Recombinant bacteria for producing 3-hydroxypropionic acid and construction method and application thereof

Abstract

The application provides a recombinant bacterium for producing 3-hydroxypropionic acid, and a construction method and application thereof. The recombinant bacterium expresses a halomonas sp. derived accA gene, accB gene and malonyl-CoA reductase. The application fully utilizes the 3HB synthesis pathway of the strain itself, cooperates with the introduced 3-HP synthesis path, realizes high-efficiency synthesis of poly(3-hydroxybutyric acid-3-hydroxypropionic acid) or poly(3,4-dihydroxybutyric acid-3-hydroxypropionic acid) without passing through the intermediate product 3-hydroxypropionic acid aldehyde, and avoids the toxic effect of the intermediate product on cells.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a recombinant bacterium for producing 3-hydroxypropionic acid, its construction method, and its application. Background Technology

[0002] Under the current global sustainable development strategy framework, developing alternatives to fossil fuels and petroleum-based chemicals has become an important research direction in the field of biomanufacturing. Among them, the sustainable conversion of biomass resources and the synthesis of bio-based chemicals have become a research hotspot at the intersection of synthetic biology and green chemistry due to their dual advantages in alleviating the pressure of petroleum resource depletion and reducing environmental footprint.

[0003] 3-Hydroxypropionic acid (3-HP), as a platform compound, possesses the unique property of having both carboxyl and hydroxyl groups in its molecular structure, making it significant for industrial applications in biodegradable materials, biofuels, green solvents, and the synthesis of high-value chemicals. Traditional petroleum-based chemical synthesis methods rely on non-renewable resources and suffer from inherent drawbacks such as harsh reaction conditions, low atom economy, and high carbon emissions. In contrast, biocatalytic pathways based on microbial cell factories, utilizing renewable carbohydrates as substrates, achieve the biosynthesis of 3-HP through optimized metabolic networks (such as glycerol / glucose metabolic flow redirection). Due to its mild reaction conditions, environmental friendliness, and adherence to carbon neutrality principles, it has gained widespread research attention in synthetic biology and metabolic engineering in recent years. However, the production of 3-HP currently faces a major problem: the intermediate product 3-hydroxypropionic aldehyde (3-HPA) exhibits certain toxicity to cells. This negative effect leads to cell growth inhibition and decreased metabolic activity, becoming a major bottleneck restricting the improvement of 3-HP biosynthetic efficiency. In addition, the precursors PDO and glycerol used in traditional methods are relatively expensive, and the route for synthesizing 3HP using glycerol as a precursor also requires the addition of expensive VB12.

[0004] Therefore, there is an urgent need to find a production route that can avoid the intermediate product 3-hydroxypropionic acid aldehyde and has a lower production cost. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a recombinant bacterium for producing 3-hydroxypropionic acid, its construction method, and its application.

[0006] This invention provides a recombinant bacterium for producing 3-hydroxypropionic acid, wherein the recombinant bacterium expresses:

[0007] (1) Halomonas source accA Gene, accB Gene;

[0008] (2) Malonyl-CoA reductase encoded by any of the following pairs of genes:

[0009] (2-1) mcrC Genes and mcrN Gene;

[0010] Compared to (2-1), (2-2) mcrC The 59th base of the gene is mutated from G to C;

[0011] Compared to (2-1), (2-3) mcrC The gene's 126th base C is mutated to A. mcrN The 289th base of the gene is mutated from A to G;

[0012] Compared to (2-1), (2-4) mcrN The 826th base of the gene is mutated from A to C;

[0013] Compared to (2-1), (2-5) mcrN The gene's 1371st base C is mutated to G;

[0014] Compared to (2-1), (2-6) mcrC The 439th base of the gene is mutated from A to C;

[0015] Compared to (2-1), (2-7) mcrC The gene has a G mutation at position 223 and a C mutation at position 694.

[0016] The mcrC The nucleotide sequence of the gene is shown in SEQ ID No. 4. mcrN The nucleotide sequence of the gene is shown in SEQ ID No. 5.

[0017] The accA The nucleotide sequence of the gene is shown in SEQ ID No. 1; accB The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0018] In some embodiments, the recombinant bacteria is any one of the genera *Halomonas*, *Escherichia*, *Klebsiella*, *Azotobacter*, *Pseudomonas*, and *Akkermansia*.

[0019] In some embodiments, the *Halomonas* genus includes or its derivatives Halomonas aydingkolgenesis Or its derivatives.

[0020] In a specific embodiment of the present invention, the *Haloxymonas* is preferably... MDF-9, LY01, LY02, LY03, LY04.

[0021] In one specific embodiment of the present invention, the *Haloxymonas* is preferably... MDF-9.

[0022] In some embodiments, the recombinant bacteria also express a polyhydroxyalkanoate synthesis module.

[0023] In some embodiments, the polyhydroxyalkanoate synthesis module includes any one or more of the following:

[0024] (1) AldD Genes and dhaT Gene;

[0025] (2) orfz Gene.

[0026] The present invention also provides a method for constructing the recombinant bacteria, comprising the following steps;

[0027] (1) The above accA Gene, accB The gene encoding malonyl-CoA reductase and the gene encoding malonyl-CoA reductase were introduced into a plasmid to obtain a recombinant plasmid.

[0028] (2) Introduce the recombinant plasmid described in step (1) into the strain.

[0029] The plasmid vector can be a high-copy plasmid or a low-copy plasmid.

[0030] In some implementations, the high-copy plasmids include pSEVA341, ColE1, pMB1, pUC (such as pUC18, pUC19, etc.), pBluescript (such as pBluescript II KS+, pBluescript II SK+, etc.), pTZ57R, pGEM, etc.

[0031] In some implementations, the low-copy plasmids include the pRE series (such as pRE112), pSEVA321, pSC101, pACYC184, pWE15, and the pET series (such as pET-15b and pET-28a).

[0032] The present invention also provides the application of the recombinant bacteria in the production of 3-hydroxypropionic acid.

[0033] The present invention also provides a method for producing 3-hydroxypropionic acid, the method comprising fermenting and culturing the recombinant bacteria.

[0034] In some embodiments, the fermentation culture temperature is 30~42℃, preferably 32~40℃, more preferably 35~38℃; the initial pH of the fermentation culture is 6~11, preferably 7~10.

[0035] The present invention also provides the application of the recombinant bacteria in the production of 3-hydroxypropionic acid derivatives.

[0036] In some embodiments, the 3-hydroxypropionic acid derivative is a polyhydroxyalkanoate (PHA).

[0037] The production of polyhydroxyalkanoates can be carried out by batch fermentation, continuous fermentation, or fed-batch fermentation. Fermentation can be optionally carried out for the desired duration, for example, from 12 to 120 hours, such as 24 hours, 48 ​​hours, 60 hours, 72 hours, etc., but is not limited to these. Those skilled in the art can appropriately select the fermentation time according to specific circumstances. These fermentation methods are well known to those skilled in the art, and they can appropriately select any fermentation method to carry out the method of the present invention according to the desired purpose and experimental conditions.

[0038] In the above-described method for producing PHA, the fermentation medium used for fermentation can be sterilized before being added to the fermentation system, or it can be added directly to the fermentation system without any treatment. Furthermore, the fermentation system can be maintained under open, sterile conditions (i.e., it can be directly exposed to the environment without the need for closed fermentation conditions to avoid contamination) or under closed conditions to avoid contamination.

[0039] The fermentation medium can be selected based on the characteristics of the bacteria and the microbial culture techniques known in the art. It can typically include nutrient sources (such as carbon and / or nitrogen sources), energy sources, and essential minerals. For example, conventional mineral media, LB medium, MM medium, MM-G medium, or beef extract peptone, etc., can be used, or media modified based on these media according to the desired purpose can be used.

[0040] The fermentation medium can be liquid, solid, or semi-solid.

[0041] The fermentation medium may contain carbon and nitrogen sources, and / or carbon and / or nitrogen sources may be added to the fermentation system during the fermentation process. Carbon sources include, but are not limited to, monosaccharides, disaccharides, organic acids, fatty acids, polyols, alcohols, hydrocarbons and their derivatives. Specific carbon sources include glucose, fructose, sucrose, lactose, xylose, arabinose, glycerol, ethanol, methanol, lactic acid, acetic acid, propionic acid, butyric acid, decanoic acid, oleic acid, palmitic acid, stearic acid, or mixtures thereof. Nitrogen sources are inorganic nitrogen sources, organic nitrogen sources, or combinations thereof, including but not limited to urea, ammonium sulfate, nitrates, ammonium chloride, peptone, yeast extract, peptone, corn steep liquor powder, amino acids, casein, and soybean meal hydrolysate.

[0042] In this invention, the accumulation of PHA is achieved by regulating the nitrogen source concentration, including induced synthesis under nitrogen-limited conditions, or PHA synthesis can still be achieved under specific nitrogen-rich conditions.

[0043] The PHA is a homopolymer or copolymer of monomers; the monomers include, but are not limited to, 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), 3-hydroxyvalerate (3-HV), 4-hydroxybutyric acid (4-HB), 4-hydroxyvalerate (4-HV), etc.

[0044] In some embodiments, the PHA is selected from the binary copolymer of 3-hydroxybutyric acid and 3-hydroxypropionic acid, P3HB3HP, and the terpolymer of 3-hydroxybutyric acid, 4-hydroxybutyric acid, and 3-hydroxypropionic acid, P34HB3HP.

[0045] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0046] (1) This invention introduces the 3-hydroxypropionic acid synthesis pathway, enabling Halomonas to synthesize 3-HP, and randomly mutates the key enzyme mcrCN gene, which significantly improves the conversion ability of malonyl-CoA to 3-hydroxypropionic acid.

[0047] (2) This invention makes full use of the strain’s own 3HB synthesis pathway, which works in synergy with the introduced 3-HP synthesis pathway to achieve efficient synthesis of poly(3-hydroxybutyric acid-3-hydroxypropionic acid) (P3HB3HP) or poly(3,4-dihydroxybutyric acid-3-hydroxypropionic acid) (P34HB3HP) without the intermediate product 3-hydroxypropionic acid aldehyde, thus avoiding the toxic effects of intermediate products on cells.

[0048] (3) The present invention does not require the addition of expensive precursors during the synthesis process, which greatly reduces the production cost and has good prospects for industrial scale-up and commercialization potential. Attached Figure Description

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is a pathway diagram of the PHA synthesis method of the present invention;

[0051] Figure 2 Example 1 of the present invention, pSEVA321 accAB-mcrCN Plasmid mapping;

[0052] Figure 3 This is Embodiment 1 of the present invention. accAB-mcrCN Fragment electrophoresis image;

[0053] Figure 4 This is Example 4 of the present invention, pRE112. accAB-mcrCN Plasmid mapping;

[0054] Figure 5 This is Embodiment 4 of the present invention. accAB-mcrCN Fragment electrophoresis image;

[0055] Figure 6 This is Example 5 of the present invention, pRE112. AldD-dhaT-orfz Plasmid mapping;

[0056] Figure 7 This is Embodiment 5 of the present invention. AldD-dhaT-orfz Fragment electrophoresis image. Detailed Implementation

[0057] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0058] MDF-9 naturally possesses the ability to synthesize 3HB, but not 3HP or 4HB. This invention addresses this by introducing endogenous 3HB from *Halomonas*. accAB Genes that enable more efficient conversion of acetyl-CoA to malonyl-CoA, followed by the introduction of genes from *Flexibrio pylori*. mcrCN The gene enables the conversion of malonyl-CoA to 3-hydroxypropionic acid. This is achieved through the... mcrCNThe gene underwent random mutations, enhancing its ability to convert malonyl-CoA to 3-hydroxypropionic acid. Based on this, the strain's own 3HB synthesis pathway was combined with the introduction of... orfz The gene enables the synthesis of the dimer P3HB3HP. Finally, by introducing... aldD - dhaT The gene cluster synthesizes 4HB, thereby achieving the synthesis of the trimer P34HB3HP.

[0059] The invention primarily introduces two exogenous pathways, one of which is the synthetic pathway of 4-hydroxybutyryl coenzyme A, from... aldD- dhaT-orfz Gene clusters control synthesis, and the second is the 3-hydroxypropionic acid synthesis pathway, which is controlled by gene clusters. accAB and mcrCN Genes control synthesis, such as Figure 1 As shown.

[0060] The present invention uses MDF-9 is disclosed in patent CN113801810A, with accession number GDMCC NO.61850. LY01 is disclosed in patent CN116396886A, with accession number GDMCCNO.62635. LY02 is disclosed in patent CN116925981A, with accession number GDMCC NO.63381. LY03 is disclosed in patent CN116970538A, with accession number GDMCC NO. 63382. LY04 is disclosed in patent CN117551585A, with accession number GDMCC NO.63383.

[0061] Example 1: Construction of a synthetic pathway for 3-hydroxypropionic acid

[0062] (1) pSEVA321 accAB-mcrCN plasmid construction

[0063] PCR amplification via overlap extension accAB-mcrCN Fragments and pSEVA321 skeleton; accAB-mcrCN The fragment and the pSEVA321 backbone were combined by Gibson ligase to form a new plasmid and labeled as pSEVA321. accAB-mcrCN Subsequently, pSEVA321 accAB-mcrCN Get the template accAB-mcrCN Fragment sequence, a portion of the product was sent to a biotechnology company for sequencing, plasmid information as follows: Figure 2 As shown. Among them, accA The nucleotide sequence is shown in SEQ ID No. 1; accBThe nucleotide sequence is shown in SEQ ID No. 2; accAB The nucleotide sequence is shown in SEQ ID No. 3; mcrC The nucleotide sequence is shown in SEQ ID No. 4; mcrN The nucleotide sequence is shown in SEQ ID No. 5; mcrCN The nucleotide sequence is shown in SEQ ID No. 6.

[0064] Amplification accAB-mcrCN Fragment and primer sequences for the pSEVA321 plasmid backbone:

[0065] accA -F: See SEQ ID No. 7;

[0066] accA -R: See SEQ ID No. 8;

[0067] accB -F: See SEQ ID No. 9;

[0068] accB -R: See SEQ ID No. 10;

[0069] mcrC -F: See SEQ ID No. 11;

[0070] mcrC -R: See SEQ ID No. 12;

[0071] mcrN -F: See SEQ ID No. 13;

[0072] mcrN -R: See SEQ ID No. 14;

[0073] V1-F: See SEQ ID No. 15;

[0074] V1-R: See SEQ ID No. 16.

[0075] The amplification system and amplification procedure are shown in Tables 1 and 2:

[0076] Table 1 Amplification System

[0077]

[0078] Table 2 Amplification Procedure

[0079]

[0080] After the PCR reaction is complete, prepare agarose gel of the appropriate concentration and perform electrophoresis to observe the size of the DNA bands. Place the gel under a UV lamp and quickly cut off the gel containing the target DNA fragment, removing as much excess gel as possible.

[0081] Gibson Assembly method linking:

[0082] The concentration of the recovered DNA was determined, and the DNA addition ratio was calculated based on the length and concentration of the target fragment and the pSEVA321 backbone. Ligation was then performed using a Gibson ligation enzyme mixture. The Gibson Assembly ligation system and procedure are shown in Tables 3 and 4.

[0083] Table 3 Gibson Assembly Connection System

[0084]

[0085] Table 4 Gibson Assembly Connector

[0086]

[0087] (2) Transformation of S17-1 Escherichia coli:

[0088] Remove the pre-prepared S17-1 Escherichia coli competent cells from -80℃ and thaw them on ice. After 5 min, allow the bacterial block to thaw. Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not shake). Incubate on ice for 30 min, then heat shock in a 42℃ water bath for 2 min, and immediately place on ice to cool for 2 min. Add 400 μL of LB medium to the centrifuge tube, mix well, and place in a 37℃ shaker at 200 rpm for 60 min to recover. Centrifuge at 5000 rpm for 5 min to collect the bacteria. Discard 350 μL of supernatant and keep 100 μL. Gently pipette and resuspend the bacterial block and spread it onto LB medium containing the appropriate antibiotic. Invert the medium and incubate at 37℃ for 12–16 h.

[0089] (3) Verification of positive monoclonal colonies

[0090] Colonies were selected from the corresponding antibiotic-resistant LB plates and colony PCR was performed for verification. PCR products with the correct band size were sent to a biotechnology company for sequencing. Single colonies with correct sequences were selected for amplification. After 12–16 h, they were conjugated with MDF-9 on 20 LB plates. After 8 h, a small number of conjugated cells were picked and plated onto 60 LB plates with the corresponding antibiotic resistance. Single colony verification was performed again after 36–48 h.

[0091] (4) Sequencing identification

[0092] The results showed that the MDF-9 strain in this embodiment was successfully transformed with accAB-mcrCN The fragment was named MDF-9-1, and the size of the target product was used for verification. Figure 3 As shown, the target fragment is 5980 bp, which is in line with the expected result.

[0093] Example 2: 3-Hydroxypropionic acid fermentation test

[0094] (1) Culture medium:

[0095] 60LB plate medium: yeast extract 0.5%; tryptone 1%; sodium chloride 60%; agar powder 1.8g / 100mL; pH 8.5.

[0096] Fermentation medium (50 mm): glucose 30 g / L; sodium chloride 50 g / L, yeast extract 1.2 g / L, urea 0.6 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5 g / L, component III: Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, component IV: ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L.

[0097] (2) Specific operating steps:

[0098] Strain activation:

[0099] The bacterial culture was taken from a laboratory freezer at -80℃. The bacterial culture was picked up with a pipette tip and streaked onto a plate solid medium (5 g / L yeast extract; 10 g / L tryptone; 60 g / L sodium chloride, pH 8.5) and incubated at 37℃ for 24 h.

[0100] Primary seed culture:

[0101] Pick a single colony and inoculate it into a 12 mL shaker tube (5 mL 60 LB medium: 5 g / L yeast extract; 10 g / L tryptone; 60 g / L sodium chloride; pH 8.5). Incubate the culture in a shaker at 37°C and 220 rpm for 12 h.

[0102] Secondary seed culture:

[0103] Take 200 μL of primary bacterial culture (1% inoculum) and inoculate it into a 150 mL Erlenmeyer flask (20 mL 60 LB medium). Incubate on a shaker at 37 °C and 220 rpm for 12 h.

[0104] (3) Preparation of fermentation medium

[0105] Fermentation medium (50 mm): glucose 30 g / L; sodium chloride 50 g / L; yeast extract 1.2 g / L; urea 1 g / L; anhydrous magnesium sulfate 0.2 g / L; potassium dihydrogen phosphate 1.5 g / L; Fe(III)-NH4-Citrate 5 g / L; CaCl2·2H2O 2 g / L; HCl 12 mol / L; ZnSO4·7H2O 0.1 g / L; MnCl2·4H2O 0.03 g / L; H2BO3 0.3 g / L; CoCl2·6H2O 0.2 g / L; CuSO4·5H2O 0.01 g / L; NiCl2·6H2O 0.02 g / L; NaMoO4·2H2O 0.03 g / L.

[0106] (4) Fermentation culture

[0107] Inoculate the seed culture at 5% (2.5 mL) into a 500 mL Erlenmeyer flask and incubate on a shaker at 37°C and 220 rpm for 48 h.

[0108] (5) Determination of cell dry weight and 3HP content

[0109] Cell dry weight (CDW): Place 30-35 mL of fermented bacterial culture into a 50 mL centrifuge tube, centrifuge at room temperature for 6 minutes at 8000 rpm, and discard the supernatant; add an appropriate amount of deionized water to restore the original volume, resuspend, and ensure that the precipitate completely disappears. Centrifuge under the same conditions and discard the supernatant; seal the centrifuge tube with sealing film and freeze at -80℃ for 2 hours; dry the centrifuge tube in a vacuum freeze dryer for 12-16 hours; weigh and calculate the cell dry weight (g / L).

[0110] Determination of 3HP content: Weigh 0.05 g of ground, fermented, dried cells and place them in a well-sealed esterification tube. Add 2 mL of chloroform, 1700 μL of methanol, and 300 μL of concentrated sulfuric acid. React in an oil bath at 100℃ for 1 h. After cooling to room temperature, add 1 mL of ddH2O, shake thoroughly to mix, and allow to stand for layering. After complete separation of the aqueous and organic phases, take the chloroform layer (usually the lower layer) and filter it through a 0.22 μm organic filter membrane into a liquid chromatography bottle for GC detection. Use a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, 30 m long, 0.25 mm inner diameter and 0.25 μm stationary phase), and flame ionization detection (FID). High-purity nitrogen was used as the carrier gas. The temperature program was set as follows:

[0111] Table 5 Program Temperature Settings

[0112]

[0113] The injection volume was 1 μL. The external standard method was used to quantitatively analyze the 3HP content, and the 3HP content was calculated based on the peak area.

[0114] For strain MDF-9-1, 50 μL of chloramphenicol (25 mg / L) needs to be added.

[0115] The fermentation test results are as follows:

[0116] Table 6. 3HP fermentation test of recombinant strains

[0117]

[0118] The fermentation data in Table 6 show that the recombinant strain MDF-9-1 successfully synthesized 3-hydroxypropionic acid, and the cell dry weight was slightly increased compared with the original strain MDF-9.

[0119] Example 3: Random Mutation Modification of Malonyl-CoA Reductase (mcrCN)

[0120] (1) mcrCN random mutations

[0121] To improve the expression efficiency of malonyl-CoA reductase, mcrCN Chemical random mutagenesis (such as dNTP, Tris-HCl mix) was performed to screen for more efficient malonyl-CoA reductase (mcrCN) mutants. The specific steps are as described in Example 1 for the construction of the 3-hydroxypropionic acid synthesis pathway.

[0122] The PCR amplification system is shown in Table 7:

[0123] Table 7 Amplification System

[0124]

[0125] The main components of the Tris-HCl mix described in Table 7 are Tris-HCl 10 mM, KCl 50 mM, and MgCl2 2 mM.

[0126] After screening and sequencing, the following mcrCN positive mutants were obtained: mcrCN-2, mcrCN-3, mcrCN-7, mcrCN-9, mcrCN-13, and mcrCN-17.

[0127] wild type mcrCN Nucleotide sequence for reference:

[0128] mcrCN-2: mcrC The 59th base of the gene is mutated from G to C.

[0129] mcrCN-3:mcrC The gene's 126th base C is mutated to A. mcrN The 289th base of the gene is mutated from A to G.

[0130] mcrCN-7: mcrN The 826th base of the gene is mutated from A to C.

[0131] mcrCN-9: mcrN The gene's 1371st base C is mutated to G.

[0132] mcrCN-13: mcrC The 439th base of the gene is mutated from A to C.

[0133] mcrCN-17: mcrC The gene's 223rd base G is mutated to T, and the 694th base C is mutated to T.

[0134] The strains transformed into each mutant were named MDF-9-2, MDF-9-3, MDF-9-4, MDF-9-5, MDF-9-6, and MDF-9-7, respectively.

[0135] (2) Fermentation test of mutant 3-hydroxypropionic acid

[0136] Fermentation tests were conducted according to the fermentation method in Example 2, wherein strains MDF-9-1, MDF-9-2, MDF-9-3, MDF-9-4, MDF-9-5, MDF-9-6, and MDF-9-7 required the addition of 50 μL of chloramphenicol (25 mg / L).

[0137] The fermentation test results are as follows:

[0138] Table 8. 3HP fermentation test of mutant strains

[0139]

[0140] Fermentation data showed that all mutants increased cell dry weight and 3HP yield compared to the non-mutated strain MDF-9-1. Among them, strain MDF-9-3 (mutant mcrCN-3) showed the greatest increase in cell dry weight and 3HP yield, with cell dry weight increasing by 6% and 3HP yield increasing by 50% compared to the non-mutated strain MDF-9-1.

[0141] Example 4: Optimal mutant genome integration and fermentation test

[0142] (1) accAB-mcrCN Genome integration at the G49 site of the gene cluster (optimal mutant)

[0143] Integral plasmid pRE112 accAB-mcrCN Build:

[0144] PCR amplification via overlap extension accAB-mcrCN Fragments and pRE112 skeleton with G49 upstream and downstream homologous arms; accAB-mcrCN The fragment and the pRE112 backbone were combined by Gibson ligase to form a new plasmid and labeled as pRE112. accAB-mcrCN Subsequently, pRE112 accAB-mcrCN Get the template accAB-mcrCN Fragment sequence, a portion of the product was sent to a biotechnology company for sequencing, plasmid information as follows: Figure 4 As shown. Refer to Example 1 for specific steps.

[0145] Amplification accAB-mcrCN The primer sequences for the fragment are described in Example 1. The primer sequences for amplifying the pRE112 plasmid backbone are as follows:

[0146] V2-F: See SEQ ID No. 17;

[0147] V2-R: See SEQ ID No. 18.

[0148] (2) Screening of genome-integrated strains:

[0149] Select single colonies with the correct sequence for propagation. After 12-16 hours, conjugate them with MDF-9 on 20LB plates. After 8 hours, pick a small amount of conjugated cells and spread them on 60LB plates with the corresponding antibiotic. Perform the first validation after 36-48 hours. If the validation is successful, remove the antibiotic.

[0150] Antibiotic removal involves subculturing in a 60LB deep-well plate at 42℃ for 3-7 generations (one generation every 24 hours; plating verification begins from generation 3). After antibiotic removal, a small amount of bacterial culture is added to 60LB (approximately 10...). 5 ~10 6 Diluted medium is spread evenly on 60 LB plates. For subculturing, transfer to fresh 60 LB medium (without antibiotics) at a ratio of 0.02%.

[0151] After single colonies grow on plates, streak them onto 60LB and 60LB (cm) plates respectively. After 12 hours of incubation, colonies that grow on 60LB plates but not on 60LB (cm) plates are considered successfully de-antibioticated. Selected colonies for PCR verification are then used for sequencing.

[0152] The results showed that the MDF-9 strain in this embodiment successfully integrated [the enzyme]. accAB-mcrCN The fragment was named MDF-9-8, and the size of the target product was used for verification. Figure 5 As shown, the target fragment is 5980bp, which is in line with the expected result.

[0153] Example 5: Recombinant strain synthesizes dimer P3HB3HP and trimer P34HB3HP

[0154] (1) Construction of the synthetic pathway of 4-hydroxybutyryl coenzyme A

[0155] AldD-dhaT-orfz Genome integration at the G51 site

[0156] Integral plasmid pRE112 AldD-dhaT-orfz Build:

[0157] PCR amplification using overlap extension AldD-dhaT-orfz Gene and pRE112 scaffold with G51 upstream and downstream homologous arms; AldD-dhaT-orfz The gene and pRE112 backbone were combined by Gibson ligase to form a new plasmid and labeled as pRE112. AldD-dhaT-orfz pRE112 AldD-dhaT-orfz Get the template AldD-dhaT-orfz Sequence, a portion of the product was sent to a biotechnology company for sequencing, plasmid information as follows: Figure 6 As shown. The specific steps are described in Example 1. Wherein, AldD The nucleotide sequence of the gene is shown in SEQ ID No. 19; dhaT The nucleotide sequence of the gene is shown in SEQ ID No. 20; orfz The nucleotide sequence of the gene is shown in SEQ ID No. 21; AldD-dhaT-orfz The nucleotide sequence of the fragment is shown in SEQ ID No. 22.

[0158] Amplification aldD-dhaT-orfz Fragment and primer sequences for the pRE112 plasmid backbone:

[0159] aldD -F: See SEQ ID No. 23;

[0160] aldD -R: See SEQ ID No. 24;

[0161] dhaT -F: See SEQ ID No. 25;

[0162] dhaT -R: See SEQ ID No. 26;

[0163] orfz -F: See SEQ ID No. 27;

[0164] orfz -R: See SEQ ID No. 28;

[0165] V3-F: See SEQ ID No. 29;

[0166] V3-R: See SEQ ID No. 30.

[0167] Select single colonies with the correct sequence for propagation. After 12-16 hours, conjugate them with MDF-9-8 on 20LB plates. After 8 hours, pick a small amount of conjugated cells and spread them on 60LB plates with the corresponding antibiotic. Perform the first validation after 36-48 hours. If the validation is successful, remove the antibiotic. Refer to Example 1 for specific steps.

[0168] The results showed that the MDF-9-8 strain in this embodiment successfully integrated [the enzyme]. AldD-dhaT-orfz The gene was used, and the strain was named MDF-9-9. Verification was performed based on the size of the target product, such as... Figure 7 As shown, the target fragment is 4076bp, which is in line with the expected result.

[0169] (2) Fermentation test of dimer and trimer of recombinant strain MDF-9-9

[0170] The specific steps for the fermentation test are as described in Example 1. The synthesis of trimer P34HB3HP also requires the addition of 5 g / L BDO.

[0171] The fermentation test results are as follows:

[0172] Table 9. Dimer and trimer fermentation tests of recombinant strain MDF-9-9

[0173]

[0174] Fermentation data showed that the recombinant strain MDF-9-9 produced dimer P3HB3HP and trimer P34HB3HP with good results, and the cell dry weight and PHA content were increased to a certain extent.

[0175] Example 6: Construction of a recombinant strain producing 3HP

[0176] 1. Constructing a recombinant Halomonas strain that produces 3HP

[0177] The plasmid pSEVA321 containing the optimal mutant was used. accAB-mcrCN Transfected with Halomonas LY01 LY02 LY03 LY04, respectively named LY01-A, LY02-A, LY03-A, and LY04-A.

[0178] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L).

[0179] The fermentation test results are as follows:

[0180] Table 10 3HP fermentation test of other Halomonas bacteria

[0181]

[0182] Fermentation test data showed that all four halophilic bacteria could synthesize 3HP through this modification strategy, with strain LY03-A showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other halophilic bacteria.

[0183] 2. Construct other recombinant strains to produce 3HP

[0184] The plasmid pSEVA321 containing the optimal mutant was used. accAB-mcrCN The bacteria were respectively introduced into Escherichia coli (EcN), Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 17699), Pseudomonas putida (ATCC 47054), and Akkermansia myxophilus (ATCCBAA-835).

[0185] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L).

[0186] The fermentation test results are as follows:

[0187] Table 11 3HP fermentation test of other recombinant strains

[0188]

[0189] Fermentation test data showed that all five strains could synthesize 3HP through this modification strategy, with Escherichia coli EcN showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other strains of non-halophilic bacteria.

[0190] Example 7: Construction of recombinant strains for producing dimers and trimers

[0191] 1. Construction of recombinant halomonas bacteria that produce dimers and trimers

[0192] The plasmid pSEVA321 containing the optimal mutant was used. accAB-mcrCN and plasmid pRE112 AldD-dhaT-orfz Simultaneously introduced Halomonas LY01 LY02 LY03 LY04, respectively named LY01-B, LY02-B, LY03-B, and LY04-B.

[0193] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L).

[0194] The fermentation test results are as follows:

[0195] Table 12 Dimer fermentation test of other Halomonas bacteria

[0196]

[0197] Table 13 Trimer fermentation test of other Halomonas bacteria

[0198]

[0199] Fermentation test data showed that all four types of halophilic bacteria could synthesize dimers and trimers through this modification strategy, with strain LY03-B showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other halophilic bacteria.

[0200] 2. Construct other recombinant strains for producing dimers and trimers.

[0201] The plasmid pSEVA321 containing the optimal mutant was used. accAB-mcrCN and plasmid pRE112 AldD-dhaT-orfz Simultaneously introduced were Escherichia coli (EcN), Klebsiella pneumoniae (ATCC 13883), Evotrophobicus roximatei (ATCC 17699), Pseudomonas putida (ATCC 47054), and Akkermansia myxophilus (ATCC BAA-835).

[0202] The specific steps for the fermentation test are as described in Example 1. Each recombinant strain needs to be supplemented with 50 μL of chloramphenicol (25 mg / L).

[0203] The fermentation test results are as follows:

[0204] Table 14 Dimer fermentation tests of other recombinant strains

[0205]

[0206] Table 15 Trimer fermentation tests of other recombinant strains

[0207]

[0208] Fermentation test data showed that all five strains could synthesize dimers and trimers through this modification strategy, with Escherichia coli EcN showing the best performance. This example illustrates that the construction method of the present invention is widely applicable to other strains of non-halophilic bacteria.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant bacterium for producing 3-hydroxypropionic acid, characterized in that, The recombinant bacteria expressed: (1) Halomonas source accA Gene, accB Gene; (2) Malonyl-CoA reductase encoded by any of the following pairs of genes: (2-1) mcrC Genes and mcrN Gene; Compared to (2-1), (2-2) mcrC The 59th base of the gene is mutated from G to C; Compared to (2-1), (2-3) mcrC The gene's 126th base C is mutated to A. mcrN The 289th base of the gene is mutated from A to G; Compared to (2-1), (2-4) mcrN The 826th base of the gene is mutated from A to C; Compared to (2-1), (2-5) mcrN The gene's 1371st base C is mutated to G; Compared to (2-1), (2-6) mcrC The 439th base of the gene is mutated from A to C; Compared to (2-1), (2-7) mcrC The gene has a G mutation at position 223 and a C mutation at position 694. The mcrC The nucleotide sequence of the gene is shown in SEQ ID No.

4. mcrN The nucleotide sequence of the gene is shown in SEQ ID No. 5; The accA The nucleotide sequence of the gene is shown in SEQ ID No.

1. accB The nucleotide sequence of the gene is shown in SEQ ID No. 2; The recombinant bacteria are Halomonas lutescens MDF-9 Halomonas sp. LY01、 Halomonas sp. LY02、 Halomonas sp. LY03、 Halomonas Any one of the following: sp. LY04, Escherichia coli EcN, Klebsiella pneumoniae ATCC 13883, Evotrophobicus loureae ATCC 17699, Pseudomonas putida ATCC 47054, and Akkermansia myxophilus ATCCBAA-835.

2. The recombinant bacteria according to claim 1, characterized in that, The recombinant bacteria also express a polyhydroxy fatty acid ester synthesis module.

3. The recombinant bacteria according to claim 2, characterized in that, The polyhydroxy fatty acid ester synthesis module includes any one or more of the following: (1) AldD Gene, dhaT Gene; (2) orfz Gene.

4. The method for constructing the recombinant bacteria according to any one of claims 1 to 3, characterized in that, Includes the following steps; (1) The method described in claim 1 accA Gene, accB The gene encoding malonyl-CoA reductase and the gene encoding malonyl-CoA reductase were introduced into a plasmid to obtain a recombinant plasmid. (2) Introduce the recombinant plasmid described in step (1) into the strain.

5. The use of the recombinant bacteria according to any one of claims 1 to 3 in the production of 3-hydroxypropionic acid.

6. A method for producing 3-hydroxypropionic acid, characterized in that, The method includes fermenting and culturing the recombinant bacteria according to any one of claims 1 to 3.

7. The method according to claim 6, characterized in that, The fermentation culture was carried out at a temperature of 30-42℃ and a pH of 6-11.

8. The use of the recombinant bacteria according to any one of claims 1 to 3 in the production of polyhydroxyalkanoates.

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