WO2023159745A1 - Genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propanediol, and construction method therefor and application thereof - Google Patents

Abstract

The present application relates to the technical field of bioengineering, and provides a genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propanediol, and a construction method therefor and an application thereof. Escherichia coli W3110 (DE3) is used as a starting strain, a genetically engineered bacterium for co-production of 3-hydroxypropionic acid and 1,3-propanediol is constructed by utilizing the technical means of genetic engineering, and the fermentation process of the constructed genetically engineered bacterium is optimized, such that efficient co-production of 3-hydroxypropionic acid and 1,3-propanediol is realized.

Description

A genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol and its construction method and application technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium co-producing 3-hydroxypropionic acid and 1,3-propanediol and its construction method and application.

Background technique

3-Hydroxypropionic acid and 1,3-propanediol are two important platform compounds in industry, which are widely used as precursors of biodegradable polymers and food additives. The production of 3-hydroxypropionic acid and 1,3-propanediol has two methods: chemical synthesis and biological method. Most of the chemical methods use non-renewable resources as raw materials. The production process consumes a lot of energy, and many by-products are difficult to separate and purify. The production process produces immeasurable environmental pollution. The biosynthesis of 3-hydroxypropionic acid or 1,3-propanediol mostly uses glucose and glycerol as substrates, and the production of 3-hydroxypropionic acid and 1,3-propanediol using glycerol as a substrate has simple steps, sufficient research, and cheap raw materials. And can solve the problem of excess glycerin.

Production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol, first, glycerol dehydratase dehydrates glycerol to produce intermediate metabolite 3-hydroxypropionaldehyde; then NAD ⁺ dependent aldehyde dehydrogenase and NAD(P)H Dependent 1,3-propanediol oxidoreductase/isoenzyme generates 3-hydroxypropionate and 1,3-propanediol, respectively. However, the production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol produces a toxic intermediate metabolite, 3-hydroxypropionaldehyde. In addition, sustainable glycerol fermentation to produce 3-hydroxypropionic acid and 1,3-propanediol still has problems such as accumulation inhibition of toxic intermediate metabolite 3-hydroxypropionaldehyde and insufficient supply of necessary cofactors.

Currently, glycerol metabolism is inhibited, limiting further intake of glycerol, resulting in low yields of 3-hydroxypropionate and 1,3-propanediol production; and excessive carbon flux to central metabolic pathways will reduce 3-hydroxypropionate and 1,3-propionate production. The yield of 3-propanediol; the above two points are the main bottlenecks for microbial fermentation of glycerol to produce 3-hydroxypropionic acid and 1,3-propanediol at this stage.

In addition, on the one hand, most of the culture media for microbial fermentation of glycerol to produce 3-hydroxypropionic acid and 1,3-propanediol are improved low-salt media, in which yeast extract is added to maintain rapid cell growth, and yeast powder is an expensive Nitrogen source, which virtually increases the cost of microbial fermentation to produce 3-hydroxypropionic acid and 1,3-propanediol; on the other hand, the pH suitable for microbial growth and target product synthesis is generally different, and this contradiction limits 3-hydroxypropionic acid Efficient production of propionic acid and 1,3-propanediol production.

Contents of the invention

Aiming at the deficiencies in the prior art, the invention provides a genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol, its construction method and application. In the present invention, using Escherichia coli Escherichia coli W3110 (DE3) as the starting strain, the technical means of genetic engineering has been used to construct the genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol, and the constructed gene The fermentation process of engineering bacteria is optimized to realize the efficient co-production of 3-hydroxypropionic acid and 1,3-propanediol.

In the present invention, at first a kind of genetically engineered bacterium co-producing 3-hydroxypropionic acid and 1,3-propanediol is provided, which is denoted as E.coli S10G; Obtained by recombination of expressed and knocked-out genes;

The expressed genes are: glycerol dehydratase and its reactivator (DhaB123-GdrAB), propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isozyme (YqhD) and membrane-bound pyridine core nucleotide transhydrogenase (PntAB);

The knockout genes are: soluble pyridine nucleotide transhydrogenase (SthA), lactate dehydrogenase (LdhA), alcohol dehydrogenase (AdhE), pyruvate formate lyase (PflB), pyruvate oxidation The genes for enzyme (PoxB), phosphoacetyltransferase-acetate kinase PTA-AckA, and inhibitor of glycerol metabolism (GlpR).

Wherein, the nucleotide sequence of the DhaB123-GdrAB is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2.

The nucleotide sequence of the GabD4 is shown in SEQ ID No: 3, and the amino acid sequence is shown in SEQ ID No: 4.

The nucleotide sequence of the YqhD is shown in SEQ ID No:5, and the amino acid sequence is shown in SEQ ID No:6.

The nucleotide sequence of the PntAB is shown in SEQ ID No: 7, and the amino acid sequence is shown in SEQ ID No: 8.

Preferably, the original UTR sequence replacing glycerol kinase (GlpK) in the genome is an artificially designed UTR sequence.

The artificially designed UTR sequence is any of the following seven sequences:

glpK-U1AGATTATACGGGACAACACGAA (SEQ ID No: 70);

glpK-U2GTTAGCTACGGGACAAGGGTTT (SEQ ID No: 71);

glpK-U3ATTCTTTACGGGACAACCATAA (SEQ ID No: 72);

glpK-U4ACGTCTAACGGGACAAAGTTCC (SEQ ID No: 73);

glpK-U5ACGGTTTACGGGACAACGCGG (SEQ ID No: 74);

glpK-U6ACCGTCTACGGGACAACGCTGT (SEQ ID No: 75);

glpK-U7CCGGTCTACGGGACAACGCTGT (SEQ ID No: 76).

The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol, specifically comprising the following steps:

(1) Construction of glycerol dehydratase and its reactivator (DhaB123-GdrAB) recombinant plasmid:

Glycerol dehydratase (DhaB123) gene and glycerol dehydratase reactivator (GdrAB) gene were amplified by PCR, and fusion PCR technology was used to form a complete glycerol dehydratase and its reactivator gene fragment DhaB123-GdrAB, and then cloned into pCDFDuet-1 Plasmid, after transforming E.coli DH5α, screening positive clones, plasmid extraction, and sequencing confirmation, the glycerol dehydratase and its reactivation factor plasmid was obtained, named pCDF-DhaB123-GdrAB.

Wherein, the glycerol dehydratase gene and the glycerol dehydratase reactivator gene are derived from Klebsiella pneumoniae.

(2) Construction of recombinant plasmids co-expressing propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isoenzyme (YqhD) and membrane tuberculosis-type pyridine nucleotide transhydrogenase (PntAB):

Propionaldehyde dehydrogenase (GabD4) gene, 1,3-propanediol oxidoreductase isoenzyme (YqhD) gene and membrane tubercle-type pyridine nucleotide transhydrogenase (PntAB) gene were amplified by PCR, and the seamless cloning technique was used to amplify The above two genes were cloned into the pRSFuet-1 plasmid, and after transforming E.coli DH5α, screening positive clones, plasmid extraction, and sequencing confirmation, the co-expressed propionaldehyde dehydrogenase (GabD4) and 1,3-propanediol oxidoreductase isoforms were obtained. enzyme (YqhD) and membrane tuberculosis-type pyridine nucleotide transhydrogenase (PntAB) recombinant plasmid, named pRSF-GabD4-YqhD-PntAB.

Among them, the source of the propionaldehyde dehydrogenase (GabD4) gene is derived from the pANY-gabD4 plasmid, the 1,3-propanediol oxidoreductase isoenzyme (YqhD) gene and the membrane tuberculosis type pyridine nucleotide transhydrogenase (PntAB) gene are derived from Escherichia coli (Escherichia coli W3110).

(3) Construction of Escherichia coli with by-product knockout:

Knockout Escherichia coli (Escherichia coli W3110(DE3)) soluble pyridine nucleotide transhydrogenase (SthA) gene, lactate dehydrogenase (LdhA) gene, alcohol dehydrogenase (AdhE ) gene, pyruvate formate lyase (PflB) gene, pyruvate oxidase (PoxB) gene, phosphoacetyltransferase-acetate kinase PTA-AckA gene and glycerol metabolism inhibitor (GlpR) gene, after PCR verification and sequencing verification , and finally the genetically deficient engineered bacteria, that is, Escherichia coli with by-product knockout.

Preferably, the Glycerol Kinase (GlpK) expression level of Escherichia coli knocked out by UTR engineering technology can be used to artificially modify, use UTR design tool, design UTR artificial sequence, use double plasmid CRIPSR CAS9 tool plasmid, replace gene defect engineering The UTR sequence of the original glycerol kinase (GlpK) gene in the bacterial genome was verified by sequencing, and the engineering bacteria with artificially modified expression of glycerol kinase (GlpK) were obtained.

Wherein, the artificially designed UTR sequence is any one of the following seven sequences:

glpK-U1 AGATTATACGGGACAACACGAA (SEQ ID No: 70);

glpK-U2 GTTAGCTACGGGACAAGGGTTT (SEQ ID No: 71);

glpK-U3 ATTCTTTACGGGACAACCATAA (SEQ ID No: 72);

glpK-U4 ACGTCTAACGGGACAAAGTTCC (SEQ ID No: 73);

glpK-U5 ACGGTTTACGGGACAACGCGG (SEQ ID No: 74);

glpK-U6 ACCGTCTACGGGACAACGCTGT (SEQ ID No: 75);

glpK-U7 CCGGTCTACGGGACAACGCTGT (SEQ ID No: 76).

(4) Construction of genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol:

Simultaneously transforming pCDF-DhaB123-GdrAB and pRSF-GabD4-YqhD-PntAB into competent cells of genetically deficient engineered bacteria to obtain highly efficient co-production genetic engineering of 3-hydroxypropionic acid and 1,3-propanediol.

Wherein, the steps (1)-(3) are in no particular order.

The present invention also provides the application of the above-mentioned genetically engineered bacteria in the co-production of 3-hydroxypropionic acid and 1,3-propanediol by fermenting glycerin.

The present invention also provides a method for co-producing 3-hydroxypropionic acid and 1,3-propanediol by fermentation of the above-mentioned genetically engineered bacteria, which specifically includes the following steps:

(1) Activate the genetically engineered bacteria E.coli S10G overnight in LB medium to obtain the primary seed liquid of E.coli S10G;

(2) Inoculate the primary seed liquid of E.coli S10G into the improved M9-CSL medium for cultivation to obtain the secondary seed liquid of E.coli S10G;

(3) Inoculate the secondary seed liquid of E.coli S10G in the improved M9-CSL medium, divide the fermentation process into a growth stage and a production stage, and control the pH value to carry out fed-batch fermentation;

During the growth stage, the pH is controlled to be 7.0, and the temperature, ventilation, and stirring rate are adjusted for fermentation and cultivation until the OD600 reaches 4, and IPTG and vitamin B12 are added to continue the cultivation;

During the production stage, the pH is controlled to be 8.0, and the temperature, ventilation, and dissolved oxygen value are adjusted for fermentation, and then fed-feed fermentation is carried out every 6 hours to co-produce 3-hydroxypropionic acid and 1,3-propanediol;

The ingredients of the supplement contained glycerin and corn steep liquor.

Wherein, in step (2), the composition of the improved M9-CSL medium is MgSO ₄ 7H ₂ O 0.5g/L, NH ₄ Cl 2.0g/L, NaCl 2.0g/L, corn steep liquor 2.5mL/L , glycerol 40g/L and 0.1M potassium phosphate buffer, pH 7.0;

The inoculum amount of the EC10S10G primary seed solution is 1% v/v, and the culture conditions are 37° C. and 220 rpm for 12 hours.

In step (3), the inoculation amount of E.coli S10G secondary seed solution is 5% v/v;

During the growth stage, the temperature is adjusted to 37°C, the initial ventilation is 2vvm, and the stirring rate is 500rpm for fermentation;

During the production stage, adjust the temperature and ventilation volume to be adjusted to 3vvm, the stirring rate is 200-800rpm, and the dissolved oxygen value is controlled to be 10% for fermentation;

The composition of described feed contains 800g/L glycerol and 50mL/L corn steep liquor;

The feeding process is as follows: feed every 6 hours to control the glycerin concentration to be maintained at 40g/L.

Compared with prior art, the beneficial effect of the present invention is:

Through the design of metabolic engineering and synthetic biology, this technology constructs a genetic engineering strain that can efficiently co-produce 3-hydroxypropionic acid and 1,3-propanediol, promotes the cycle of essential cofactors through cofactor engineering, and knocks out byproducts pathways, inactivating metabolic inhibitors, and controlling central metabolic pathways to enhance 3-hydroxypropionate and 1,3-propanediol pathway flux. The constructed genetically engineered bacteria can efficiently co-produce 3-hydroxypropionic acid and 1,3-propanediol through two-stage pH-controlled fed-batch fermentation, using biodiesel waste glycerol as a substrate.

In the present invention, the traditional method of producing 3-hydroxypropionic acid or 1,3-propanediol alone is abandoned, and the co-production of 3-hydroxypropionic acid is combined with cofactor engineering, and UTR engineering is applied to metabolic flux balance, which has important The theoretical and practical significance of the constructed genetic engineering E.coli S10G can efficiently produce 3-hydroxypropionic acid and 1,3-propanediol, and has the potential for large-scale industrial production.

Moreover, the engineered bacteria constructed in the present invention can efficiently transform and metabolize the intermediate metabolite 3-hydroxypropanal, and generate the final products 3-hydroxypropionic acid and 1,3-propanediol; through cofactor engineering, the necessary cofactors and cyclic regeneration can be promoted to solve the problem of 3 The problem of insufficient supply of essential cofactors in the production of -hydroxypropionic acid and 1,3-propanediol; moreover, the elimination of by-product production pathways and the weakening of central metabolic pathways make the production of 3-hydroxypropionic acid and 1,3-propanediol Production has higher production yield and less accumulation of by-products, and has broad application prospects and practical significance.

Description of drawings

Fig. 1 is the plasmid map of pCDF-DhaB123-GDRAB in genetically engineered bacteria E.coli S10G.

Figure 2 is a map of the pRSF-GabD4-YqhD-PntAB plasmid in the genetically engineered bacterium E.coli S10G.

Figure 3 shows the relative expression level and metabolites of GlpK in genetically engineered E.coli UTR-GlpK.

Fig. 4 is a graph showing the yield results of the two-stage pH-controlled fermentation of the present invention.

Fig. 5 is a diagram of the fermentation of genetically engineered bacteria E.coli S10G co-producing 3-hydroxypropionic acid and 1,3-propanediol through two-stage pH-controlled fed-batch fermentation according to the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

Those who do not indicate the specific conditions in the following examples are all carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market. Unless otherwise specified, what the present invention adopts is the prior art in this field.

Embodiment 1: Construction of glycerol dehydratase and its reactivator (DhaB123-GdrAB) recombinant plasmid

(1) Utilize Oligo7.0 software to design primers according to the glycerol dehydratase and its reactivator (DhaB123-GdrAB) gene sequence of Klebsiella pneumoniae:

DhaB F: GTTTAACTTTAATAAGGAGATATACCatgaaaagatcaaaacgatttgcagtactg (SEQ ID No: 9);

GdrA R: CGGCCCCCTCGTTAACACttaattcgcctgaccggccag (SEQ ID No: 10);

GrdB F: CTGGCCGGTCAGGCGAATTAAgtgttaacgagggggccgtc (SEQ ID No: 11);

GrdB R: TTATGCGGCCGCAAGCTTGTCGACtcagtttctctcacttaacggcaggac (SEQ ID No: 12).

(2) Use DhaB F and GdrA R primer pair to amplify DhaB123-GdrA gene, use GdrB F and GdrB R primer pair to amplify GdrB gene, PCR reaction parameters: pre-denaturation, 98°C for 3min; denaturation, 98°C for 10s; annealing, 55°C for 10s; extension at 72°C for 1.5min; final extension at 72°C for 5min; after 33 cycles, DhaB123-GdrA gene and GdrB gene were obtained.

(3) Use the obtained DhaB123-GdrA gene and GdrB gene fragments as templates, and use DhaB F and GdrB R primer pairs to perform fusion PCR. PCR reaction parameters: pre-denaturation, 98°C for 3min; denaturation, 98°C for 10s; annealing, 55°C 10s; extension, 72°C for 1.5min; final extension, 72°C for 5min; after 33 cycles, the complete DhaB123-GdrAB gene was obtained.

(4) The pCDFDuet-1 plasmid (Miaoling Bio) was double digested with NcoI and BlnI, and the reaction condition was 37°C for 30 minutes; after the reaction, the linearized pCDFDuet-1 plasmid backbone was obtained. Then the DhaB123-GdrAB gene and the linearized pCDFDuet-1 plasmid backbone were subjected to Gibson assembly using 2×MultiF Seamless Assembly Mix to obtain recombinant plasmids. The reaction conditions are: 50°C for 30 minutes;

Transform the recombinant plasmid obtained above into E.coli DH5α competent (Miaoling Biology) by standard heat shock method. The specific steps of transformation are:

Add the recombinant plasmid after 5 min in ice bath, 30 min on ice, 45 s at 42 °C, 2 min on ice, add 1 mL of antibiotic-free LB medium, incubate at 37 °C for 1 h, and spread LB plates with 50 μg/mL spectinomycin; pick an appropriate amount A single colony was cultured and the recombinant plasmid was extracted and sent to Suzhou Jinweizhi for sequencing verification. The verified correct recombinant plasmid was named pCDF-DhaB123-GdrAB, its nucleotide sequence is shown in SEQ ID No: 13, and the plasmid map is shown in Figure 1.

Figure 1 is the map of the pCDF-DhaB123-GDRAB plasmid in the genetically engineered bacteria E.coli S10G. It can be seen from the figure that the recombinant plasmid contains the origin of replication of ColDF3, and the gene expression is driven by the T7 promoter.

Embodiment 2: the construction of co-expressing propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isozyme (YqhD) and membrane tuberculosis type pyridine nucleotide transhydrogenase (PntAB) recombinant plasmid

(1) According to the gene sequence of propionaldehyde dehydrogenase (GabD4) on plasmid pANY-gabD4, the gene sequence of 1,3-propanediol oxidoreductase isoenzyme (YqhD) in Escherichia coli and membrane tubercle-type pyridine nucleotide transhydrogenase (PntAB) gene sequence and sequence characteristics of pRSFDuet-1 plasmid, using Oligo7.0 software to design primers:

GabD4 F: GTTTAACTTTAAGAAGGAGATACCatgtaccaagatctggcact (SEQ ID No: 14);

GabD4 R: TTATGCGGCCGCAAGCTTGTCGACttacgcttgggtgatgaact (SEQ ID No: 15);

Backbone1 F: attagttaagtataagaaggagatatacat (SEQ ID No: 16);

Backbone1 R: gtggcagcagcctaggttaa (SEQ ID No: 17);

YqhD F: ATTAGTTAAGTATAAGAAGGAGATATACATatgaacaactttaatctgcacac (SEQ ID No: 18);

YqhD R: GTGGCAGCAGCCTAGGTTAAttagcgggcggcttcgtat (SEQ ID No: 19);

Backbone2 F: ttacgcttgggtgatgaacttg (SEQ ID No: 20);

Backbone2 R: gtcgacaagcttgcggc (SEQ ID No: 21);

PntAB F: ACCAAGTTCATCACCCAAGCGTAAaaccgatggaagggaatatcatgc (SEQ ID No: 22);

PntAB R: GCGGCCGCAAGCTTGTCGACttacagagctttcaggattgcatccac (SEQ ID No: 23).

(2) The GabD4 gene was amplified using the GabD4F and GabD4R primer pair, the YqhD gene was amplified using the YqhD F and YqhDR primer pair, and the PntAB gene was amplified using the PntAB F and PntA R primer pair. PCR reaction parameters: pre-denaturation, 98°C 3min; denaturation, 98°C 10s; annealing, 55°C 10s; extension, 72°C 1.5min; final extension, 72°C 5min; GabD4, YqhD and PntAB genes were obtained after 33 cycles.

(3) Digest the pRSFDuet-1 plasmid with NcoI and BlnI, the reaction conditions are: 37°C, 30min; after the reaction, the linearized pRSFDuet-1 plasmid backbone is obtained, and then the GabD4 gene and the linearized pRSFDuet-1 plasmid backbone are Perform Gibson assembly to obtain recombinant plasmid pRSF-GabD4.

Using the obtained recombinant plasmid pRSF-GabD4 as a template, reverse PCR was performed on Backbone1F and Backbone1R using primers to obtain the backbone of the recombinant plasmid pRSF-GabD4; Gibson assembly was performed on the backbone and the YqhD gene to obtain the recombinant plasmid pRSF-GabD4-YqhD.

Using the obtained recombinant plasmid pRSF-GabD4-YqhD as a template, reverse PCR was performed on Backbone2F and Backbone2R using primers to obtain the backbone of the recombinant plasmid pRSF-GabD4-YqhD; Gibson assembly was performed on the backbone and the PntAB gene to obtain the recombinant plasmid pRSF- GabD4-YqhD-PntAB is a recombinant plasmid for co-expressing propionaldehyde dehydrogenase (GabD4), 1,3-propanediol oxidoreductase isoenzyme (YqhD) and membrane tubercle-type pyridine nucleotide transhydrogenase (PntAB). The nucleotide sequence of the pRSF-GabD4-YqhD-PntAB is shown in SEQ ID No: 24, and the plasmid map is shown in Figure 2.

Figure 2 is a map of the pRSF-GabD4-YqhD-PntAB plasmid, the recombinant plasmid contains the RSF replication origin, the expression of the GabD4 gene and the PntAB gene is driven by T7, and the YqhD gene is driven by a separate T7 promoter.

Embodiment 3: the construction of genetically deficient engineered bacteria:

(1) According to the soluble pyridine nucleotide transhydrogenase (SthA) gene sequence, use sgRNAcas9 to select a suitable target to generate a suitable sgRNA sequence; according to the sequence characteristics of the above gene sequence and ptargetF plasmid, use Oligo7.0 software to design for Primers for SthA gene knockout:

sgRNA-sthA F: CCGCGCCATGTACTTATCTAgttttagagctagaaatagc (SEQ ID No: 25);

sgRNA-sthAR: TAGATAAGTACATGGCGCGGactagtattatacctaggac (SEQ ID No: 26);

sthA-up F: agttcgtctacgtcgcggaaatgc (SEQ ID No: 27);

sthA-up R: GCCATTTCGATAAAGTTTTTAcatggtagggcttacctgt (SEQ ID No: 28);

sthA-down F: AACAGGTAAGCCCTACCATGtaaaactttatcgaaatggccatc (SEQ ID No: 29);

sthA-down R: tcctcgcgctggtgaaaga (SEQ ID No: 30);

(2) Using the pTargetF plasmid as a template, use the primer pair sgRNA-sthA F and sgRNA-sthA R to amplify the pTargetF backbone targeting the SthA gene; use Gibson assembly to make it self-ligated to obtain the recombinant plasmid pTargetF-SthA.

(3) Using the Escherichia coli E.coli W3110 genome (NC_000913.3) as a template, use the primer pair sthA-up F and sthA-up R to amplify the upstream sequence of the SthA gene; use the primer pair sthA-down F and sthA-down R Amplify the downstream sequence of the SthA gene; and use the upstream sequence of the SthA gene and the downstream sequence of the SthA gene as templates, and use the primer pair sthA-up F and sthA-down R to amplify the upstream and downstream homology arms of the SthA gene.

(4) Transform the pCas9 plasmid (Miaoling Biotechnology) into E.coli W3110(DE3) (E.coli W3110 was purchased from Biobiology, and lysogenized it with a lysogenization kit to obtain E.coli W3110 (DE3)), the E.coli W3110(DE3) containing the pCas9 plasmid was obtained, and then induced by 0.1M L-arabinose to make it competent for electroporation. Then, 100 ng of the obtained pTargetF-SthA plasmid and 400 ng of the upstream and downstream homology arms of the SthA gene were transformed into E.coli W3110 (DE3) containing the pCas9 plasmid using an electroporator. Mycin and 50 μg/mL kanamycin on LB plates, cultured at 30°C for 24-48h. The electroporation parameters are: voltage 1800V, resistance 200Ω, capacitance 25μF, electroporation cup 1mm, and shock time 1.5ms.

Pick a single colony after culture, use primers to sthA up F and sthA down R for colony PCR verification, and send the suspected genetically defective engineering bacteria to Suzhou Jinweizhi for sequencing verification, and verify that the correct genetically defective engineering bacteria are preserved for future use.

(5) For the above-mentioned genetically deficient engineering bacteria verified by sequencing, it is cultivated in LB liquid medium containing 50 to 50 μg/mL kanamycin, induced by 0.5mM IPTG, and the pTargetF-SthA plasmid is removed. The engineering strain of the pTargetF-SthA plasmid is named E.coli pCas9△sthA.

(6) Based on E.colipCas9△sthA, refer to steps (1) to (5) to knock out the lactate dehydrogenase (LdhA) gene, alcohol dehydrogenase (AdhE) gene, pyruvate formate lyase ( PflB) gene, pyruvate oxidase (PoxB) gene, phosphoacetyltransferase-acetate kinase (PTA-AckA) gene and glycerol metabolism inhibitor (GlpR) gene.

Wherein, the primers for knocking out the lactate dehydrogenase (LdhA) gene are as follows:

sgRNA-ldhA F: CGACAAGAAGTACCTGCAACgttttagagctagaaatagc (SEQ ID No: 31);

sgRNA-ldhAR: GTTGCAGGTACTTCTTGTCGactagtattatacctaggac (SEQ ID No: 32);

ldhA-up F: tttaactttttcgccctga (SEQ ID No: 33);

ldhA-up R: GCAGGGGAGCGGCAAGATTAcataagactttctccagtgatg (SEQ ID No: 34);

ldhA-down F: TCACTGGAGAAAGTCTTATGtaatcttgccgctcccctgc (SEQ ID No: 35);

ldhA-down R: taaaagcgtcgatgtccagt (SEQ ID No: 36).

The primers for knocking out the alcohol dehydrogenase (AdhE) gene are as follows:

sgRNA-adhE F: TCGAATCCCACTCGCGAAAAgttttagagctagaaatagc (SEQ ID No: 37);

sgRNA-adhE R: TTTTCGCGAGTGGGATTCGAactagtattatacctaggac (SEQ ID No: 38);

adhE-up F: taccaaaaagttgtagaatcgtg (SEQ ID No: 39);

adhE-up R: CCAGACAGCGCTACTGATTAcataatgctctcctgataatgt (SEQ ID No: 40);

adhE-down F: ATTATCAGGAGAGCATTATGtaatcagtagcgctgtctgg (SEQ ID No: 41);

adhE-down R: cagcacagtttcgctctg (SEQ ID No: 42).

The primers for knocking out the pyruvate formate lyase (PflB) gene are as follows:

sgRNA-pflB F: ATGAAAAGTTAGCCACAGCCgttttagagctagaaatagc (SEQ ID No: 43);

sgRNA-pflB R: GGCTGTGGCTAACTTTTCATactagtattatacctaggac (SEQ ID No: 44);

pflB-up F: atttgcttctctctggggctga (SEQ ID No: 45);

pflB-up R: ATTTCAGTCAAATCTAATTAcatgtaacacctaccttct (SEQ ID No: 46);

pflB-down F: AAGAAGGTAGGTGTTACatgtaattagatttgactgaaatcgt (SEQ ID No: 47);

pflB-down R: cgtagcggatccacaccttc (SEQ ID No: 48).

The primers for knocking out the pyruvate oxidase (PoxB) gene are as follows:

sgRNA-poxB F: TATCGCCAAAACACTCGAATgttttagagctagaaatagc (SEQ ID No: 49);

sgRNA-poxB R: ATTCGAGTGTTTTGGCGATAactagtattatacctaggac (SEQ ID No: 50);

poxB-up F: aaccgtccacaggccgatgt (SEQ ID No: 51);

poxB-up R: GGGAAATGCCACCCTTTTTAcatggttctccatctcctga (SEQ ID No: 52);

poxB-down F: TCAGGAGATGGAGAACCATgtaaaaagggtggcatttcccgtc (SEQ ID No: 53);

poxB-down R: ccagcgaatggcacgtt (SEQ ID No: 54);

The primers for knocking out the phosphoacetyltransferase-acetate kinase (PTA-AckA) gene are as follows:

sgRNA-pta-ackA F: AATAAACAGGAAGCGGCTTTgttttagagctagaaatagc (SEQ ID No: 55);

sgRNA-pta-ackAR: AAAGCCGCTTCCTGTTTATTactagtattatacctaggac (SEQ ID No: 56);

pta-ackA-up F: aacacctgtccagactcct (SEQ ID No: 57);

pta-ackA-up R: TGCGGATGATGACGAGATTAcatggaagtacctataattga (SEQ ID No: 58);

pta-ackA-down F: CAATTATAGGTACTTCCATgtaatctcgtcatcatccgcag (SEQ ID No: 59);

pta-ackA-down R: ttctcccataccaaataccg (SEQ ID No: 60).

Knockout glycerol metabolism inhibitor (GlpR) gene primers are as follows:

sgRNA-glpRF: CGTAACGCGATGGTCAATATgttttagagctagaaatagc (SEQ ID No: 61);

sgRNA-glpR R: ATATTGACCATCGCGTTACGactagtattatacctaggac (SEQ ID No: 62);

glpR-up F: cgtggtggtgtttattgcc (SEQ ID No: 63);

glpR-up R: AGCACAGCTCCAGTTGAAtcatttataaatccctggaattatt (SEQ ID No: 64);

glpR-down F: AATAATTCCAGGGATTTATAAATGattcaactggagctgtg (SEQ ID No: 65);

glpR-down R: tgaagagaaaaccttttaccc (SEQ ID No: 66).

(7) After all the above-mentioned gene defects are completed, the genetically deficient engineering bacteria obtained are E.coli pCas9△sthA△ldhA△adhE△pflB△poxB△pta△ackA△glpR.

Embodiment 4: the construction of the engineered bacterium that the expression level of glycerol kinase (GlpK) is artificially modified

(1) According to the genome sequence of Escherichia coli E.coli W3110 (DE3), it can be seen that the original UTR sequence of the glycerol kinase (GlpK) gene is: TATGACTACGGGACAATTAAAC (SEQ ID No: 67), and the N-terminal 35bp sequence of the GlpK gene is: ATGACTGAAAAAAAATATATCGTTGCGCTCGACCA (SEQ ID No:68), following the UTR design principles, the artificially designed UTR sequence format should be NNNNNN TACGGGACAA NNNNNN, where TACGGGACAA is the E.coli W3110 ribosome binding site, and N represents any base. In order to make the expression of GlpK reach the expected range, 7 UTR sequences with strength ranging from strong to weak were designed, as shown in Table 1.

Table 1. Different UTRs and their predicted expression strengths

name 5'-UTR sequence (5'-3') Predicted expression (a.u.) glP TATGACTACGGGACAATTAAAC (SEQ ID No: 69) 162064.41 glpK-U1 AGATTATACGGGACAACACGAA (SEQ ID No: 70) 137030.54 glpK-U2 GTTAGCTACGGGACAAGGGTTT (SEQ ID No: 71) 100744.62 glpK-U3 ATTCTTTACGGGACAACCATAA (SEQ ID No: 72) 70038.41 glpK-U4 ACGTCTAACGGGACAAAGTTCC (SEQ ID No: 73) 30268.08 glpK-U5 ACGGTTTACGGGACAACGCGG (SEQ ID No: 74) 10170.17 glpK-U6 ACCGTCTACGGGACAACGCTGT (SEQ ID No: 75) 5198.13 glpK-U7 CCGGTCTACGGGACAACGCTGT (SEQ ID No: 76) 2008.70

(2) According to the glycerol kinase (GlpK) gene sequence, use sgRNAcas9 to select a suitable target to generate a suitable sgRNA sequence; according to the above gene sequence and the sequence characteristics of the ptargetF plasmid (Miaoling Biology), use Oligo7.0 software to design to replace the GlpK gene Primers for UTR:

sgRNA-glpKUTR F: CTTCGCTGTAATATGACTACgttttagagctagaaatagc (SEQ ID No: 77);

sgRNA-glpKUTR R: GTAGTCATATTACAGCGAAGactagtattatacctaggac (SEQ ID No: 78);

glpK-up F: cgcagttgagatggtgattaccg (SEQ ID No: 79);

glpK-up R: ttacagcgaagctttttgttc (SEQ ID No: 80);

glpK-down F: atgactgaaaaaaaatatatcgttgcg (SEQ ID No: 81);

glpK-down R: atccacttcactttggtgcca (SEQ ID No: 82);

glpK-up R1: TTCGTGTTGTCCCGTATAATCTttacagcgaagctttttgttc (SEQ ID No: 83);

glpK-up R2: AAACCCTTGTCCCGTAGCTAACttacagcgaagctttttgttc (SEQ ID No: 84);

glpK-up R3: TTATGGTTGTCCCGTAAAGAATttacagcgaagctttttgttc (SEQ ID No: 85);

glpK-up R4: GGAACTTTGTCCCGTTAGACGTttacagcgaagctttttgttc (SEQ ID No: 86);

glpK-up R5: CCGCGTTGTCCCGTAAACCGTttacagcgaagctttttgttc (SEQ ID No: 87);

glpK-up R6: ACAGCGTTGTCCCGTAGACGGTttacagcgaagctttttgttc (SEQ ID No: 88);

glpK-up R7: ACAGCGTTGTCCCGTAGACCGGttacagcgaagctttttgttc (SEQ ID No: 89);

glpK-down F1: AGATTATACGGGACAACACGAAatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 90);

glpK-down F2: GTTAGCTACGGGACAAGGGTTTatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 91);

glpK-down F3: ATTCTTTACGGGACAACCATAAatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 92);

glpK-down F4: ACGTCTAACGGGACAAAGTTCCatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 93);

glpK-down F5: ACGGTTTACGGGACAACGCGGatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 94);

glpK-down F6: ACCGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 95);

glpK-down F7: CCGGTCTACGGGACAACGCTGTatgactgaaaaaaaatatatcgttgcg (SEQ ID No: 96).

(4) Using the pTargetF plasmid as a template, use the primer pair sgRNA-glpKUTR F and sgRNA-glpKUTR R to amplify the pTargetF backbone targeting the UTR sequence of the GlpK gene; use Gibson assembly to make it self-ligated, and obtain the recombinant plasmid pTargetF-GlpKUTR.

(5) Using the Escherichia coli E.coli W3110 genome as a template, use the primer pair glpK-up F and glpK-up R to amplify the upstream sequence of the GlpK gene; use the primer pair glpK-down F and glpK-down R to amplify the downstream of the GlpK gene sequence.

Obtaining the upstream and downstream homology arms of the GlpK gene containing UTR-U1:

Using the upstream sequence of the GlpK gene as a template, use the primer pair glpK-up F and glpK-up R1 to amplify the upstream sequence of the GlpK gene containing UTR-U1;

Using the downstream sequence of the GlpK gene as a template, use the primer pair glpK-down F1 and glpK-down R to amplify the downstream sequence of the GlpK gene containing UTR-U1;

Using the gene fragments of the upstream sequence of the GlpK gene containing UTR-U1 and the downstream sequence of the GlpK gene containing UTR-U1 as a template, use glpK-down F and glpK-down R to perform fusion PCR to obtain the upstream and downstream of the GlpK gene containing UTR-U1 homology arm.

Obtaining the upstream and downstream homology arms of the GlpK gene containing UTR-U2:

Using the upstream sequence of the GlpK gene as a template, use the primer pair glpK-up F and glpK-up R2 to amplify the upstream sequence of the GlpK gene containing UTR-U2;

Using the downstream sequence of the GlpK gene as a template, use the primer pair glpK-down F2 and glpK-down R to amplify the downstream sequence of the GlpK gene containing UTR-U2;

Using the gene fragments of the upstream sequence of the GlpK gene containing UTR-U2 and the downstream sequence of the GlpK gene containing UTR-U2 as templates, use glpK-down F and glpK-down R to perform fusion PCR to obtain the upstream and downstream of the GlpK gene containing UTR-U2 homology arm.

(6) Combine 100ng pTargetF-GlpKUTR plasmid and 400ng GlpK gene upstream and downstream homology arms containing UTR-U1, UTR-U2, UTR-U3, UTR-U4, UTR-U5, UTR-U6 and UTR-U7 Transformed into E.coli pCas9△sthA△ldhA△adhE△pflB△poxB△pta△ackA△glpR electroporation competent, incubated, coated and sequenced, and finally for the engineering bacteria that successfully replaced the UTR sequence, that is, glycerol kinase ( GlpK) artificially modified engineering bacteria, respectively named as E.coli pCas9 UTR1-GlpK, E.coli pCas9 UTR2-GlpK, E.coli pCas9 UTR3-GlpK, E.coli pCas9 UTR4-GlpK, E.coli pCas9 UTR5 -GlpK, E.coli pCas9 UTR6-GlpK and E.coli pCas9 UTR7-GlpK.

(7) For the above-mentioned engineered bacteria successfully modified by the GlpK gene, they were placed in LB liquid medium without antibiotics and cultured at 37°C for 12 hours to eliminate the pCas9 plasmid, and finally obtained E.coli UTR1-GlpK, E.coli UTR2-GlpK , E.coli UTR3-GlpK, E.coli UTR4-GlpK, E.coli UTR5-GlpK, E.coli UTR6-GlpK and E.coli UTR7-GlpK.

Example 5: Construction of genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol:

The pCDF-DhaB123-GdrAB recombinant plasmid and pRSF-GabD4-YqhD-PntAB recombinant plasmid were simultaneously transformed into E.coli UTR1-GlpK, E.coli UTR2-GlpK, E.coli UTR3-GlpK, E.coli UTR4-GlpK, Among the E.coli UTR5-GlpK, E.coli UTR6-GlpK, and E.coli UTR7-GlpK genetically engineered bacteria, the genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol were obtained, and they were named E.coli U1, E. coli U2, E. coli U3, E. coli U4, E. coli U5, E. coli U6, and E. coli U7.

Example 6: Coproduction of 3-hydroxypropionic acid and 1,3-propanediol:

The genetically engineered bacteria containing the co-production of 3-hydroxypropionic acid and 1,3-propanediol obtained in Example 5 were placed in LB containing 50 μg/mL spectinomycin and 100 μg/mL kanamycin respectively. Activate on the plate at 37°C for 18-24h, then pick a single colony with good growth and activate it in LB liquid medium containing 50μg/mL spectinomycin and 100μg/mL kanamycin for 12h at 37°C, and the activated culture Transfer to M9 liquid medium (MgSO ₄ 7H ₂ O 0.5 g/L, NH ₄ Cl 2.0 g/L, NaCl 2.0 g/L, yeast extract 1.0g, 0.1M potassium phosphate buffer (pH 7.0) and cultured at 37°C for 12h to obtain E.coli U1, E.coli U2, E.coli U3, E.coli U4, E.coli U5, Fermented seed liquors of E.coli U6 and E.coli U.

Then inoculate the fermented seed liquid obtained above into M9 medium containing 30g/L glycerol at 1% inoculation amount, culture at 37°C and 220rpm until the _OD600 reaches 0.8, add 0.05mM IPTG to induce the recombinant plasmid pCDF-DhaB123-GdrAB and gene expression on pRSF-GabD4-YqhD-PntAB, while adding 50 μM vitamin B12 to activate glycerol dehydratase activity, and then switch the culture temperature to 35°C for 36 hours of fermentation to obtain fermentation metabolites.

The fermentation metabolites were determined by liquid chromatography, and the specific measurement conditions were: chromatographic column Aminex HPX-87H column (Bio-Rad, 300×7.8mm), mobile phase 0.5mM sulfuric acid, flow rate 0.4mL/min, column temperature 65 °C, UV detector wavelength 210nm, differential refractive index detector 45 °C, detection time 40min.

The test results show that regardless of the expression level of GlpK, glycerol can be fermented to produce 3-hydroxypropionic acid and 1,3-propanediol at the same time, as shown in Figure 3. Among them, E.coli U3 has the best production performance, and it is further named E.coli U3. .coli S10G.

In this example, the fermentation medium of E. coli S10G was also improved, and the nitrogen source in the fermentation medium was replaced with cheap corn steep liquor. Specifically, the yeast extract in the fermentation culture M9 was replaced with different concentrations of corn steep liquor, the content of which was 0.05%, 0.1%, 0.25% and 0.5% (v/v), and the remaining fermentation medium components were: MgSO ₄ . 7H ₂ O 0.5g/L, NH ₄ Cl 2.0g/L, NaCl 2.0g/L and 0.1M potassium phosphate buffer, pH 7.0.

The test results show that the medium containing 0.25% corn steep liquor is the optimal concentration, under this concentration, not only the cost of fermentation is reduced, but the yield of 3-hydroxypropionic acid and 1,3-propanediol remains unchanged, and the concentration of by-product acetic acid is reduced. , the improved medium was named M9-CSL.

In this example, the fermentation and production conditions of E.coli S10G are also discussed. Specifically, the entire fermentation process is divided into a growth stage and a production stage, and a two-stage pH control fermentation method is designed for it. Specifically, the large intestine is used in the growth stage The optimal pH of bacilli is 7.0 for biomass accumulation; different pHs are used for testing in the production stage to explore the optimal pH for the production stage.

The results showed that pH 8.0 was the optimum pH in the production stage, and the yields of 3-hydroxypropionic acid and 1,3-propanediol were significantly higher at pH 8.0 than at pH 7.0. Therefore, subsequent fermentation tests were carried out at pH 7.0 in the growth stage and pH 8.0 in the production stage.

(7) Finally, the fed-batch fermentation of E.coli E.coli S10G was carried out in a 5L fermenter using the above-mentioned two-stage pH control method, and the supplemented ingredients contained 800g/L glycerin and 50mL/L corn steep liquor. First, use the fresh seed liquid activated in the M9-CSL medium to inoculate in a 5L fermenter with a 5% inoculum size (the actual liquid volume is 2L), the temperature is 37°C, the initial ventilation is 2vvm, and the stirring rate is 500rpm. The pH was controlled at 7.0 with 10M NaOH, and when the OD600 reached 4, 0.05mM IPTG and 50μM vitamin B12 were added, and the temperature was changed to 35°C; when the growth of the engineered bacteria entered a stable period (OD600 was about 45), the pH was adjusted to 8.0 , the ventilation volume was adjusted to 3vvm, the dissolved oxygen value was controlled to 10%, and metabolites were detected and fed every 6h thereafter, so that the glycerin concentration was maintained at about 40g/L.

The test results are shown in Figure 5, the engineered bacteria E.coli E.coli S10G was fermented for 66h, and it produced 77.34g/L of 3-hydroxypropionic acid and 63.16g/L of 1,3-propanediol, with a total output of 140.5g /L.

The described embodiment is a preferred implementation of the present invention, but the present invention is not limited to the above-mentioned implementation, without departing from the essence of the present invention, any obvious improvement, replacement or modification that those skilled in the art can make Modifications all belong to the protection scope of the present invention.

Claims (13)

A kind of genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol is characterized in that, said genetically engineered bacterium expresses glycerol dehydratase and its reactivation on the basis of Escherichia coli E.coli W3110 (DE3) Factors DhaB123-GdrAB, propionaldehyde dehydrogenase GabD4, 1,3-propanediol oxidoreductase isoenzyme YqhD and membrane-bound pyridine nucleotide transhydrogenase PntAB were obtained. The genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol according to claim 1 is characterized in that, one or more groups in the following gene combinations have also been knocked out in the genetically engineered bacterium: (1) the gene of soluble pyridine nucleotide transhydrogenase SthA; (2) genes of lactate dehydrogenase LdhA, alcohol dehydrogenase AdhE, pyruvate formate lyase PflB, pyruvate oxidase PoxB, phosphate acetyltransferase-acetate kinase PTA-AckA; (3) Gene of glycerol metabolism inhibitor GlpR. The genetically engineered bacterium of claim 1 or 2, which co-produces 3-hydroxypropionic acid and 1,3-propanediol, is characterized in that, in the genetically engineered bacterium, the original UTR sequence of glycerol kinase GlpK is also replaced by an artificially designed UTR sequence, the artificially designed UTR sequence is any of the following seven sequences: glpK-U1 AGATTATACGGGACAACACGAA (SEQ ID No: 70); glpK-U2 GTTAGCTACGGGACAAGGGTTT (SEQ ID No: 71); glpK-U3 ATTCTTTACGGGACAACCATAA (SEQ ID No: 72); glpK-U4 ACGTCTAACGGGACAAAGTTCC (SEQ ID No: 73); glpK-U5 ACGGTTTACGGGACAACGCGG (SEQ ID No: 74); glpK-U6 ACCGTCTACGGGACAACGCTGT (SEQ ID No: 75); glpK-U7 CCGGTCTACGGGACAACGCTGT (SEQ ID No: 76). The genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol according to claim 1 is characterized in that, the amino acid sequence of the DhaB123-GdrAB is as shown in SEQ ID No: 2, and its nucleus is encoded The nucleotide is shown in SEQ ID No:1; The amino acid sequence of the GabD4 is shown in SEQ ID No: 4, and the nucleotide encoding it is shown in SEQ ID No: 3; The amino acid sequence of the YqhD is shown in SEQ ID No: 6, and the nucleotide encoding it is shown in SEQ ID No: 5; The amino acid sequence of the PntAB is shown in SEQ ID No: 8, and the nucleotide encoding it is shown in SEQ ID No: 7. The preparation method of the genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol according to claim 1 is characterized in that, comprising: (1) Construction of glycerol dehydratase and its reactivation factor DhaB123-GdrAB recombinant plasmid: Glycerol dehydratase DhaB123 gene and glycerol dehydratase reactivator GdrAB gene were amplified by PCR, and fusion PCR technology was used to form a complete glycerol dehydratase and its reactivator gene fragment DhaB123-GdrAB, and then cloned into pCDFDuet-1 plasmid, transformed E.coli DH5α, screening positive clones, plasmid extraction, and sequencing confirmation, obtained glycerol dehydratase and its reactivation factor plasmid, named pCDF-DhaB123-GdrAB; (2) Construction of recombinant plasmids that co-express propionaldehyde dehydrogenase GabD4, 1,3-propanediol oxidoreductase isoenzyme YqhD and membrane tuberculosis-type pyridine nucleotide transhydrogenase PntAB: Propionaldehyde dehydrogenase GabD4 gene, 1,3-propanediol oxidoreductase isoenzyme YqhD gene and membrane tubercle-type pyridine nucleotide transhydrogenase PntAB gene were amplified by PCR, and the above two genes were cloned into pRSFuet-1 plasmid, after transforming E.coli DH5α, screening positive clones, plasmid extraction, and sequencing confirmation, the co-expression of propionaldehyde dehydrogenase GabD4, 1,3-propanediol oxidoreductase isoenzyme YqhD and membrane tubercle-type pyridinic nucleus were obtained The recombinant plasmid of nucleotide transhydrogenase PntAB, named pRSF-GabD4-YqhD-PntAB; (3) Construction of genetically engineered bacteria that co-produce 3-hydroxypropionic acid and 1,3-propanediol: Simultaneously transform pCDF-DhaB123-GdrAB and pRSF-GabD4-YqhD-PntAB into competent cells of Escherichia coli W3110 (DE3) to obtain genetic engineering for efficient co-production of 3-hydroxypropionic acid and 1,3-propanediol; Wherein, steps (1) and (2) are in no particular order. The preparation method of the genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol according to claim 2 is characterized in that, the method for knocking out is: use double-plasmid CRIPSR CAS9 tool plasmid to knock out Escherichia coli The following genes of E.coli W3110(DE3): soluble pyridine nucleotide transhydrogenase SthA, lactate dehydrogenase LdhA, alcohol dehydrogenase AdhE, pyruvate formate lyase PflB, pyruvate oxidase PoxB, phosphoric acid Acetyltransferase-acetate kinase PTA-AckA and glycerol metabolism inhibitor GlpR. The preparation method of the genetically engineered bacterium that co-produces 3-hydroxypropionic acid and 1,3-propanediol according to claim 3 is characterized in that, the original UTR sequence of glycerol kinase GlpK is replaced by the UTR sequence of artificial design. The steps are: The steps are: using UTR engineering technology to artificially modify the expression level of glycerol kinase GlpK of Escherichia coli knocked out by-products, using UTR design tools to design UTR artificial sequences, and using double-plasmid CRIPSR CAS9 tool plasmids to replace genetically defective engineering bacteria The UTR sequence of the original glycerol kinase GlpK gene in the genome was verified by sequencing, and the engineering bacteria with artificially modified expression of glycerol kinase GlpK were obtained; The artificially designed UTR sequence is any of the following seven sequences: glpK-U1 AGATTATACGGGACAACACGAA (SEQ ID No: 70); glpK-U2 GTTAGCTACGGGACAAGGGTTT (SEQ ID No: 71); glpK-U3 ATTCTTTACGGGACAACCATAA (SEQ ID No: 72); glpK-U4 ACGTCTAACGGGACAAAGTTCC (SEQ ID No: 73); glpK-U5 ACGGTTTACGGGACAACGCGG (SEQ ID No: 74); glpK-U6 ACCGTCTACGGGACAACGCTGT (SEQ ID No: 75); glpK-U7 CCGGTCTACGGGACAACGCTGT (SEQ ID No: 76). The application of the genetic engineering bacteria described in claim 1 in the co-production of 3-hydroxypropionic acid and 1,3-propanediol by fermenting glycerol. The application of the genetic engineering bacteria described in claim 2 in the co-production of 3-hydroxypropionic acid and 1,3-propanediol by fermenting glycerol. The application of the genetically engineered bacteria described in claim 3 in the co-production of 3-hydroxypropionic acid and 1,3-propanediol by fermenting glycerol. The application according to any one of claims 8, 9 or 10, characterized in that it comprises: (1) activating the genetically engineered bacteria overnight in LB medium to obtain the primary seed liquid of the genetically engineered bacteria; (2) inoculating the primary seed liquid of the genetically engineered bacteria into the improved M9-CSL medium for cultivation to obtain the secondary seed liquid of the genetically engineered bacteria; (3) Inoculate the secondary seed liquid of the genetically engineered bacteria into the improved M9-CSL medium, divide the fermentation process into a growth stage and a production stage, and control the pH value to carry out fed-batch fermentation; During the growth stage, the pH is controlled to be 7.0, and the temperature, ventilation, and stirring rate are adjusted for fermentation and cultivation until the OD600 reaches 4, and IPTG and vitamin B12 are added to continue the cultivation; During the production stage, the pH is controlled to be 8.0, and the temperature, ventilation, and dissolved oxygen value are adjusted for fermentation, and then fed-feed fermentation is carried out every 6 hours to co-produce 3-hydroxypropionic acid and 1,3-propanediol; The ingredients of the supplement contained glycerin and corn steep liquor. The application according to claim 11, characterized in that, in step (2), the composition of the improved M9-CSL medium is MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ Cl 2.0g/L, NaCl 2.0g/L, 2.5mL/L corn steep liquor, 40g/L glycerol and 0.1M potassium phosphate buffer, pH 7.0; The inoculum amount of the primary seed liquid of the genetically engineered bacteria is 1% v/v, and the culture conditions are 37° C. and 220 rpm for 12 hours. The application according to claim 11, characterized in that, in step (3), the inoculation amount of the secondary seed liquid is 5% v/v; During the growth stage, the temperature is adjusted to 37°C, the initial ventilation is 2vvm, and the stirring rate is 500rpm for fermentation; During the production stage, adjust the temperature and ventilation volume to be adjusted to 3vvm, the stirring rate is 200-800rpm, and the dissolved oxygen value is controlled to be 10% for fermentation; The composition of described feed contains 800g/L glycerol and 50mL/L corn steep liquor; The feeding process is as follows: feed every 6 hours to control the glycerin concentration to be maintained at 40g/L.

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