CN114276970A

CN114276970A - Gene engineering bacterium for producing 1, 3-propylene glycol

Info

Publication number: CN114276970A
Application number: CN202111660305.1A
Authority: CN
Inventors: 谭天伟; 李明达
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-05
Anticipated expiration: 2041-12-31
Also published as: CN114276970B

Abstract

The invention relates to a gene engineering bacterium for producing 1, 3-propylene glycol. The genetically engineered bacterium is obtained by introducing a gene panD for coding aspartate decarboxylase, a gene bauA for coding beta-alanine-pyruvate aminotransferase, a gene ydfG for coding 3-hydroxy acid dehydrogenase, a gene pdup for coding aldehyde dehydrogenase subjected to codon optimization, a gene yqhD for coding alcohol dehydrogenase and a gene Mseed-1456 for coding 3-hydroxypropionyl coenzyme A synthetase subjected to codon optimization by using escherichia coli as a host cell, and is the genetically engineered bacterium with high 1,3-propanediol yield.

Description

Gene engineering bacterium for producing 1, 3-propylene glycol

Technical Field

The invention belongs to the technical field of biology, relates to a genetic engineering bacterium for producing 1,3-propanediol, and particularly relates to a genetic engineering bacterium for producing 1,3-propanediol and application thereof in producing 1, 3-propanediol.

Background

1,3-propanediol is an important chemical substance, and has been widely applied to the fields of textiles, resins, medicines and the like, in particular to being used as a synthetic monomer of polymer polytrimethylene terephthalate (PTT). According to Global 1,3-Propanediol (PDO) published by TechNavio (Infiniti Research Ltd.), Market 2020-.

The production method of the 1, 3-propylene glycol mainly comprises a chemical synthesis method and a biological synthesis method, wherein the chemical synthesis method comprises an acrolein method and an ethylene oxide method as representative processes, but the pollution is serious, the number of byproducts is large, the biological synthesis method is produced by microbial fermentation, the pollution is low, and the biological method for synthesizing the 1,3-PDO becomes a strategy with the highest potential for replacing the chemical synthesis.

Therefore, research and development of a1, 3-PDO biosynthesis technology which has high conversion rate and good economical efficiency and is easy for industrial production are needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the 1,3-propanediol genetically engineered bacterium which is a genetically engineered bacterium with high 1,3-propanediol yield, and the 1,3-propanediol produced by the genetically engineered bacterium has high conversion rate, good economy and easy industrial production.

Therefore, the invention provides a genetic engineering bacterium for producing 1,3-propanediol, and the pathway for synthesizing the 1,3-propanediol is as follows:

(1) the glucose generates oxaloacetate under the action of glycolytic pathway of the microorganism;

(2) oxaloacetate generates L-aspartic acid under the action of aspartate aminotransferase;

(3) the L-aspartic acid generates beta-alanine under the catalysis of aspartate decarboxylase;

(4) beta-alanine generates malonic semialdehyde under the catalytic action of beta-alanine-pyruvic acid aminotransferase;

(5) the malonic semialdehyde generates 3-hydroxypropionic acid under the catalytic action of 3-hydroxy acid dehydrogenase;

(6) 3-hydroxypropionic acid generates 3-hydroxypropionyl coenzyme A under the catalytic action of 3-hydroxypropionyl coenzyme A synthetase;

(7) 3-hydroxypropionyl coenzyme A is catalyzed by aldehyde dehydrogenase to generate 3-hydroxypropionaldehyde;

(8) the 3-hydroxypropionaldehyde generates 1,3-propanediol under the catalytic action of alcohol dehydrogenase.

According to the invention, the genetically engineered bacterium is a recombinant host bacterium comprising a gene panD coding for aspartate decarboxylase, a gene bauA coding for beta-alanine-pyruvate aminotransferase, a gene ydfG coding for 3-hydroxyacid dehydrogenase, a gene pdup coding for aldehyde dehydrogenase, a gene yqhD coding for alcohol dehydrogenase, and a gene Mseed _1456 coding for 3-hydroxypropionyl-CoA synthetase.

In some embodiments of the invention, the gene panD coding for aspartate decarboxylase is derived from the Bacillus subtilis strain 168 (Bacillus subtilis strain 168), the sequence of which is shown in SEQ No. 1.

In some embodiments of the invention, the gene bauA encoding β -alanine-pyruvate aminotransferase is derived from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1), the sequence of which is shown in SEQ No. 2.

In some embodiments of the invention, the gene ydfG encoding the 3-hydroxy acid dehydrogenase is derived from Escherichia coli K12 strain (Escherichia coli strain K12), the sequence of which is shown in SEQ No. 3.

In some embodiments of the present invention, the gene coding for aldehyde dehydrogenase pdup is derived from Salmonella enterica (Salmonella enterica), and the sequence of the codon-optimized gene coding for aldehyde dehydrogenase pdup is shown in SEQ No. 4.

In some embodiments of the invention, the gene yqhD encoding alcohol dehydrogenase is derived from Escherichia coli K12 strain (Escherichia coli strain K12), the sequence of which is shown in SEQ No. 5.

In some embodiments of the invention, the gene Mseed _1456 encoding 3-hydroxypropionyl-CoA synthetase is derived from Metallococcus DSM 5348(Metallosphaera sedula DSM 5348), and the codon-optimized gene Mseed _1456 encoding 3-hydroxypropionyl-CoA synthetase has the sequence shown in SEQ No. 6.

According to the invention, the host bacteria comprise Escherichia coli, Corynebacterium glutamicum, yeast, and modified bacteria and fungi.

In some preferred embodiments of the invention, the host bacterium is escherichia coli.

In some further preferred embodiments of the invention, the host bacterium is escherichia coli BW 25113.

The invention also provides an application of the genetic engineering bacteria in the production of 1, 3-propanediol.

According to the invention, the application comprises the steps of inoculating the genetic engineering bacteria for producing the 1,3-propanediol into a fermentation culture medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain the 1, 3-propanediol.

In some embodiments of the invention, the fermentation culture conditions are: the fermentation culture time is 72h, when the thallus grows to OD600 is 0.6-0.8, IPTG is added, after the addition of the IPTG, the fermentation temperature is changed from 37 ℃ to 30 ℃, and the induction concentration of the IPTG is 0.2 mM; further preferably, aspartic acid is added in vitro, and the amount of aspartic acid added is 2-10g/L, more preferably 5-10 g/L.

In some embodiments of the present invention, the isolation and purification of the obtained fermentation broth comprises:

step S1, carrying out first centrifugal separation on the fermentation culture solution to obtain first supernatant;

step S2, diluting the supernatant fluid I by 1000 times with acetonitrile, and uniformly mixing to obtain a solution A; adding 10 mul of trichloro-acetyl isocyanate into 200 mul of the solution A for derivatization, and uniformly mixing; adding 190 microliter of ultrapure water into the derivatized solution, and uniformly mixing to obtain a second supernatant; step S3, filtering the second supernatant with 0.22 μm organic phase filter membrane to obtain 1, 3-propanediol.

The present inventors obtained a recombinant host bacterium by introducing a gene panD encoding aspartate decarboxylase, a gene bauA encoding beta-alanine-pyruvate aminotransferase, a gene ydfG encoding 3-hydroxy acid dehydrogenase, a gene pdup encoding aldehyde dehydrogenase codon-optimized, a gene yqhD encoding alcohol dehydrogenase, and a gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase codon-optimized into E.coli as a host cell. The strain is a genetic engineering strain with high yield of 1,3-propanediol, and the genetic engineering strain is used for producing the 1,3-propanediol, so that the conversion rate is high, the economy is good, and the industrial production is easy.

Drawings

The invention is described in further detail below with reference to the attached drawing figures:

FIG. 1 shows the reaction mechanism for the biosynthesis of 1, 3-propanediol;

FIG. 2 is a graph showing the yield of 1,3-propanediol produced by fermentation of strain BW-PA-pby-PR-pyM in a 250ml shake flask;

FIG. 3 is a graph showing the yield of 1,3-propanediol produced by fermentation of strain BW-PA-pby-PR-pyM in a 5L fermentor.

The strain BW-PA-pby-PR-pyM carries two plasmids, one of which is PACYC-pby plasmid, which comprises a gene panD which is derived from Bacillus subtilis strain 168 (Bacillus subtilis strain 168) and codes aspartate decarboxylase, a gene bauA which is derived from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1) and codes beta-alanine-pyruvate aminotransferase, and a gene ydfG which is derived from Escherichia coli K12 strain (Escherichia coli strain K12) and codes 3-hydroxy acid dehydrogenase; another is the PRSF-pyM plasmid, which includes pdup, a codon-optimized gene encoding aldehyde dehydrogenase derived from Salmonella enterica (Salmonella enterica), yqhD, a gene encoding alcohol dehydrogenase derived from Escherichia coli K12 strain (Escherichia coli strain K12), Mseed-1456, a gene encoding 3-hydroxypropionyl-CoA synthetase derived from Metallococcus DSM 5348(Metallosphaera sedula DSM 5348), and codon-optimized.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "genetically engineered bacterium" as used herein refers to a bacterium or fungus, such as Escherichia coli, which is transformed by introducing a desired gene into a host organism (i.e., a host cell or a basal disc microorganism or a bacterial body) to express the gene, or by modifying the bacterial body to produce a desired protein by a basal disc microorganism including the knock-out or attenuation of genes involved in the enhanced and competitive metabolic pathways of the precursor synthesis pathway. The core technology of genetic engineering is the recombination technology of DNA, therefore, the genetically engineered bacteria are also called recombinant microorganisms in the invention.

The term "recombinant" as used herein refers to the construction of a transgenic organism that utilizes the genetic material of a donor organism or an artificially synthesized gene, which is cleaved with restriction enzymes in vitro or ex vivo and then ligated with a suitable vector to form a recombinant DNA molecule, which is then introduced into a recipient cell or a recipient organism to construct a transgenic organism that exhibits a certain property of another organism according to a human blueprint that has been previously designed.

Embodiments II

In order to overcome the defects of the prior art and realize the aim of efficiently biosynthesizing the 1,3-propanediol, the inventor conducts a great deal of research on the process technology for biosynthesizing the 1, 3-propanediol. The inventor finds that a genetic engineering bacterium for producing 1,3-propanediol with high yield is successfully constructed and obtained by introducing a gene panD for coding aspartate decarboxylase, a gene bauA for coding beta-alanine-pyruvate aminotransferase, a gene ydfG for coding 3-hydroxy acid dehydrogenase, a gene pdup for coding aldehyde dehydrogenase after codon optimization, a gene yqhD for coding alcohol dehydrogenase and a gene Mseed _1456 for coding 3-hydroxypropionyl-CoA synthetase after codon optimization by using escherichia coli as a host cell, and the genetic engineering bacterium for producing 1,3-propanediol has high conversion rate, good economy and easy industrial production. The present invention was thus obtained.

Therefore, the invention provides a new way for synthesizing 1,3-propanediol, which realizes the high-efficiency synthesis of 1,3-propanediol by using aspartate as a precursor through a genetic engineering bacterium for high yield of 1, 3-propanediol.

Specifically, the pathway for synthesizing 1,3-propanediol by the genetically engineered bacteria for producing 1,3-propanediol is shown in fig. 1, and comprises the following steps:

It will be appreciated by those skilled in the art that in pathway (1) above, glucose is subject to the glycolytic pathway of the microorganism itself to produce oxaloacetate, and TCA can be supplemented to grow it.

In order to realize the technical scheme, the invention provides a host strain capable of producing 1,3-propanediol, which expresses genes in a1, 3-propanediol synthesis path in original or modified bacterial and fungal cells to prepare a host capable of synthesizing 1, 3-propanediol.

In an embodiment of the present invention, the present invention provides a genetically engineered bacterium producing 1,3-propanediol, which is a recombinant host bacterium expressing a gene in the 1,3-propanediol synthesis pathway.

The reaction mechanism for biosynthesis of 1,3-propanediol in the present invention is shown in FIG. 1, and it can be understood from FIG. 1 that the genes in the 1,3-propanediol synthesis pathway include a gene panD encoding aspartate decarboxylase, a gene bauA encoding beta-alanine-pyruvate aminotransferase, a gene ydfG encoding 3-hydroxy acid dehydrogenase, a gene pdup encoding aldehyde dehydrogenase through codon optimization, a gene yqhD encoding alcohol dehydrogenase, and a gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase through codon optimization.

As will be readily understood from the above, the 1, 3-propanediol-producing genetically engineered bacterium of the present invention is a recombinant host bacterium comprising a gene panD encoding aspartate decarboxylase, a gene bauA encoding beta-alanine-pyruvate aminotransferase, a gene ydfG encoding 3-hydroxy acid dehydrogenase, a codon-optimized gene pdup encoding aldehyde dehydrogenase, a gene yqhD encoding alcohol dehydrogenase, and a codon-optimized gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase.

In some embodiments of the invention, the nucleotide sequence of the gene panD (GenBank: CAB14157.1) encoding aspartate decarboxylase derived from Bacillus subtilis strain 168 (Bacillus subtilis strain 168) is shown as SEQ No. 1.

In some embodiments of the invention, the nucleotide sequence of the gene bauA (GenBank: AAG03522.1) encoding beta-alanine-pyruvate aminotransferase from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1) is shown in SEQ No. 2.

In some embodiments of the invention, the nucleotide sequence of the gene ydfG (GenBank: BAA15241.1) encoding 3-hydroxy acid dehydrogenase derived from Escherichia coli strain K12 (Escherichia coli strain K12) is shown in SEQ No. 3.

In some embodiments of the invention, the codon optimized gene encoding aldehyde dehydrogenase, pdup, has the sequence shown in SEQ No. 4; the GenBank accession number of the pdup sequence of the gene encoding aldehyde dehydrogenase is EBO5705941.1, which is derived from Salmonella enterica (Salmonella enterica).

In some embodiments of the invention, the nucleotide sequence of the gene yqhD (GenBank: BAE77068.1) encoding alcohol dehydrogenase derived from Escherichia coli K12 strain (Escherichia coli strain K12) is shown in SEQ No. 5.

In some embodiments of the invention, codon optimized gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase has the sequence shown in SEQ No. 6; GenBank accession number ABP95613.1 of the Mseed-1456 sequence of the gene encoding 3-hydroxypropionyl-CoA synthetase is derived from the metal coccus DSM 5348(Metallosphaera sedula DSM 5348).

In some further preferred embodiments of the invention, by efficiently expressing in a host bacterium the genes for enzymes involved in the 1,3-propanediol synthesis pathway, including the gene panD encoding aspartate decarboxylase from Bacillus subtilis strain 168 (Bacillus subtilis strain 168), the gene bauA encoding beta-alanine-pyruvate aminotransferase from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1), the gene ydfG encoding 3-hydroxy acid dehydrogenase from Escherichia coli K12 strain (Escherichia coli strain K12), the gene ydfG encoding aldehyde dehydrogenase from Salmonella enterica (Salmonella enterica) and codon-optimized, the gene qhD encoding alcohol dehydrogenase from Escherichia coli K12 strain (Escherichia coli strain K12), the gene MsqhD encoding hydroxy acid dehydrogenase from Metallococcus DSM 5348 (Metallococcus DSM 5348) and codon-optimized for propionyl 3A-hydroxy acid 1456, realizes the synthesis of 1,3-propanediol by using aspartic acid as a precursor.

According to the invention, the host bacteria comprise escherichia coli, corynebacterium glutamicum, yeast, and modified bacteria and fungi; preferably, the host bacterium is escherichia coli; further preferably, the host bacterium is escherichia coli BW25113 (beijing washington biotechnology limited).

In the invention, the type of the expression plasmid has no special requirement, and can be correspondingly adjusted according to the selection of a host, and the construction method for expressing the target gene in the escherichia coli can adopt various methods commonly used in the field, for example, the target gene and the expression vector are connected after enzyme digestion treatment, and the details are not repeated.

In some further preferred embodiments, the E.coli strain Trans10 (Beijing holotype gold organism) is used for vector construction and E.coli BW25113 (Beijing Huayuyo Biotech Co., Ltd.) is used as the fermentation strain.

The genetically engineered bacteria producing 1,3-propanediol according to the embodiment of the present invention are escherichia coli BW25113 capable of expressing panD, a gene bauA encoding β -alanine-pyruvate aminotransferase, ydfG, a gene ydfG encoding 3-hydroxy acid dehydrogenase, pdup, a gene yqhD encoding alcohol dehydrogenase, and Msed, a gene Msed-1456, a gene encoding 3-hydroxypropionyl-coa synthetase, all of which are codon-optimized genes.

In some further preferred examples, genetically engineered bacteria were constructed using the genes panD, bauA, ydfG, pdup (codon optimized), yqhD, Msed-1456 (codon optimized) and the plasmids PACYC-Ptac, PRSF-Ptac, the relevant primers for the construction of recombinant plasmids are shown in Table 1, and the corresponding sequences are shown in SEQ ID Nos. 7-22.

TABLE 1 primers related to the construction of recombinant plasmids

The invention adopts escherichia coli as a host cell, and successfully constructs and obtains the genetic engineering bacteria for high yield of 1,3-propanediol by introducing and expressing a gene panD for coding aspartate decarboxylase, a gene bauA for coding beta-alanine-pyruvate aminotransferase, a gene ydfG for coding 3-hydroxy acid dehydrogenase, a gene pdup for coding aldehyde dehydrogenase after codon optimization, a gene yqhD for coding alcohol dehydrogenase and a gene Mseed _1456 for coding 3-hydroxypropionyl coenzyme A synthetase after codon optimization.

The use of the genetically engineered bacterium of the present invention as described above for the production of 1,3-propanediol according to the present invention is understood to be a method for producing 1,3-propanediol by using the genetically engineered bacterium of the present invention as described above.

In some embodiments of the present invention, inoculating the genetically engineered bacterium that produces 1,3-propanediol into a fermentation medium for fermentation culture comprises: inoculating the gene engineering bacteria producing 1,3-propanediol into a fermentation culture medium, performing fermentation culture at 200rpm, adding the final concentration of 0.2mM when OD600 is between 0.6 and 0.8, adding IPTG (isopropyl-beta-thiogalactoside), changing the fermentation temperature from 37 ℃ to 30 ℃, and culturing for 72 hours to obtain a fermentation culture solution; aspartic acid is added into the culture medium when needed, and the addition amount of the aspartic acid is 2-10g/L, preferably 5-10 g/L.

In other embodiments of the present invention, the isolation and purification of the obtained fermentation broth comprises:

step S2, diluting the supernatant fluid I by 1000 times with acetonitrile, and uniformly mixing to obtain a solution A; adding 10 mul of trichloro-acetyl isocyanate into 200 mul of the solution A for derivatization, and uniformly mixing; adding 190 microliter of ultrapure water into the derivatized solution, and uniformly mixing to obtain a second supernatant;

step S3, filtering the second supernatant with 0.22 μm organic phase filter membrane to obtain 1, 3-propanediol.

The fermentation medium in the present invention is not particularly limited as long as it is a fermentation medium for producing 1,3-propanediol, and preferably, the M9 fermentation medium has a formula of: glucose 20g/L, Yeast powder 3g/L, M9 salt (10X) 100mL, 1M MgSO₄ 1mL，1M CaCl₂0.3mL, 1g/L biotin 1mL, 1g/L thiamine 1mL, M9 trace elements (100X) 10mL (1L system); wherein, M9 salt (M)Salt 9) (10 ×) composition: na (Na)₂HPO₄ 67.8g/L，KH₂PO₄ 30g/L，NaCl 5g/L，NH₄Cl 10 g/L; the trace elements (100 ×) of M9 are: EDTA 5g/L, FeCl₂·6H₂O 0.83g/L,ZnCl₂ 84mg/L，CuCl₂·2H₂O 13mg/L，CoCl₂·2H₂O 10mg/L，H₃BO₃ 10mg/L MnCl₂·4H₂O 1.6mg/L。

III example

The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.

Example 1:

the primers used in this example are shown in table 1 above.

The large Gene of Wahua was entrusted with the gene panD (GenBank: CAB14157.1, nucleotide sequence shown in SEQ No. 1) encoding aspartate decarboxylase derived from Bacillus subtilis strain 168 (Bacillus subtilis strain 168), the gene bauA (GenBank: AAG03522.1 nucleotide sequence shown in SEQ No. 2) encoding β -alanine-pyruvate aminotransferase derived from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1), the gene pdup (GenBank: EBO5705941.1 nucleotide sequence shown in SEQ No. 4) encoding aldehyde dehydrogenase derived from Salmonella enterica (Salmonella enterica) and codon-optimized, the gene Mseed _1456(GenBank: ABP95613.1 nucleotide sequence shown in SEQ No. 6) encoding 3-hydroxypropionyl CoA synthetase derived from Metallococcus DSM 5348(Metallosphaera sedula 5348) and codon-optimized, and the genes respectively, and the genes panD and BAD were obtained, pUC57 plasmid containing pdup and Mseed _1456 gene. Amplification of the target genes panD, bauA, ydfG, pdup, yqhD, ysed _1456 and plasmid fragments PAC-YCtac and PRSF-tac were carried out using panD-F/panD-R, bauA-F/bauA-R, ydfG-F/ydfG-R, pdup-F/pdup-R, yqhD-F/yqhD-R, Msed-1456-F/Mseed _1456-R, PACYC-tac-F/PACYC-tac-R, PRSF-tac-F/PRSF-tac-R as primers and pUC57-panD, pUC57-bauA, BW25113 genome, pUC57-pdup, BW25113 genome, 57-Mseed _1456, PACYCpUC-tac, PRSF-tac as templates, respectively. Inserting panD, bauA and ydfG into PACYC-tac by a Gibson ligation method to obtain a plasmid PACYC-pby; pdup, yqhD, and Msed _1456 were inserted into PRSF-tac to obtain plasmid PRSF-pyM (see table 2).

Example 2:

competent cells of E.coli BW25113 were prepared and dispensed in 100. mu.L 1.5mL centrifuge tubes for electroporation. 2-4. mu.L of the constructed PACYC-pby and PRSF-pyM recombinant plasmids are added into a 1.5mL centrifuge tube containing 100. mu.L of competent cells and mixed uniformly. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the electrotransfer was completed, LB medium [ peptone 10g/L, yeast powder 5g/L, NaCl 5g/L ] was added rapidly, and the mixture was transferred to a 1.5mL centrifuge tube and allowed to resuscitate at 37 ℃ for 1 h. Then, the bacterial solution was applied to a plate containing the corresponding antibiotic and cultured at 37 ℃ for 12 hours. The strain BW-PA-pby-PR-pyM (see Table 2) for producing 1,3-propanediol was prepared.

TABLE 2 plasmids and strains

Example 3:

(1) shake flask culture of engineering strain for producing 1,3-propanediol gene

Picking single colony on a plate of a strain BW-PA-pby-PR-pyM for producing 1,3-propanediol, inoculating the single colony into 4mL of liquid LB with resistance, culturing at 37 ℃ for 12h, then transferring the bacterial liquid into 20mL of fermentation medium according to the inoculation amount of 5%, adding aspartic acid with corresponding concentration into the medium if necessary, adding IPTG with the final concentration of 0.2mM for induction when the bacterial body grows to OD600 of 0.6-0.8, and after adding the IPTG, changing the fermentation temperature from 37 ℃ to 30 ℃, rotating speed of 200rpm and culturing time of 72 h.

(2) Biological quantity measurement

Adding appropriate amount of sterile distilled water into the fermentation liquid, diluting until OD600 is 0.2-0.8, placing 200 μ L diluted fermentation liquid into 96-well plate, and measuring absorbance at 600nm wavelength with microplate reader (Thermo).

(3) Sample processing and detection

Carrying out first centrifugal separation on the fermentation culture solution to obtain first supernatant;

The fermentation broth was centrifuged at 12000rpm for 10min at 4 ℃. Adding acetonitrile 900 mu L into 100 mu L of supernatant, diluting by 10 times, mixing uniformly (obtaining solution A), adding acetonitrile 990 mu L into 10 mu L of solution A, and diluting by 100 times. After uniformly mixing, adding 10 mu L of trichloro-acetyl isocyanate into 200 mu L of diluted solution supernatant for derivatization, and uniformly mixing; 190 μ L of ultrapure water was added to the derivatized solution, mixed well, and passed through a 0.22 μm organic phase filter. Then, quantitative detection is carried out by using liquid chromatography-mass spectrometry (QTRAP 5500, AB SCIEX). The final yield of the strain BW-PA-pby-PR-pyM is shown in FIG. 2 and FIG. 3, wherein FIG. 2 is a graph of the yield of 1,3-propanediol produced by fermentation of the strain BW-PA-pby-PR-pyM in a 250ml shake flask; FIG. 3 is a graph showing the yield of 1,3-propanediol produced by fermentation of strain BW-PA-pby-PR-pyM in a 5L fermentor. The test results show that the yield of the de novo synthesis reaches 162mg/L, and the yield in the 5L fermentation tank is 1002.5 mg/L.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Sequence listing

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<213> (Gene ydfG encoding 3-hydroxy acid dehydrogenase)

<400> 3

atgatcgttt tagtaactgg agcaacggca ggttttggtg aatgcattac tcgtcgtttt 60

attcaacaag ggcataaagt tatcgccact ggccgtcgcc aggaacggtt gcaggagtta 120

aaagacgaac tgggagataa tctgtatatc gcccaactgg acgttcgcaa ccgcgccgct 180

attgaagaga tgctggcatc gcttcctgcc gagtggtgca atattgatat cctggtaaat 240

aatgccggcc tggcgttggg catggagcct gcgcataaag ccagcgttga agactgggaa 300

acgatgattg ataccaacaa caaaggcctg gtatatatga cgcgcgccgt cttaccgggt 360

atggttgaac gtaatcatgg tcatattatt aacattggct caacggcagg tagctggccg 420

tatgccggtg gtaacgttta cggtgcgacg aaagcgtttg ttcgtcagtt tagcctgaat 480

ctgcgtacgg atctgcatgg tacggcggtg cgcgtcaccg acatcgaacc gggtctggtg 540

ggtggtaccg agttttccaa tgtccgcttt aaaggcgatg acggtaaagc agaaaaaacc 600

tatcaaaata ccgttgcatt gacgccagaa gatgtcagcg aagccgtctg gtgggtgtca 660

acgctgcctg ctcacgtcaa tatcaatacc ctggaaatga tgccggttac ccaaagctat 720

gccggactga atgtccaccg tcagtaa 747

<210> 4

<211> 1395

<212> DNA

<213> (codon-optimized gene coding for aldehyde dehydrogenase, pdup)

<400> 4

atgaacacct ctgaactgga aaccctgatc cgtaccatcc tgtctgaaca gctgaccacc 60

ccggctcaga ccccggttca gccgcagggt aaaggtatct tccagtctgt ttctgaagct 120

atcgacgctg ctcaccaggc tttcctgcgt taccagcagt gcccgctgaa aacccgttct 180

gctatcatct ctgctatgcg tcaggaactg accccgctgc tggctccgct ggctgaagaa 240

tctgctaacg aaaccggtat gggtaacaaa gaagacaaat tcctgaaaaa caaagctgct 300

ctggacaaca ccccgggtgt tgaagacctg accaccaccg ctctgaccgg tgacggtggt 360

atggttctgt tcgaatactc tccgttcggt gttatcggtt ctgttgctcc gtctaccaac 420

ccgaccgaaa ccatcatcaa caactctatc tctatgctgg ctgctggtaa ctctatctac 480

ttctctccgc acccgggtgc taaaaaagtt tctctgaaac tgatctctct gatcgaagaa 540

atcgctttcc gttgctgcgg tatccgtaac ctggttgtta ccgttgctga accgaccttc 600

gaagctaccc agcagatgat ggctcacccg cgtatcgctg ttctggctat caccggtggt 660

ccgggtatcg ttgctatggg tatgaaatct ggtaaaaaag ttatcggtgc tggtgctggt 720

aacccgccgt gcatcgttga cgaaaccgct gacctggtta aagctgctga agacatcatc 780

aacggtgctt ctttcgacta caacctgccg tgcatcgctg aaaaatctct gatcgttgtt 840

gaatctgttg ctgaacgtct ggttcagcag atgcagacct tcggtgctct gctgctgtct 900

ccggctgaca ccgacaaact gcgtgctgtt tgcctgccgg aaggtcaggc taacaaaaaa 960

ctggttggta aatctccgtc tgctatgctg gaagctgctg gtatcgctgt tccggctaaa 1020

gctccgcgtc tgctgatcgc tctggttaac gctgacgacc cgtgggttac ctctgaacag 1080

ctgatgccga tgctgccggt tgttaaagtt tctgacttcg actctgctct ggctctggct 1140

ctgaaagttg aagaaggtct gcaccacacc gctatcatgc actctcagaa cgtttctcgt 1200

ctgaacctgg ctgctcgtac cctgcagacc tctatcttcg ttaaaaacgg tccgtcttac 1260

gctggtatcg gtgttggtgg tgaaggtttc accaccttca ccatcgctac cccgaccggt 1320

gaaggtacca cctctgctcg taccttcgct cgttctcgtc gttgcgttct gaccaacggt 1380

ttctctatcc gttaa 1395

<210> 5

<211> 1164

<212> DNA

<213> (Gene yqhD encoding alcohol dehydrogenase)

<400> 5

atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60

ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120

gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180

gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240

gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300

accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360

caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420

gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480

caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540

tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600

gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660

ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720

cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780

ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840

cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900

cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960

gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020

acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080

gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140

cgtatatacg aagccgcccg ctaa 1164

<210> 6

<211> 1986

<212> DNA

<213> (codon-optimized Gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase)

<400> 6

atgttcatgc gttacatcat ggttgaagaa cagaccctga aaaccggttc tcaggaactg 60

gaagaaaaag ctgactacaa catgcgttac tacgctcacc tgatgaaact gtctaaagaa 120

aaaccggctg aattctgggg ttctctggct caggacctgc tggactggta cgaaccgtgg 180

aaagaaacca tgcgtcagga agacccgatg acccgttggt tcatcggtgg taaaatcaac 240

gcttcttaca acgctgttga ccgtcacctg aacggtccgc gtaaattcaa agctgctgtt 300

atctgggaat ctgaactggg tgaacgtaaa atcgttacct accaggacat gttctacgaa 360

gttaaccgtt gggctaacgc tctgcgttct ctgggtgttg gtaaaggtga ccgtgttacc 420

atctacatgc cgctgacccc ggaaggtatc gctgctatgc tggcttctgc tcgtatcggt 480

gctatccact ctgttatctt cgctggtttc ggttctcagg ctatcgctga ccgtgttgaa 540

gacgctaaag ctaaagttgt tatcaccgct gacgcttacc cgcgtcgtgg taaagttgtt 600

gaactgaaaa aaaccgttga cgaagctctg aactctctgg gtgaacgttc tccggttcag 660

cacgttctgg tttaccgtcg tatgaaaacc gacgttaaca tgaaagaagg tcgtgacgtt 720

ttcttcgacg aagttggtaa ataccgttac gttgaaccgg aacgtatgga ctctaacgac 780

ccgctgttca tcctgtacac ctctggtacc accggtaaac cgaaaggtat catgcactct 840

accggtggtt acctgaccgg taccgctgtt atgctgctgt ggtcttacgg tctgtctcag 900

gaaaacgacg ttctgttcaa cacctctgac atcggttgga tcgttggtca ctcttacatc 960

acctactctc cgctgatcat gggtcgtacc gttgttatct acgaatctgc tccggactac 1020

ccgtacccgg acaaatgggc tgaaatcatc gaacgttacc gtgctaccac cttcggtacc 1080

tctgctaccg ctctgcgtta cttcatgaaa tacggtgacg aatacgttaa aaaccacgac 1140

ctgtcttcta tccgtatcat cgttaccaac ggtgaagttc tgaactactc tccgtggaaa 1200

tggggtctgg aagttctggg tggtggtaaa gttttcatgt ctcaccagtg gtggcagacc 1260

gaaaccggtg ctccgaacct gggttacctg ccgggtatca tctacatgcc gatgaaatct 1320

ggtccggctt ctggtttccc gctgccgggt aacttcgttg aagttctgga cgaaaacggt 1380

aacccgtctg ctccgcgtgt tcgtggttac ctggttatgc gtccgccgtt cccgccgaac 1440

atgatgatgg gtatgtggaa cgacaacggt gaacgtctga aaaaaaccta cttctctaaa 1500

ttcggttctc tgtactaccc gggtgacttc gctatggttg acgaagacgg ttacatctgg 1560

gttctgggtc gtgctgacga aaccctgaaa atcgctgctc accgtatcgg tgctggtgaa 1620

gttgaatctg ctatcacctc tcacccgtct gttgctgaag ctgctgttat cggtgttccg 1680

gactctgtta aaggtgaaga agttcacgct ttcgttgttc tgaaacaggg ttacgctccg 1740

tcttctgaac tggctaaaga catccagtct cacgttcgta aagttatggg tccgatcgtt 1800

tctccgcaga tccacttcgt tgacaaactg ccgaaaaccc gttctggtaa agttatgcgt 1860

cgtgttatca aagctgttat gatgggttct tctgctggtg acctgaccac catcgaagac 1920

gaagcttcta tggacgaaat caaaaaagct gttgaagaac tgaaaaaaga actgaaaacc 1980

tcttaa 1986

<210> 7

<211> 32

<212> DNA

<213> (primer PACYC-tac-F)

<400> 7

tccaccgtca gtaactcgag caccaccacc ac 32

<210> 8

<211> 29

<212> DNA

<213> (primer PACYC-tac-R)

<400> 8

tatctcctgt cgacacccat ttgctgtcc 29

<210> 9

<211> 46

<212> DNA

<213> (primer panD-F)

<400> 9

tgggtgtcga caggagatat accatgtatc gaacaatgat gagcgg 46

<210> 10

<211> 55

<212> DNA

<213> (primer panD-R)

<400> 10

gcggctggtt catggtatat ctcctgcggc cgcctacaaa attgtacggg ctggt 55

<210> 11

<211> 25

<212> DNA

<213> (primer bauA-F)

<400> 11

atataccatg aaccagccgc tcaac 25

<210> 12

<211> 34

<212> DNA

<213> (primer bauA-R)

<400> 12

atatctccta agctttcagg cgatgccgtt gagc 34

<210> 13

<211> 51

<212> DNA

<213> (primer ydfG-F)

<400> 13

gcctgaaagc ttaggagata taccatgatc gttttagtaa ctggagcaac g 51

<210> 14

<211> 29

<212> DNA

<213> (primer ydfG-R)

<400> 14

tcgagttact gacggtggac attcagtcc 29

<210> 15

<211> 30

<212> DNA

<213> (primer PRSF-tac-F)

<400> 15

aaaacctctt aagcggccgc actcgagcac 30

<210> 16

<211> 47

<212> DNA

<213> (primer PRSF-tac-R)

<400> 16

gttcagaggt gttcatggta tatctcctgg atccgcgacc catttgc 47

<210> 17

<211> 24

<212> DNA

<213> (primer Pdup-F)

<400> 17

accatgaaca cctctgaact ggaa 24

<210> 18

<211> 37

<212> DNA

<213> (primer Pdup-R)

<400> 18

cctgaattct taacggatag agaaaccgtt ggtcaga 37

<210> 19

<211> 57

<212> DNA

<213> (primer yqhD-F)

<400> 19

tctctatccg ttaagaattc aggagatata ccatgaacaa ctttaatctg cacaccc 57

<210> 20

<211> 57

<212> DNA

<213> (primer YqhD-R)

<400> 20

tgtaacgcat gaacatggta tatctcctgt cgacttagcg ggcggcttcg tatatac 57

<210> 21

<211> 27

<212> DNA

<213> (primer Msed _ 1456-F)

<400> 21

taccatgttc atgcgttaca tcatggt 27

<210> 22

<211> 39

<212> DNA

<213> (primer Msed _ 1456-R)

<400> 22

ggccgcttaa gaggttttca gttctttttt cagttcttc 39

Claims

1. A genetically engineered bacterium for producing 1,3-propanediol is synthesized by the following route:

2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is a recombinant host bacterium comprising a gene panD encoding aspartate decarboxylase, a gene bauA encoding β -alanine-pyruvate aminotransferase, a gene ydfG encoding 3-hydroxy acid dehydrogenase, a gene pdup encoding aldehyde dehydrogenase codon-optimized, a gene yqhD encoding alcohol dehydrogenase, and a gene Mseed-1456 encoding 3-hydroxypropionyl-CoA synthetase codon-optimized.

3. The genetically engineered bacterium of claim 2, wherein the gene panD encoding aspartate decarboxylase is derived from Bacillus subtilis strain 168 (Bacillus subtilis strain 168), and the sequence thereof is shown in SEQ No. 1; and/or the gene bauA for coding the beta-alanine-pyruvate aminotransferase is derived from Pseudomonas aeruginosa PAO1(Pseudomonas aeruginosa PAO1), and the sequence of the gene bauA is shown as SEQ No. 2.

4. The genetically engineered bacterium of claim 2, wherein the gene ydfG encoding 3-hydroxy acid dehydrogenase is derived from Escherichia coli K12 strain (Escherichia coli strain K12), and the sequence thereof is shown in SEQ No. 3.

5. The genetically engineered bacterium of claim 2, wherein the gene pdup encoding aldehyde dehydrogenase is derived from Salmonella enterica (Salmonella enterica), and the sequence of the codon-optimized gene pdup encoding aldehyde dehydrogenase is shown in SEQ No. 4.

6. The genetically engineered bacterium of claim 2, wherein the gene yqhD encoding alcohol dehydrogenase is derived from Escherichia coli K12 strain (Escherichia coli strain K12), and the sequence thereof is shown in SEQ No. 5.

7. The genetically engineered bacterium of claim 2, wherein the gene Mseed _1456 encoding 3-hydroxypropionyl-CoA synthetase is derived from Metallococcus DSM 5348(Metallosphaera sedula DSM 5348), and the codon-optimized gene Mseed _1456 encoding 3-hydroxypropionyl-CoA synthetase has the sequence shown in SEQ No. 6.

8. The genetically engineered bacterium of any one of claims 2 to 7, wherein the host bacterium comprises Escherichia coli, Corynebacterium glutamicum, yeast, and modified bacteria, fungi; preferably, the host bacterium is escherichia coli; further preferably, the host bacterium is escherichia coli BW 25113.

9. The application of the genetically engineered bacterium of any one of 1 to 8 in producing 1, 3-propanediol; preferably, the application comprises the steps of inoculating the genetic engineering bacteria for producing the 1,3-propanediol into a fermentation culture medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain the 1, 3-propanediol; further preferably, the fermentation culture conditions are: the fermentation culture time is 72h, when the thallus grows to OD600 is 0.6-0.8, IPTG is added, after the addition of the IPTG, the fermentation temperature is changed from 37 ℃ to 30 ℃, and the induction concentration of the IPTG is 0.2 mM; particularly preferably, aspartic acid is added in vitro in an amount of 2 to 10g/L, more particularly 5 to 10 g/L.

10. The use according to claim 9, wherein the isolation and purification of the obtained fermentation broth comprises: