes is merely a preferred embodiment. Those skilled in the art can reasonably expect that the technical effect of producing 3-AP can still be achieved in strains that have not knocked out the aforementioned genes. For example, wild-type Klebsiella pneumoniae strains that knock out or reduce any one of the puuc, dhaT, and yqhD genes or any combination thereof can also produce 3-AP.

[0014] Based on this, in order to increase the yield of 3-AP and reduce costs, this application also carried out a series of optimizations on the strain:

[0015] In a second aspect, the invention further includes constructing an L-alanine cycling pathway in the 3-hydroxypropanal-producing microorganism. Since an amino donor is required for the ω-TA13 transamination-catalyzed synthesis of 3-AP from 3-HPA, L-alanine is used as the amino donor in the embodiments of the invention. However, L-alanine is converted to pyruvate after transamination by ω-TA13. To ensure an effective supply of L-alanine, the inventors considered recycling the transamination-generated pyruvate using alanine dehydrogenase (alaD) to convert it back into L-alanine; however, this process requires the NADH cofactor. Therefore, the inventors also constructed a formate dehydrogenase (FDH) pathway for the regeneration of the NADH cofactor, thereby establishing a complete cycling system that allows the strain to continuously rely on self-circulating L-alanine as an amino donor during fermentation.

[0016] In a specific embodiment, the inventors introduced two genes, alaD (whose nucleotide sequence is shown in SEQ ID NO: 5) and FDH (whose nucleotide sequence is shown in SEQ ID NO: 6), into the second expression frame of an expression plasmid containing the ω-TA13 coding sequence, alaD, and FDH genes, obtaining a recombinant expression plasmid (e.g., pUC57-ω-TA13-alaD-FDH expression plasmid). This plasmid was then introduced into the 3HPAL028 substrate bacteria to obtain the recombinant strain 3HPAL404. The recombinant strain 3HPAL404 contains a complete L-alanine cycle system, and can still maintain 3-AP production even when the L-alanine supply is reduced. For example, in a 2L parallel reactor with other fermentation conditions unchanged, when the L-alanine amino donor amount was adjusted from the initial 7.5 g / L to 1 g / L, the 3-AP production of recombinant strain 3HPAL404 after fermentation was 2.0 g / L, higher than that of recombinant strain 3HPAL403.

[0017] In a third aspect of the invention, for the purpose of reducing costs, a dual-carbon-source metabolic pathway of glycerol and glucose is constructed in the 3-hydroxypropanal-producing microorganism. For example, in a fermentation medium using Klebsiella pneumoniae for one-step fermentation to produce 3-AP, the carbon source is glycerol. For economic reasons, the inventors wish to reduce costs by using a portion of glucose as a carbon source to cultivate the cells; therefore, it is necessary to relieve the inhibition of glucose on delayed-release carbon sources. Numerous studies have reported that when glucose and other carbon sources are present in the culture medium, glucose is preferentially utilized, leading to the degradation of phosphate groups in the PTS (phosphoric acid-dependent saturated starch ... Glc Cascaded transport between transport proteins increases PTS protein dephosphorylation. Unphosphorylated EIIA Glc Proteins inhibit the transport and phosphorylation of carbon sources such as lactose, maltose, and glycerol by binding to their respective transport proteins or kinases. Other extracellular carbon sources cannot be transported into the cell and therefore cannot be absorbed and utilized.

[0018] In a specific embodiment of the present invention, recombinant strain 3HPAL028 was used as the starting strain. The PTS-type glucose transport system in Klebsiella pneumoniae was disrupted. The ptsG gene (its nucleotide sequence is shown in SEQ ID NO: 7), which encodes a component of the glucose-specific PTS transport system in Klebsiella pneumoniae, was knocked out. Furthermore, the glucose-specific non-PTS transport system-related gene glf (its nucleotide sequence is shown in SEQ ID NO: 9) was overexpressed (the principle of which can be found in "Research Progress on the Composition and Function of Bacterial Phosphotransferase System (PTS)"). This resulted in recombinant strain 3HPAL405, thereby relieving the inhibition of glucose on delayed carbon sources and enabling Klebsiella pneumoniae to use some glucose as a carbon source, thus saving economic costs.

[0019] In a further embodiment of the present invention, in order to efficiently convert glycerol and alleviate the feedback inhibition of glycerol kinase glpK by fructose-1,6-bisphosphate, a point mutation was performed on the glpK gene using 3HPAL405 as the starting strain (the nucleotide sequence of glpK* is shown in SEQ ID NO: 8) to obtain the recombinant strain 3HPAL406, which improved the utilization rate of the carbon source glycerol, thereby enabling Klebsiella pneumoniae to grow in a dual carbon source medium of glycerol and glucose and produce 3-HPA. Subsequently, a recombinant expression plasmid containing the ω-TA13, alaD, and FDH genes (e.g., pUC57-ω-TA13-alaD-FDH expression plasmid) was introduced into 3HPAL406, finally obtaining the recombinant strain 3HPAL407, which can produce 3-AP at a yield of 2.5 g / L when the carbon source 40 g / L glycerol in the fermentation broth is replaced with 15 g / L glucose and 10 g / L glycerol while keeping other conditions unchanged. This yield is higher than the 3-AP yield of recombinant strains 3HPAL403 and 3HPAL404.

[0020] In summary, this invention provides a recombinant microorganism for the production of 3-aminopropanol and its construction method. Specifically, this invention uses gene editing technology to knock out the encoding gene of an enzyme involved in the catabolism of 3-hydroxypropanol in *Klebsiella pneumoniae*, obtaining the recombinant strain 3HPAL028. The encoding gene of an ω-transaminase, such as ω-TA13, is introduced into the recombinant strain 3HPAL028 to obtain the recombinant strain 3HPAL403. This strain, after inducing TA enzyme expression, can produce 1.8 g / L of 3-aminopropanol in the fermentation broth via fermentation. Furthermore, an L-alanine cycling pathway is constructed on the recombinant strain 3HPAL403, resulting in strain 3HPAL404 containing a complete L-alanine cycling system, which can maintain 3-AP production even when the L-alanine supply is reduced. A dual-carbon-source metabolic pathway of glycerol and glucose was constructed for the recombinant strain 3HPAL404 to obtain strain 3HPAL407. This strain was finally fermented in a 2L parallel reactor in one step to produce 2.5 g / L of 3-aminopropanol.

[0021] Specifically, the present invention provides the following technical solutions:

[0022] 1. A method for constructing recombinant microorganisms for producing 3-aminopropanol, characterized in that an encoding gene for ω-transaminase is introduced into a microorganism producing 3-hydroxypropanal, wherein the ω-transaminase is capable of catalyzing the conversion of 3-hydroxypropanal to 3-aminopropanol in the presence of an amino donor.

[0023] 2. The construction method according to Project 1, wherein the amino acid sequence of the ω-transaminase is as shown in SEQ ID NO:10, 11 or 12.

[0024] 3. The construction method according to Project 1 or 2, wherein the 3-hydroxypropanal-producing microorganism includes naturally occurring 3-hydroxypropanal-producing microorganisms or genetically engineered 3-hydroxypropanal-producing recombinant microorganisms; preferably, the 3-hydroxypropanal-producing microorganism is Klebsiella pneumoniae or Lactobacillus reuteri; more preferably, the 3-hydroxypropanal-producing microorganism is Klebsiella pneumoniae.

[0025] 4. The construction method according to Project 1 or 2 further includes blocking or weakening the 3-hydroxypropanal degradative metabolic pathway in the 3-hydroxypropanal-producing microorganisms;

[0026] Preferably, when the microorganism producing 3-hydroxypropanal is Klebsiella pneumoniae, any one of the puuc, dhaT, or yqhD genes, or any combination thereof, is knocked out or reduced in the Klebsiella pneumoniae.

[0027] 5. The construction method according to Project 1 or 2, wherein the amino donor is L-alanine, and the construction method further includes constructing an L-alanine cycling pathway in the 3-hydroxypropanal-producing microorganism;

[0028] Preferably, the alaD and FDH genes are introduced or overexpressed in the 3-hydroxypropionaldehyde-producing microorganism;

[0029] Preferably, the nucleotide sequence of the alaD is as shown in SEQ ID NO: 5, and the nucleotide sequence of the FDH is as shown in SEQ ID NO: 6.

[0030] 6. The construction method according to Project 1 or 2 further includes constructing a dual carbon source metabolic pathway of glycerol and glucose in the 3-hydroxypropanal-producing microorganism;

[0031] Preferably, the ptsG gene is knocked out or knocked down in the 3-hydroxypropanal-producing microorganism, and the glf gene is overexpressed; wherein the nucleotide sequence of the ptsG gene is as shown in SEQ ID NO: 7, and the nucleotide sequence of the glf gene is as shown in SEQ ID NO: 9.

[0032] 7. Recombinant microorganisms obtained according to the construction method of any one of items 1-6.

[0033] 8. Application of the recombinant microorganisms described in Project 7 in the production of 3-aminopropanol.

[0034] 9. A method for producing 3-aminopropanol, comprising culturing the recombinant microorganisms described in item 7 in a culture medium;

[0035] Preferably, the culture medium further includes an amino donor;

[0036] Preferably, the amino donor is L-alanine.

[0037] Beneficial effects of the technical solution of this invention

[0038] This invention introduces a transaminase with high transaminase activity against 3-HPA into Klebsiella pneumoniae based on gene editing, molecular cloning and other technologies, to obtain a recombinant Klebsiella pneumoniae capable of one-step fermentation to achieve full-pathway biosynthesis of 3-aminopropanol, thus pioneering a completely new process route for the full-pathway biosynthesis of 3-aminopropanol.

[0039] Furthermore, through further optimization and modification, such as blocking or weakening the 3-hydroxypropionaldehyde decomposition pathway in microorganisms, constructing the L-alanine cycle pathway, and constructing a dual carbon source metabolic pathway of glycerol and glucose, the present invention has further achieved the following advantages: (1) The key genes puuc, dhaT, and yqhD in the two degradation pathways of 3-HPA are missing, thus reducing the degradation of 3-HPA and achieving the accumulation of 3-HPA, providing substrate accumulation for the next transaminase transamination reaction; (2) The alaD and FDH genes required for the L-alanine cycle are introduced, so that when using L-alanine as the amino donor for transamination, there is a complete L-alanine cycle system, and the production of 3-AP can still be maintained when the L-alanine supply is reduced; (3) The ptsG gene is knocked out and the glf gene is introduced or overexpressed, so that the strain can use glucose as a carbon source for growth and fermentation, saving economic costs; (4) The glpK gene in the strain is point-mutated, which promotes the effective conversion of glycerol, improves the utilization rate of carbon source glycerol, and further saves economic costs. Attached Figure Description

[0040] Figure 1 The in vivo metabolic pathway for 3-AP production in Klebsiella pneumoniae was shown;

[0041] Figure 2 The pEcCas plasmid used in the examples is shown in the diagram. Detailed Implementation

[0042] The present invention is further illustrated by the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope or embodiments of the invention. The scope of the invention is limited by the appended claims. Based on this specification and general knowledge in the art, those skilled in the art can clearly understand the scope of the claims. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications and alterations to the technical solutions of the invention, and such modifications and alterations are also included within the scope of the invention.

[0043] Unless otherwise stated, the experimental methods used in the following examples are conventional methods, such as those described in Molecular Cloning: A Laboratory Manual by J. Sambrook et al., and gene editing technologies such as CRISPR-Cas9; unless otherwise stated, all reagents and materials used are commercially available.

[0044] In some implementations, the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae, etc.

[0045] The strains and their related characteristics involved in the embodiments of this disclosure are listed below:

[0046] 3HPAL028 strain: wild-type Klebsiella pneumoniae with the puuc, dhaT, and yqhD genes knocked out, prepared in Example 1;

[0047] 3HPAL403 strain: The gene for ω-TA13 enzyme was transferred into the 3HPAL028 strain and prepared in Example 2;

[0048] 3HPAL404 strain: ω-TA13 coding sequence, alaD and FDH genes were introduced or overexpressed in 3HPAL028 strain, which was prepared in Example 4;

[0049] 3HPAL405 strain: The ptsG gene was knocked out and the glf gene, a glucose-specific non-PTS transport system-related gene, was introduced or overexpressed in the 3HPAL028 strain, which was prepared in Example 6.

[0050] 3HPAL406 strain: The glpK gene was point-mutated in the 3HPAL405 strain (i.e., the ptsG gene was knocked out in the 3HPAL028 strain and the glucose-specific non-PTS transport system-related gene glf was introduced or overexpressed and the glpK gene was point-mutated), which was prepared in Example 6.

[0051] 3HPAL407 strain: ω-TA13 coding sequence, alaD and FDH genes were introduced or overexpressed in 3HPAL406, which was prepared in Example 6.

[0052] Example 1: Construction of recombinant bacteria 3HPAL028

[0053] 1. Construct the pTarget-puuc-N20 plasmid for knocking out the puuc gene.

[0054] 1.1 Design of sgRNA targeting puuc and construction of pTarget-puuc-gRNA

[0055] Using the website http: / / chopchop.cbu.uib.no / #, enter the target gene puuc sequence (its nucleotide sequence is shown in SEQ ID NO: 1) as prompted by the website, and select the highest score sequence as the reference sequence for primer design.

[0056] The results showed that the optimal N20 sequence was: GAAGTTCCACGGCACCACCG.

[0057] The original 20 bp sequence (TGTCGCTGTCTTTAAAGACG) on the pTarget plasmid was mutated to the N20 sequence selected above. The primers were designed as follows.

[0058] puuc-gRNA-F:CTAGTGAAGTTCCACGGCACCACCGGTTTTAGAGCTAGAAATAGCAAGTT

[0059] puuc-gRNA-R:AAAACCGGTGGTGCCGTGGAACTTCACTAGTATTATACCTAGGACTGAGC

[0060] 1.2 Using pTarget plasmid (purchased from Hongxun Biotechnology) as a template and puuc-gRNA-F / R as primers, amplification was performed, and the amplified product was the pTarget-puuc-gRNA linear vector (2118bp).

[0061] The amplification system and procedure are as follows (refer to the instructions of the enzyme preparation used for the amount of each component and the amplification procedure; the total amplification system is generally 50 μL * 4 = 200 μL).

[0062] Table 1 PCR reaction system

[0063]

[0064] Table 2 PCR reaction procedure

[0065]

[0066] The denaturation, annealing, and extension steps are repeated 30 times.

[0067] 1.3 Digestion of residual circular plasmid template in PCR products using DpnI restriction endonuclease

[0068] Since pTarget template plasmid may still remain in the PCR reaction system, it needs to be digested. The system, reaction temperature, and time are as follows: Add 1 µL of DpnI to 50 µL of system, 37℃, 2 h.

[0069] 1.4 Preparation of pTarget-puuc-N20 plasmid

[0070] (1) The digestion products undergo direct chemical transformation.

[0071] Take 2 µL (or increase the amount as needed) of the digested product (i.e., the pTarget-puuc-gRNA linear vector) and add it to 100 µL of DH5α competent cells (purchased from Qingke Biotechnology). Place on ice for 30 min, then heat shock at 42℃ for 60 s. Immediately place on ice for 3-5 min, add 600 µL of LB liquid medium, and incubate at 37℃ and 200 rpm for 45-60 min. Take 200 µL and spread it on LB+SD (spectinomycin hydrochloride, abbreviated as SD) plates. Incubate overnight upside down and verify by sequencing.

[0072] (2) Pick about 5 single clones that grow on the plate and culture them in 5 mL LB + 5 µL SD stock solution. Send the bacterial culture and primers pTarget seq-F / R (F: GGCCTTTTGCTCACATGTTC; R: TAGCACGATCAACGGCACTG) (or select only one of them) for sequencing. Alternatively, the pTarget seq-F / R primer pair can be used to perform colony PCR amplification (465bp). Send the PCR product and primers for sequencing. According to the sequencing results, it was determined that N20 targeting puuc successfully replaced the original sequence.

[0073] (3) Select the correct sequenced single clone, culture it overnight in a shake flask, extract the plasmid, and use the kit purchased from Shanghai Sangon Biotech to obtain the pTarget-puuc-N20 plasmid containing the N20 fragment of puuc.

[0074] 2. Design and Construction of Homologous Arms: Upstream and Downstream Donor Homologous Fragments

[0075] To repair the gaps created by Cas9 protein cleavage of the genome, repair is necessary. The pEcCas plasmid contains a gene for a Red homologous recombinant protein that promotes repair. Therefore, a repair template is required to prevent random repair and mutations. Approximately 800 bp upstream and downstream of the puuc gene from the genome of *Klebsiella pneumoniae* DSM2026 (purchased from Beina Biotechnology) was ligated together as the donor. Primer design is shown in the table below:

[0076] Table 3. Primer sequence list of upstream and downstream homologous arms

[0077]

[0078] 2.1 Using the genome of Klebsiella DSM2026 as a template and puuc-UF / UR as primers, the amplified product was the upstream homologous arm fragment puuc-UP (830bp), and the amplification system used is shown in Table 4; using puuc-DF / DR as primers, the amplified product was the downstream homologous arm fragment puuc-DOWN (804bp), and the amplification system used is shown in Table 5; the amplification program is shown in Table 6 (the amount of each component and the amplification program are based on the instructions of the enzyme preparation used. Generally, the total amplification volume is 50 μL * 4 = 200 μL).

[0079] Table 4 PCR reaction system

[0080]

[0081] Table 5 PCR reaction system

[0082]

[0083] Table 6 PCR reaction procedure

[0084]

[0085] The denaturation, annealing, and extension steps are repeated 30 times.

[0086] 2.3 Upstream and downstream homologous arm connection

[0087] The products obtained from the PCR were purified by gel extraction using a kit purchased from Shanghai Sangon Biotech. The resulting upstream and downstream homologous fragments were puuc-UP and puuc-DOWN, respectively. Using these as templates and puuc-UF and puuc-DR as primers, the upstream homologous arm fragment, Donor-puuc-UP / DOWN (1614 bp), was amplified. The amplification system and procedure are as follows (the amounts of each component and the amplification procedure are based on the instructions for the enzyme preparation used; generally, the total amplification volume is 50 μL * 4 = 200 μL).

[0088] Table 7 PCR Reaction System

[0089]

[0090] Table 8 PCR reaction procedure

[0091]

[0092] The denaturation, annealing, and extension steps are repeated 30 times.

[0093] The PCR product was purified by gel extraction to obtain the upstream homologous arm fragment Donor-puuc-UP / DOWN.

[0094] 3. Transform the pEcCas plasmid into chassis DSM2026 competent cells.

[0095] (1) Preparation of DSM2026 competent cells

[0096] 1) DSM2026 cells (purchased from Beina Biotechnology) were seeded in 5 mL LB tubes and cultured overnight on a shaker at 37°C.

[0097] 2) Inoculate 1% of the culture into a 50 mL LB medium shake flask and incubate at 37 ℃ for 3-4 h. The bacterial biomass OD600 should reach about 0.6 before competent cells can be prepared.

[0098] 3) Pour the mixture into a 50 mL sterile centrifuge tube that has been pre-cooled at 4 ℃, balance it, and centrifuge at 6000 rpm for 5 min.

[0099] 4) Discard the supernatant culture medium, add 30 mL of pre-cooled 10% glycerol (4℃) to suspend the bacterial cells, and centrifuge at 6000 rpm for 5 min. After centrifugation, quickly discard the supernatant (the bacterial cells are easily soluble in 10% glycerol). Repeat this step twice. Add 500-600 μL of 10% glycerol to the centrifuge tube, mix well with a pipette, and dispense 100 μL / tube for immediate use.

[0100] (2) Take the pEcCas plasmid preserved in the laboratory (its pattern can be found in the image). Figure 2 Add approximately 1 μL of 50 ng to 100 µL of LDSM2026 competent cells, place on ice for 30 min, then heat shock at 42°C for 60 s, and immediately place on ice for 3-5 min. Add 600 µL of LB liquid medium and incubate at 37°C and 200 rpm for 45-60 min. Spread 200 µL onto an LB+Kan plate and incubate upside down overnight.

[0101] (3) Sequencing verification: Select about 5 single clones that grow on the plate and culture them in 5 mL LB + 5 µL kan stock solution. Perform colony PCR amplification on the bacterial culture and pEcCas plasmid universal primers (F: tacttttcatactcccgccattcag; R: tgcccgaacagcaactcag). Send the PCR products and primers for sequencing.

[0102] 4. Preparation of puuc gene knockout strains

[0103] 4.1 Preparation of electrocompetent cells

[0104] 1) Inoculate a single clone of the original bacteria or metabolically engineered bacteria to be prepared (i.e., the DSM2026 bacteria transformed with pEcCas plasmid obtained in step 3) into a 5 mL LB test tube (containing pEcCas plasmid, and Kan resistance needs to be added) and culture overnight in a shaker at 37°C.

[0105] 2) Inoculate 1% of the culture into a 50 mL LB medium shake flask, add Kan resistance (50 ng / μL) and 500 μL of 1 M L-arabinose (to induce Cas9 protein expression), and incubate at 37 ℃ for 3-4 h. The competent cells can be prepared when the cell biomass OD600 reaches about 0.6.

[0106] 3) Pour the mixture into a 50 mL sterile centrifuge tube that has been pre-cooled at 4 ℃, balance it, and centrifuge at 6000 rpm for 5 min.

[0107] 4) Discard the supernatant culture medium, add 30 mL of pre-cooled 10% glycerol (4℃) to suspend the bacterial cells, and centrifuge at 6000 rpm for 5 min. After centrifugation, quickly discard the supernatant (the bacterial cells are easily soluble in 10% glycerol). Repeat this step twice. Add 500-600 μL of 10% glycerol to the centrifuge tube, mix well with a pipette, and dispense 100 μL / tube for immediate use.

[0108] 4.2 Electroconversion Experiment

[0109] 1) Prepare the electric transfer cup in advance. If it is a new electric transfer cup, it can be used directly. If it is a reusable electric transfer cup, it is usually stored in alcohol. It needs to be taken out in the laminar flow hood in advance, dried under the ultraviolet lamp of the laminar flow hood, and then pre-cooled before use.

[0110] 2) Add 500 ng of the Donor-puuc-UP / DOWN fragment obtained in step 2.3 and 100 ng of the pTarget-puuc-N20 plasmid obtained in step 1.4 to the prepared electrocompetent cells, mix well with a pipette, and place on ice for 20-30 min.

[0111] 3) Add the ice-baked receptive state to the pre-cooled electrode cup and cover it. This step should be done quickly and on ice, away from fire sources.

[0112] 4) Set the voltage of the electroplaster to 2.5 kV in advance, quickly remove the electrode cup from the ice and wipe the water off the cup wall with absorbent paper (to prevent electrical breakdown), place it on the electroplaster for electric shock, and the electric shock time is generally 5-5.8 ns.

[0113] 5) Immediately after the electric shock, add 0.9 mL of LB medium to the clean bench and incubate at 37°C in a shaker for 2-2.5 h.

[0114] 6) Remove the bacterial culture and centrifuge at 6000 rpm for 3 min to collect the bacteria. In a clean bench, discard 700-800 μL of supernatant with a pipette and resuspend the bacterial cells. Add 100-200 μL of bacterial culture to a solid plate, spread it evenly with a cooled spreader, and incubate overnight at 37 ℃.

[0115] 7) Pick 10 single clones and pipette them into 200ul of LB medium containing kan+SD for colony PCR verification.

[0116] Table 9 PCR Reaction System

[0117]

[0118] Table 10 PCR reaction procedure

[0119]

[0120] The denaturation, annealing, and extension steps are repeated 30 times.

[0121] The obtained PCR product was sequenced, and the sequencing results showed that the puuc gene was successfully knocked out.

[0122] 5. To achieve scarless editing and avoid introducing foreign DNA fragments into the genome, pEcCas and PTarget plasmid loss experiments were performed in this step.

[0123] (1) Select DSM2026 strains with the correct puuc gene knockout and inoculate them with kan, final concentration 10mM L-rhamnose, and incubate overnight at 37℃ and 220r / min.

[0124] (2) Transfer the cells to LB medium without any antibiotics, 37°C, 220 r / min, for 2 h.

[0125] (3) Spread the cells evenly on LB solid medium plates containing sucrose (10 g / L) and incubate overnight at 37°C.

[0126] (4) Select 30 single colonies and screen them on LB and kan+SD plates respectively. Colonies that grow only on LB plates are single clones that have lost pEcCas plasmid and PTarget plasmid. The experimental results show that there are 3 single clones that have lost plasmids, namely DSM2026-△puuc strain.

[0127] 6. The knockout steps for dhaT and yqhD genes are the same as above. After knocking out the three genes, the chassis strain 3HPAL028 is obtained.

[0128] The primer sequences used in the dhaT and yqhD gene knockout process are shown in the table below:

[0129] Table 11 Primer Sequences

[0130]

[0131] Example 2: Construction of BL21(DE3) / pUC57-ω-TA13

[0132] (1) The coding sequence of transaminase ω-TA13 (SEQ ID NO: 4) was placed in the pUC57-kana vector (purchased from Youkang Biotechnology), and PCR amplification was performed using ω-TA13-F and ω-TA13-R as primers to obtain the ω-TA13 fragment.

[0133] Table 12 List of ω-TA13 upper and lower primer sequences

[0134]

[0135] The PCR product was purified by gel extraction to obtain a fragment of 107.3 ng / ul (1389 bp). The gel extraction kit used was purchased from Shanghai Sangon Biotech.

[0136] (2) Double enzyme digestion of linearized vector pUC57-kana

[0137] Following the enzyme digestion system in the table below, the pUC57-kana plasmid (purchased from Youkang Biotechnology) was linearized by double enzyme digestion to obtain the linearized vector pUC57-kana.

[0138] Table 13 Double enzyme digestion reaction system

[0139]

[0140] The double enzyme digestion product was purified by gel recovery to obtain a linearized vector of 56.3 ng / ul.

[0141] (3) The ω-TA13 fragment was ligated to the linearized vector pUC57-kana using the seamless cloning kit (Novizan ClonExpress II One Step Cloning Kit).

[0142] Table 14 Connection System

[0143]

[0144] Prepare the reaction system according to the table above, react at 37°C for 30 minutes, then cool to 4°C or immediately place on ice to cool.

[0145] (4) Take 20 µl of recombinant product and add it to 100 µl of BL21(DE3) competent cells (purchased from Sangon Biotech). Place on ice for 30 min, then heat shock at 42℃ for 90 s. Quickly place on ice for 3-5 min, add 600 µL of LB liquid medium, and incubate at 37℃ and 200 rpm for 45-60 min. Then take 200 µL and spread it on a KANA resistant plate and incubate it upside down overnight.

[0146] (5) Eight single colonies were selected and PCR was performed using primers JD-pUC57-F / R (F: CCCTGATTCTGTGGATAACCGTATTACCG; R: CTGAGAGTGCACCATATGCGGT). The correct colony amplification product size was 1811bp. One correct single colony was selected and the plasmid pUC57-ω-TA13 was extracted by shaking. The plasmid pUC57-ω-TA13 was electroporated into strain 3HPAL028, and the resulting strain was named 3HPAL403.

[0147] Following the above method, the coding genes for ω-TA2 and ω-TA12 were introduced into strain 3HPAL028, respectively, to obtain strains 3HPAL403-2 and 3HPAL403-3.

[0148] The upstream and downstream primer sequences used in the process of introducing the ω-TA2 and ω-TA12 encoding genes are shown in the table below.

[0149]

[0150] Example 3: Fermentation of engineered strain 3HPAL403 in a 2L parallel reactor to produce 3-AP

[0151] (1) Seed culture: 100 ml of seed culture medium was prepared in a 500 ml Erlenmeyer flask and sterilized at 121°C for 20 min. After cooling, recombinant Klebsiella pneumoniae 3HPAL403 was inoculated into the seed culture medium at an inoculation rate of 1% (V / V) and cultured at 37°C and 220 rpm for 12 hours to obtain seed liquid, which was used for inoculation of fermentation medium.

[0152] (2) Fermentation culture: The volume of fermentation medium in 2L is 600mL, and it is sterilized at 121°C for 20min. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (V / V), and cultured at 37°C with a stirring speed of 180rpm, an aeration rate of 0.2vvm, and a pH of 7.0.

[0153] The seed culture medium consists of: 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0154] The fermentation medium consists of: 6 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate heptahydrate, 0.4 g / L citric acid monohydrate, 1 g / L yeast extract, 5 mL / L metal salt, 40 g / L glycerol, and 0.26 g / L sodium sulfate. No vitamin B12 is required. Kana antibiotic is added at a ratio of 0.1%. When the bacterial OD is approximately 0.6, 0.2 mM IPTG, 5 mM / L PLP, and 7.5 g / L L-alanine are added.

[0155] Fermentation results: 3HPAL403 produced 1.8 g / L of 3-aminopropanol after fermentation in a 2L parallel reactor for 24 h; 3HPAL403-2 and 3HPAL403-3 produced 1.5 g / L and 1.6 g / L of 3-aminopropanol, respectively.

[0156] Example 4: Construction of engineered bacteria 3HPAL404

[0157] In this embodiment, the strain was further optimized. Specifically, since an amino donor is required for the ω-TA13 transamination-catalyzed synthesis of 3-AP from 3-HPA, L-alanine is used as the amino donor in this invention. To ensure an effective supply of L-alanine, the alaD and FDH genes are introduced or overexpressed in the strain in this embodiment, thereby establishing a complete L-alanine cycling system, enabling the strain to continuously rely on self-circulating L-alanine as an amino donor during fermentation.

[0158] (1) The gene fragments alaD (the nucleotide sequence of which is shown in SEQ ID NO: 5) and FDH (the nucleotide sequence of which is shown in SEQ ID NO: 6) were amplified by PCR, and the primers used are shown in the table below.

[0159] Table 15 Primer Sequence List

[0160]

[0161] The PCR products were purified by gel extraction to obtain alaD and FDH gene fragments with concentrations of 83 ng / ul and 102.4 ng / ul, respectively (the gel extraction kit used was purchased from Shanghai Sangon Biotech). The two fragments were then subjected to overlap PCR using primers alaD-F / FDH-R to obtain overlapping fragments.

[0162] (2) Double enzyme digestion of linearized vector pUC57-ω-TA13

[0163] The pUC57-ω-TA13 plasmid prepared in Example 2 was linearized by double enzyme digestion according to the enzyme digestion system in the table below.

[0164] Table 16 Double Enzyme Digestion Reaction System

[0165]

[0166] The double enzyme digestion product was purified by gel recovery to obtain the linearized vector pUC57-ω-TA13 with a concentration of 78.3 ng / ul.

[0167] (3) Using a seamless cloning kit, the overlapping fragment obtained in step (1) was ligated to the linearized vector pUC57-ω-TA13 to obtain the recombinant product (i.e., plasmid pUC57-ω-TA13-alaD-FDH).

[0168] Table 17 Connection System

[0169]

[0170] Prepare the reaction system according to the table above, react at 37°C for 30 minutes, then cool to 4°C or immediately place on ice to cool.

[0171] (4) Take 20 µl of the recombinant product obtained in step (3) above and add it to 100 µl of BL21(DE3) competent cells (purchased from Sangon Biotech). Place it on ice for 30 min, then heat shock it at 42℃ for 90 s. Quickly place it on ice for 3-5 min, add 600 µL of LLB liquid medium, and incubate at 37℃ and 200 rpm for 45-60 min. Then take 200 µL and spread it on a KANA resistant plate and incubate it upside down overnight.

[0172] (5) Eight single colonies were selected and PCR verification was performed using primers JD-pUC57-F / R (F: CCCTGATTCTGTGGATAACCGTATTACCG; R: CTGAGAGTGCACCATATGCGGT). The size of the amplified product of the correct colony was 3811bp. The single colonies with the correct bands were selected for sequencing. The colonies with the correct sequence were shaken and the plasmid pUC57-ω-TA13-alaD-FDH was extracted and electroporated into strain 3HPAL028. The resulting strain was named 3HPAL404.

[0173] Example 5: Fermentation of engineered strain 3HPAL404 in a 2L parallel reactor to produce 3-AP

[0174] (1) Seed culture: 100 ml of seed culture medium was prepared in a 500 ml Erlenmeyer flask and sterilized at 121°C for 20 min. After cooling, recombinant Klebsiella pneumoniae 3HPAL404 was inoculated into the seed culture medium at an inoculation rate of 1% (V / V) and cultured at 37°C and 220 rpm for 12 hours to obtain seed liquid, which was used for inoculation of fermentation medium.

[0175] (2) Fermentation culture: The volume of fermentation medium in 2L is 600mL, and it is sterilized at 121°C for 20min. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (V / V), and cultured at 37°C with a stirring speed of 180rpm, an aeration rate of 0.2vvm, and a pH of 7.0.

[0176] The seed culture medium consists of: 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract.

[0177] The fermentation medium consists of: 6 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate heptahydrate, 0.4 g / L citric acid monohydrate, 1 g / L yeast extract, 5 mL / L metal salt, 40 g / L glycerol, and 0.26 g / L sodium sulfate. No vitamin B12 is required. Kana antibiotic is added at a ratio of 0.1%. When the bacterial OD is approximately 0.6, 0.2 mM IPTG, 5 mM / L PLP, and 1.0 g / L L-alanine are added.

[0178] Fermentation results: 3HPAL404 was fermented in a 2L parallel reactor (with the concentration of amino donor L-alanine at 1.0 g / L) for 24 h to produce 2.0 g / L of 3-aminopropanol.

[0179] Example 6: Construction of engineered bacteria 3HPAL405, 3HPAL406, and 3HPAL407

[0180] (1) Using strain 3HPAL028 as the starting strain, the glucose PTS system-specific transporter gene ptsG (its nucleotide sequence is shown in SEQ ID NO: 7) was knocked out, and the glf gene (its nucleotide sequence is shown in SEQ ID NO: 9) was inserted at that site to obtain the modified strain 3HPAL405. The purpose of knocking out the ptsG gene and inserting the glf gene is to relieve the inhibition of glucose on delayed carbon sources, enabling Klebsiella pneumoniae to use glucose as a carbon source and saving economic costs. The knockout step is the same as in Example 1, and the sequences used are shown in the table below.

[0181] Table 18 Primer Sequences

[0182]

[0183] (2) In addition, in order to effectively convert glycerol, alleviate the feedback inhibition of glycerol kinase glpK by fructose-1,6-bisphosphate, and improve the utilization rate of carbon source glycerol, the glpK gene was modified by G304S point mutation using 3HPAL405 as the starting strain to obtain the mutant glpK* gene. The sequence of the glpK* gene is shown in SEQ ID NO: 8. The specific operation is as follows:

[0184] First, a portion of the glpk fragment containing the G304 site was knocked out, and the CM resistance gene was inserted into this region to construct the glpk-CM fragment and glpk*-donor, as follows: Using the DSM2026 genome as a template, the upstream glpk fragment was amplified using primers glpk-UP-F / glpk-UP-R, and the downstream glpk fragment was amplified using primers glpk-down-F / glpk-down-R. Using the fragment containing the CM resistance gene as a template, the CM fragment was amplified using primers cm-F / cm-R. After gel electrophoresis and recovery, the upstream glpk fragment, the CM fragment, and the downstream glpk fragment were subjected to overlap PCR using primers glpk-UP-F / glpk-down-R to obtain glpk-CM. Using the DSM2026 genome as a template, the upstream fragment of glpk* was amplified using primers glpk*-UP-F / glpk-UP*-R, and the downstream fragment was amplified using primers glpk*-down-F / glpk*-down-R. The upstream and downstream fragments of glpk* were then subjected to overlap PCR using primers glpk*-up-F / glpk*-down-R to obtain the glpk*-donor. The target band was recovered from the overlap PCR using a gel. The primer sequences involved are shown in the table below.

[0185] Table 19 Primer Sequences

[0186]

[0187] (3) Then, competent strains were prepared using 3HPAL405 as the starting strain, and pEcCas plasmid was electroporated into them to prepare competent strains containing pEcCas plasmid. Subsequently, the glpk-CM fragment obtained in step (2) above was electroporated into the strain to obtain strains containing glpk-CM fragment. The electroporation and verification steps were the same as in Example 1.

[0188] (4) Construct the pTarget-cm-N20 plasmid. The steps are the same as in Example 1.4, except that the primers are different. The primer sequences used in this step include:

[0189] Cm-N20-F:CTAGTTAATGAAATAAGATCACTACGTTTTAGAGCTAGAAATAGCAAGTT;

[0190] Cm-N20-R: GCTCAGTCCTAGGTATAATACTAGTTAATGAAATAAGATCACTACGTTTT.

[0191] (5) Using the strain containing the glpk-CM fragment obtained in step (3) above as the starting strain, competent cells were prepared. Then, the glpk*-donor and pTarget-cm-N20 plasmids obtained in step (2) above were electroporated. The electroporation, verification and plasmid loss operation steps were the same as in Example 1, and the modified strain 3HPAL406 was obtained.

[0192] (6) Electroporate the pUC57-ω-TA13-alaD-FDH plasmid constructed in Example 4 into strain 3HPAL406, and the resulting strain is named 3HPAL407.

[0193] Example 7: Fermentation of engineered strain 3HPAL407 in a 2L parallel reactor to produce 3-AP

[0194] (1) Seed culture medium components: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract

[0195] (2) Fermentation medium components: 6 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate heptahydrate, 0.4 g / L citric acid monohydrate, 1 g / L yeast extract, 5 mL / L metal salt, 15 g / L glucose, 10 g / L glycerol, and 0.26 g / L sodium sulfate. No VB12 is needed. Add Kana antibiotic at a ratio of 0.1%. When the bacterial OD is approximately 0.6, add 0.2 mM IPTG, 5 mM / L PLP, and 1.0 g / L L-alanine.

[0196] (3) Seed culture: 100 ml of seed culture medium was prepared in a 500 ml Erlenmeyer flask and sterilized at 121°C for 20 min. After cooling, recombinant Klebsiella pneumoniae 3HPAL407 was inoculated into the seed culture medium at an inoculation rate of 1% (V / V) and cultured at 37°C and 220 rpm for 12 hours to obtain seed liquid, which was used for inoculation of fermentation medium.

[0197] (4) Fermentation culture: The volume of fermentation medium in 2L is 600mL, and it is sterilized at 115°C for 30min. The seed liquid is inoculated into the fermentation medium at an inoculation rate of 5% (V / V), and cultured at 37℃±0.5℃, with a stirring speed of 180±10rpm, an aeration rate of 0.2±0.05vvm, a pH of 7.0±0.5, and a fermentation time of 22-25h.

[0198] Fermentation results: 3HPAL407 fermented in a 2L parallel reactor for 24 h to produce 2.5 g / L of 3-aminopropanol.

[0199] Sequence List:

[0200] The nucleotide sequence of the Puuc gene: SEQ ID NO:1:

[0201]

[0202] Nucleotide sequence of the dhaT gene: SEQ ID NO:2:

[0203]

[0204] Nucleotide sequence of the yqhD gene: SEQ ID NO:3:

[0205]

[0206] The coding sequence of the TA13 enzyme: SEQ ID NO:4:

[0207]

[0208] Nucleotide sequence of the alaD gene: SEQ ID NO:5:

[0209]

[0210] The nucleotide sequence of the FDH gene: SEQ ID NO:6:

[0211]

[0212] Nucleotide sequence of the ptsG gene: SEQ ID NO:7:

[0213]

[0214] Nucleotide sequence of the glpK* gene: SEQ ID NO:8:

[0215]

[0216] The nucleotide sequence of the glf gene: SEQ ID NO:9:

[0217]

[0218] SEQ ID NO: 10, Amino acid sequence of ω-TA13 enzyme

[0219] MNMITNHMPTAELQALDAAHHIHPFTTQDDLTAKGARIITRATGVTLTDSEGTEILDAMAGLWCVNIGYGRDELAEVAARQMRELPYYNTFFQTTHIPAIALSAKLAELAPGDLN HVFYAGSGSEANDTNMRMVRTYWAQKGKPEKKIIISRKNAYHGSTMAGASLGGMTPMHEQGGLPIPDVHHIDQPHWYSEGGDMSREEFGLQRAQELEKAILELGEDKVAAFIGEPI QGAGGVVIPPATYWPEIQRICDKYEILLIADEVICGFGRTGNWFGSETVGIKPHIMTIAKGLSSGYAPIGGSIVCDEVAEVIGACEFNHGYTYSGHPVAAAVALENLRILEEEGI VDRVREETAPYLAEKWSSLADHPLVGEARSVGLMGTLALTPNKETRASFAGDAGTIGYICREFCFANNLVMRHVGDRMIISPPLVITKAEIDTLVERARLALDLTLEKIKADGLYK

[0220] SEQ ID NO. 11: Sequence of ω-TA-2 transaminase with accession number 5G09_A

[0221] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0222] SEQ ID NO. 12: Sequence of ω-TA-12 transaminase with accession number WP_076629700.1

[0223] MPAITNHLPTSELQALDAAHHMHPFTAGGELAAKGARVITRANGVFLHDSEGNEILDGMAGLWCVNIGYGRGELADVAARQMRELPYYNTFFQTTHVPAIALTQKIAELAPGDLNHVFFAGSGSEANDTNLRMVRTYWAIKGKPDKHIVISRKNAYHGSSVGSGSLGGMTAMHAQGGLPIPGIVHIDQPNWWAEGGSMSREEFGVSRAKQLEVAILEHGEDKVAAFIAEPIQGAGGVIIPPETYWPEIQRICDKYDILLIADEVICGFGRTGNWFGSQTLGIKPHIMTIAKGLSSGYQPIGGSIVCDEVAEVIGSGEFNHGYTYSGHPVAAAVALENLRILEEENVLDHVRDVAMPALHEMWHGLADHPLVGETTITGMMGSLALTPHKDSRAKFAMDAGTAGFMCRERCFANNLVMRHVYDRMVISPPLIITPDEIAEIGRRARTALDECYVQLKDGDMLKPAA

Claims

1. A method for constructing a recombinant microorganism for producing 3-aminopropanol, characterized by, Introduce the gene encoding ω-transaminase into a microorganism that produces 3-hydroxypropanal, wherein the ω-transaminase is capable of catalyzing the conversion of 3-hydroxypropanal to 3-aminopropanol in the presence of an amino donor.

2. The construction method of claim 1, wherein, The amino acid sequence of the ω-transaminase is shown in SEQ ID NO:10, 11 or 12.

3. The construction method according to claim 1 or 2, wherein, The 3-hydroxypropanal-producing microorganisms include naturally occurring 3-hydroxypropanal-producing microorganisms or genetically engineered recombinant 3-hydroxypropanal-producing microorganisms; preferably, the 3-hydroxypropanal-producing microorganisms are Klebsiella pneumoniae or Lactobacillus reuteri; more preferably, the 3-hydroxypropanal-producing microorganisms are Klebsiella pneumoniae.

4. The construction method according to claim 1 or 2, further comprising blocking or weakening the 3-hydroxypropanal degradative metabolic pathway in the 3-hydroxypropanal-producing microorganism; Preferably, when the microorganism producing 3-hydroxypropanal is Klebsiella pneumoniae, any one of the puuc, dhaT, or yqhD genes, or any combination thereof, is knocked out or reduced in Klebsiella pneumoniae.

5. The construction method according to claim 1 or 2, wherein, The amino donor is L-alanine, and the construction method further includes constructing an L-alanine cycling pathway in the 3-hydroxypropanal-producing microorganism. Preferably, the alaD and FDH genes are introduced or overexpressed in the 3-hydroxypropionaldehyde-producing microorganism; Preferably, the nucleotide sequence of the alaD is as shown in SEQ ID NO: 5, and the nucleotide sequence of the FDH is as shown in SEQ ID NO:

6.

6. The construction method according to claim 1 or 2, further comprising constructing a dual-carbon-source metabolic pathway of glycerol and glucose in the 3-hydroxypropanal-producing microorganism; Preferably, the ptsG gene is knocked out or knocked down and the glf gene is overexpressed in said 3-hydroxypropionaldehyde-producing microorganism; wherein, The nucleotide sequence of the ptsG gene is shown in SEQ ID NO: 7, and the nucleotide sequence of the glf is shown in SEQ ID NO:

9.

7. The recombinant microorganism obtained by the construction method according to any one of claims 1-6.

8. The use of the recombinant microorganism according to claim 7 in the production of 3-aminopropanol.

9. A method for producing 3-aminopropanol, comprising culturing the recombinant microorganism of claim 7 in a culture medium; Preferably, the culture medium further includes an amino donor; Preferably, the amino donor is L-alanine.

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