Engineering bacterium as well as construction method and application thereof

Abstract

The invention discloses an engineering bacterium as well as a construction method and application thereof. Glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase are subjected to overexpression in the engineering bacteria. The invention develops a production method of glycerol and / or 1, 3-propylene glycol, designs a brand new transformation strategy, overexpresses glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, realizes synthesis of an intermediate product glycerol from glucose, further synthesizes 1, 3-propylene glycol from glycerol through glycerol dehydratase and 1, 3-propylene glycol reductase endogenous to a strain, and improves the yield of the glycerol and / or 1, 3-propylene glycol. Meanwhile, NADPH-dependent glycerol-3-phosphate dehydrogenase derived from archaea is utilized to control the consumption of NADH in cells so as to realize high yield of glycerol and 1, 3-propylene glycol.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and relates to an engineering bacterium and a construction method and application thereof, and in particular to an engineering bacterium for producing glycerol and / or 1,3-propylene glycol and a construction method and application thereof. Background Art

[0002] 1,3-Propanediol (PDO) is an intermediate widely used in food, lubricants, drugs, biodegradable plastics, and personal care products. Especially as a key monomer in the production of high-performance polyester polytrimethylene terephthalate (PTT), it has huge market demand and has received widespread attention.

[0003] At present, the production methods of 1,3-propylene glycol mainly include chemical method and biological method. The chemical method usually uses propylene oxide or propylene as raw materials to synthesize 1,3-propylene glycol through a complex catalytic process. The disadvantages of the chemical synthesis method are that there are many by-products, poor selectivity, high temperature and high pressure operating conditions, huge equipment investment, and the raw materials are non-renewable resources.

[0004] Compared with chemical synthesis, the advantages of biological production of 1,3-propylene glycol are high selectivity and mild operating conditions. The biological production of 1,3-propylene glycol currently mainly includes two technical routes: one is to use glycerol as a raw material to produce 1,3-propylene glycol using natural microorganisms; the other is to use glucose as a raw material to produce 1,3-propylene glycol using recombinant microorganisms. At present, no natural microorganisms have been found to be able to produce 1,3-propylene glycol directly from glucose. All methods for producing 1,3-propylene glycol from glucose by modifying strains through the expression of glycerol synthesis pathways use yeast-derived NADH-dependent glycerol-3-phosphate dehydrogenase, which consumes NADH in the process of synthesizing glycerol, resulting in an imbalance in the redox state in the cell, limiting the increase in 1,3-propylene glycol production. For example, CN1244217A discloses a method for producing 1,3-propylene glycol using a recombinant organism, comprising: transforming a suitable host organism with a transformation cassette, the The transformation box contains at least one of the following genes: (a) a gene encoding glycerol-3-phosphate dehydrogenase activity; (b) a gene encoding glycerol-3-phosphatase activity; (c) a gene encoding dehydratase activity; (d) a gene encoding 1,3-propanediol oxidoreductase activity; CN114107145A discloses a recombinant microorganism and its use in the production of 1,3-propanediol, the recombinant microorganism overexpresses a fusion protein of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase compared to the starting strain, the amino acid sequence of the fusion protein is shown in SEQ ID No.1; the starting strain is Corynebacterium glutamicum.

[0005] In summary, the development of a method for efficiently producing 1,3-propylene glycol is of great significance for the application field of 1,3-propylene glycol. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides an engineered bacterium and a construction method and application thereof, and in particular relates to an engineered bacterium for producing glycerol and / or 1,3-propylene glycol and a construction method and application thereof.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an engineered bacterium, wherein glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase are overexpressed in the engineered bacterium, and the amino acid sequence of the glycerol-3-phosphate dehydrogenase comprises:

[0009] (1) the sequence shown in SEQ ID NO.1, or

[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or,

[0011] (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0012] The present invention is devoted to developing a method for producing glycerol and 1,3-propanediol, utilizing NADPH-dependent glycerol-3-phosphate dehydrogenase derived from archaea to control the consumption of intracellular NADH to achieve high yield of glycerol and 1,3-propanediol.

[0013] In the present invention, it is discovered for the first time that NADPH-dependent glycerol-3-phosphate dehydrogenase from archaea can reduce the consumption of intracellular NADH, and the use of it to construct engineered bacteria has the potential to achieve high production of glycerol and 1,3-propanediol. It can be understood that based on the present invention, based on the amino acid sequence of glycerol-3-phosphate dehydrogenase as shown in SEQ ID NO.1, the common transformation means in the field such as substitution, deletion or addition of one or at least two amino acid residues are used to transform, and a mutant with the same or similar function as the original enzyme is obtained. It can be expected that the mutant can also be applied to the scheme of the present invention to achieve the same or similar effect.

[0014] SEQ ID NO.1:

[0015] MIVSILGAGAMGSALSVPLVDNGNEVRIWGTEFDTEILKSISAGREHPRLGVKLNGVEIFWPEQLEKCLENAEVVLLGVSTDGVLPVMSRILPYLKDQYIVLISKGLIDFDNSVLTVPEAVWRLKHDLRERTVAITGPAIAREVAKRMPTTVVFSSPSESSANKMKEI FETEYFGVEVTDIIGTEITSALKNVYSIAIAWIRGYESRKNVEMSNAKGVIATRAINEMAELIEILGGDRETAFGLSGFGDLIATFRGGRNGMLGELLGKGLSIDEAMEELERRGVGVVEGYKTAEKAYRLSSKINADTKLLDSIYRVLYEGLKVEEVLFELATFK.

[0016] Preferably, the amino acid sequence of the glycerol-3-phosphatase includes the sequence shown in SEQ ID NO.2.

[0017] SEQ ID NO.2:

[0018] MSSASTPWGSPSSSSTSVATQNYLPPVDMVVDGCLFDLDGTLMLSTECVEMFWHDFGVQHGIDSHKLLQTSHGRRTIDILKIWKPEMANKQISGELEEATIPVRWASFATPVPGVRELHDALPPSQ WGIVTSGTLPMATGWLKHFLKFAPPQVFITAEDVEEGKPDPAGYKLGAERLGLEKFLVFEDAPAGIKAGKAAGATVIGMATTYTPDIVKAAGADIVIRDLHSVKVNSFDPVTKKLSLTFSNIVA.

[0019] Preferably, the starting strain of the engineered bacteria is selected from any one of Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Streptomyces, Methylobacterium, Salmonella, Bacillus, Pseudomonas, Klebsiella, Lactobacillus, Enterobacter, Citrobacter, Dark Bacillus, Silva or Clostridium.

[0020] Preferably, the engineered bacteria also lacks any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase.

[0021] In the present invention, by deleting any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase, the synthesis pathway of lactic acid, ethanol or acetic acid is deleted, so as to regulate the metabolic flow in the engineered bacteria and further increase the yield of 1,3-propanediol.

[0022] Preferably, the gene encoding any one or a combination of at least two of lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase in the engineered bacteria is deleted or the expression level is reduced.

[0023] In a second aspect, the present invention provides a method for preparing the engineered bacteria according to the first aspect, the method comprising:

[0024] The genes encoding glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase were overexpressed in the starting strain.

[0025] Preferably, the overexpression method comprises inserting the coding genes of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a starting strain.

[0026] Preferably, the method further comprises the step of modifying the starting strain to lack any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase.

[0027] Preferably, the modified starting strain lacks any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase, specifically comprises deleting or reducing the expression level of the coding gene of any one or a combination of at least two of lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase.

[0028] It is understood that in the present invention, the starting strain can be modified by using common genetic engineering strategies in the art, such as gene overexpression, knockout and expression interference, etc. to obtain the desired engineered bacteria, and the specific method does not need to be particularly limited.

[0029] In a third aspect, the present invention provides use of the engineered bacteria described in the first aspect in the preparation of glycerol and / or 1,3-propylene glycol.

[0030] In a fourth aspect, the present invention provides a method for producing glycerol and / or 1,3-propylene glycol, the method comprising:

[0031] The engineered bacteria described in the first aspect are cultured, the culture fluid is collected, and the product is purified.

[0032] It is to be understood that the methods for purifying products from fermentation medium commonly used in the art are applicable to the present invention without any special limitation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention develops a method for producing glycerol and / or 1,3-propanediol, designs a completely new transformation strategy, overexpresses glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, and realizes the synthesis of intermediate glycerol from glucose. Glycerol is further synthesized into 1,3-propanediol through endogenous glycerol dehydratase and 1,3-propanediol reductase of the strain, and simultaneously utilizes NADPH-dependent glycerol-3-phosphate dehydrogenase derived from archaea to control the consumption of intracellular NADH, so as to achieve high yield of glycerol and 1,3-propanediol. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the construction strategy of the engineering bacteria of the present invention;

[0036] Figure 2 HPLC test results of glycerol and 1,3-propylene glycol;

[0037] Figure 3 These are the GC-MS test results of glycerol and 1,3-propylene glycol. DETAILED DESCRIPTION

[0038] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] The present invention designs a new engineering transformation strategy to develop a fermentation pathway for producing glycerol and 1,3-propanediol using NADPH-dependent glycerol-3-phosphate dehydrogenase from archaea, which reduces the consumption of intracellular NADH and has the potential to achieve high yields of glycerol and 1,3-propanediol. The schematic diagram of the pathway for synthesizing glycerol and 1,3-propanediol using NADPH-dependent glycerol-3-phosphate dehydrogenase is shown in the figure. Figure 1As shown, the present invention realizes the synthesis of intermediate glycerol from glucose by overexpressing glycerol-3-phosphate dehydrogenase (encoded by gpsA gene) and glycerol-3-phosphatase (encoded by gpp2 gene), and glycerol is further synthesized into 1,3-propanediol by endogenous glycerol dehydratase and 1,3-propanediol reductase of the strain. Subsequently, by destroying the ldh gene, the activity of lactate dehydrogenase is lost, so the synthesis pathway of lactic acid is inactivated; by destroying the adhE gene, the activity of bifunctional acetaldehyde / ethanol dehydrogenase is lost, so the synthesis pathway of ethanol is inactivated; by destroying the pta gene, the activity of acetylphosphotransferase is lost, so the synthesis pathway of acetic acid is inactivated; in order to regulate the metabolic flow and further increase the yield of 1,3-propanediol.

[0041] Example 1

[0042] In this example, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase were overexpressed in Klebsiella oxytoca PDL-0.

[0043] The wild fungus is Klebsiella oxytoca PDL-0, which was deposited in China Center for Type Culture Collection on April 8, 2016, with the registration number of CCTCC M 2016184, and the address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0044] (1) Primers were designed based on the gpsA gene sequence (as shown in SEQ ID NO. 3): gpsA-F (EcoRI): AATTGAATTCATGAACGCGCTTAACGCTG and gpsA-R (PstI): TTAACTGCAGTTAGTTGCTACGCTCGTCCTTG; PCR amplification was performed using the synthesized gpsA gene fragment as a template to obtain the gpsA gene fragment, and the obtained PCR fragment was digested using restriction endonucleases EcoRI and PstI.

[0045] The expression plasmid pBAD322C (John E. Cronan, A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control, Plasmid, Volume 55, Issue 2, 2006, Pages 152-157) was digested with restriction endonucleases EcoRI and PstI to obtain a linearized plasmid pBAD322C. The gene fragment gpsA and the linearized plasmid pBAD322C were ligated using T4 ligase to obtain pBAD322C-gpsA.

[0046] (2) Primers were designed according to the gpp2 gene sequence (as shown in SEQ ID NO.4): gpp2-F (EcoRI): AATTGAATTCATGAGCTCTGCCAGTACTCCG and gpp2-R (PstI): TTAACTGCAGTTAGGCCACGATGTTGCTAAAGGT; PCR amplification was performed using the synthesized gpp2 gene fragment as a template, and the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, recovered and purified to obtain the gpsA gene fragment, and the obtained PCR fragment was digested with restriction endonucleases EcoRI and PstI, and the gene fragment gpp2 and the linearized plasmid pBAD322C were ligated with T4 ligase to obtain pBAD322C-gpp2.

[0047] (3) According to the promoter sequence of the vector ptac (as shown in SEQ ID NO.5), primers Ptac-F (PstI): TTAACTGCAGCTGTGGTATGGCTGTGCAGGTC and gpp2-R (HindIII): CTAAAAGCTTTTAGGCCACGATGTTGCTAAAGGT were designed; PCR amplification was performed using the pBAD322C-gpp2 plasmid as a template to obtain P tac -gpp2 gene fragment, and the obtained PCR fragment was digested with restriction endonucleases EcoRI and PstI.

[0048] The expression plasmid pBAD322C-gpsA was digested with restriction endonucleases PstI and HindIII to obtain a linearized plasmid pBAD322C-gpsA. The gene fragment P was ligated using T4 ligase. tac -gpp2 and linearized plasmid pBAD322C-gpsA were ligated to obtain pBAD322C-gpsA-gpp2.

[0049] (4) The constructed vector pBAD322C-gpsA-gpp2 was transformed into Klebsiella oxytoca PDL-0 to obtain strain PDO-gpsA.

[0050] Example 2

[0051] In this example, the lactic acid synthesis pathway of Klebsiella oxytoca was knocked out.

[0052] According to the ldhA gene sequence (as shown in SEQ ID NO.6), primers were designed for PCR amplification of upstream and downstream homologous fragments of the ldhA gene. The genomic DNA of Klebsiella oxytoca PDL-0 was used as a template, and primers ldhA-UP-F: GGCCGAATTCAAGCGGTTGCACATATTAC and primers ldhA-UP-R: CCAAGGATCCTCACAAACCACCCTCGACAA were used. The ldhA gene fragment was obtained by PCR amplification using the genomic DNA of Klebsiella oxytoca PDL-0 as a template; the upstream primer ldhA-Down-F CCATGGATCCCTTGTCGTACTGTTTCG and primer ldhA-down-RAACCCTGCAGGCATCGTATACGCTGGCTTA were used. The downstream fragment of the ldhA gene was obtained by PCR amplification using the genomic DNA of Klebsiella oxytoca PDL-0 as a template.

[0053] After the above two PCR products were purified, 1 μl of each was taken as a template and inserted into the vector pKR6K using seamless cloning technology to obtain the knockout vector pKR6K-ΔldhA.

[0054] The knockout vector pKR6K-ΔldhA was transformed into Klebsiella oxytoca PDL-0, and the ldhA gene was knocked out by homologous recombination knockout principle. The primers selected were ldhA-UP-F: GGCCGAATTCAAGCGGTTGCACATATTAC, and ldhA-down-R: AACCCTGCAGGCATCGTATACGCTGGCTTA. The obtained strain with ldhA gene defect was Klebsiella oxytoca PDL-ΔldhA. The vector pBAD322C-gpsA-gpp2 was transformed into Klebsiella oxytoca PDL-ΔldhA to obtain strain PDO-A1.

[0055] Example 3

[0056] This example knocks out the ethanol synthesis pathway of Klebsiella oxytoca.

[0057] Primers were designed according to the adhE gene sequence (as shown in SEQ ID NO.7) to PCR amplify upstream and downstream homologous fragments of the adhE gene. Using the genomic DNA of Klebsiella oxytoca PDL-0 as a template, PCR amplification was performed using primers adhE-UP-F ACATGATTACGAATTCATGGCTGTTACTAATGTCGC and primer adhE-UP-R TGCTGTCTGTTGGCGTTACGGGTCTTCAGG to obtain the upstream homologous fragment.

[0058] PCR amplification was performed using primer adhE-down-F: CGTAACGCCAACAGACAGCATTCAGCCAGT and primer adhE-down-R: TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT to obtain the downstream homologous fragment.

[0059] After the two PCR products were purified, 1 μl of each was taken as a template and inserted into the vector pKR6K using seamless cloning technology to obtain the knockout vector pKR6K-ΔadhE.

[0060] The knockout vector pKR6K-ΔadhE was transformed into Klebsiella oxytoca PDL-ΔldhA, and the ldhA gene was knocked out by homologous recombination knockout principle. The primers selected were adhE-UP-F ACATGATTACGAATTCATGGCTGTTACTAATGTCGC, and adhE-down-R: TACCGAGCTCGAATTCTTAAGCGGATTTTTTCGCTT. The adhE gene-deficient strain obtained was Klebsiella oxytoca PDL-ΔldhAΔadhE. The vector pBAD322C-gpsA-gpp2 was transformed into Klebsiella oxytoca PDL-ΔldhAΔadhE to obtain strain PDO-A2.

[0061] Example 4

[0062] In this example, the acetate synthesis pathway of Klebsiella oxytoca was knocked out.

[0063] Primers were designed according to the ackA-pta gene sequence (as shown in SEQ ID NO.8), and upstream and downstream homologous fragments of the ackA-pta gene were amplified by PCR. Using the genomic DNA of Klebsiella oxytoca PDL-2 as a template, PCR amplification was performed using primers ackA-pta-UP-F: ACATGATTACGAATTCATGTCGAGTAAGTTAGTAC and primer ackA-pta-UP-R: CACGCGCGGTCCTCAGCGATACCGATCAG to obtain the upstream homologous fragment; PCR amplification was performed using primers ackA-ptadown-F: ATCGCTGAGGACCGCGCGTGGCCATGCTCT-3 and primer ackA-pta down-R: TACCGAGCTCGAATTCTTATGCTTGCTGCTGGGACG to obtain the downstream homologous fragment.

[0064] After the two PCR products were purified, 1 μl of each was taken as a template and inserted into the vector pKR6K using seamless cloning technology to obtain the knockout vector pKR6K-ΔackA-pta.

[0065] The knockout vector pKR6K-ΔackA-ptaE was transformed into Klebsiella oxytoca PDL-ΔldhAΔadhE, and the ldhA gene was knocked out by homologous recombination knockout principle. The primers selected were ackA-pta-UP-F: ACATGATTACGAATTCATGTCGAGTAAGTTAGTAC and ackA-pta down-R: TACCGAGCTCGAATTCTTATGCTTGCTGCTGGGACG. The obtained strain with ackA-pta gene defect was Klebsiella oxytoca PDL-ΔldhAΔadhEΔackA-pta. The vector pBAD322C-gpsA-gpp2 was transformed into Klebsiella oxytoca PDL-ΔldhAΔadhEΔackA-pta to obtain strain PDO-A3.

[0066] SEQ ID NO.3

[0067]

[0068] SEQ ID NO.4

[0069] atgagctctgccagtactccgtggggcagccctagcagcagtagtactagcgtggccacccagaattatctgccgccggtggatatggtggttgatggctgcctgtttgatctggatggcaccctgatgctgagtaccgaatgcgtggaaatgttctggcatgattttggcgtgcagcatggcatcgatagccataaactgctccagaccagccatggtcgtcgcaccatcgatatcctgaaaatctggaaaccggaaatggccaacaaacagatcagcggcgaactggaagcaaccattccggttcgttgggccagttttgccacccctgtgccgggtgtgcgtgaactgcatgatgccctgccgcctagtcaatggggtattgtgaccagcggtactctgcctatggcaaccggttggctgaaacactttctgaaatttgccccgccgcaggtttttatcaccgccgaagatgtggaagaaggcaaacctgatccggccggttataaactgggtgccgaacgtctgggcctggaaaaatttctggtgtttgaagatgccccggccggtattaaagcaggtaaagccgccggtgcaaccgtgattggtatggccaccacctacaccccggatatcgtgaaagcagccggtgccgatattgtgatccgcgatctgcatagcgtgaaagtgaacagctttgatccggtgaccaaaaaactgagcctgacctttagcaacatcgtggcctaa。

[0070] SEQ ID NO.5

[0071] ctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattctgaaatgagctgttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaatttcaccaagagatatacat。

[0072] SEQ ID NO.6

[0073] atgaaaatcgctgtatacagtacgaaacagtacgacaagaagtatctgcagcacgtcaatgatgcatacggctttgaactggaattttttgacttcctgctaaccgaaaagaccgccaaaaccgccaacggctgtgaagcggtgtgcatcttcgtaaacgatgacggtagccgcccggtactcgaagaactgaaagcccacggcgtgaagtacatcgcgctgcgctgcgcgggcttcaacaacgttgatctcgatgccgcgaaagagctgggcctgcgcgtggtgcgcgtcccggcctactcgccggaagcggtcgcagagcacgcgatcggcatgatgatgtcgctgaaccgccgtattcatcgcgcctatcagcgcacccgcgacgcgaacttctcccttgaaggactaaccggtttcaccatgcacggtaaaaccgccggcgttatcggcaccggtaaaatcggcgtcgccgcgctgcgcattctgaaaggcttcggtatgcgtctgctggcgtttgatccctacccaagcgccgccgcgctggatatgggcgtggagtatgtcgatcttgaaaccctgtaccgggagtctgacgttatctccctgcactgcccgctgaccgatgaaaactaccatttgttgaaccatgccgcgttcgatcgcatgaaagacggggtgatgatcatcaacaccagccgcggcgcgctcatcgattcgcaggcagcaatcgacgcccttaagcatcagaaaattggcgcgctggggatggacgtgtatgagaacgaacgcgatctgttctttgaagataagtctaatgacgtgattcaggatgatgtgttccgccgtctctctgcctgccataacgtcctgtttaccggtcaccaggcgtttctgaccgcggaagcgctgatcagcatttcacaaaccaccctcgacaacctgcgtcaggtggatgctggcgaaacctgccctaacgcgctggtctga。

[0074] SEQ ID NO.7

[0075]

[0076] SEQ ID NO.8

[0077]

[0078] Example 5

[0079] In this example, shake flask fermentation of Klebsiella oxytoca PDO-gpsA was carried out.

[0080] 5.1. Seeds and fermentation medium

[0081] The seed culture medium is LB medium (components are peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L)

[0082] Fermentation medium ingredients: 10g / L dipotassium hydrogen phosphate trihydrate, 5mg / L cobalt chloride hexahydrate, 30mg / L ferric chloride hexahydrate, 0.1g / L magnesium sulfate heptahydrate, 1g / L ammonium chloride, 2g / L potassium dihydrogen phosphate, 5g / L yeast powder, 20g / L glucose.

[0083] 5.2. Shake flask fermentation process

[0084] The Klebsiella oxytoca PDO-gpsA seeds constructed in the example were cultured overnight, transferred to a 250 ml conical flask at a 2% (v / v) inoculation amount, and the liquid volume was 50 ml of fermentation medium for shake flask fermentation. The fermentation time was 12 h and the culture temperature was 37° C. After sampling 1 mL, centrifugation was performed at 12000 rpm for 5 min, and filtered with a 0.22 μm aqueous phase filter membrane, and detected by high performance liquid chromatography.

[0085] HPLC detection method

[0086] Column model: Aminex HPX-87H.

[0087] Mobile phase: 0.005 mol / L sulfuric acid solution.

[0088] Flow rate: 0.5mL / min.

[0089] Column temperature: 55.0℃.

[0090] Injection volume: 10.00 μL.

[0091] Detector: differential detector.

[0092] Detector temperature: 55°C

[0093] According to the test results ( Figure 2 ), after 36 hours of shake flask fermentation, the maximum OD of the strain 620 was 2.3, the maximum glycerol production in the fermentation broth was 1.25 g / L, and the maximum 1,3-propylene glycol production was 0.12 g / L.

[0094] 5.4GS-MS verification method

[0095] 1 mL of fermentation broth was sampled and centrifuged at 12000 rpm for 5 min. The supernatant was extracted with an equal volume of ethyl acetate. The GC / MS instrument was an Agilent Technologies 6850 / 5975C system equipped with an Agilent HP-5ms capillary column (0.25 μm, 0.25 μm×30 m). The carrier gas was helium, the injector and detector temperatures were both 260 °C, the column oven temperature was 120 °C, and the flow rate was maintained at 0.5 mL / min.

[0096] The results are as follows Figure 3 As shown, it is further demonstrated that glycerol and 1,3-propanediol were synthesized.

[0097] Example 6

[0098] In this example, shake flask fermentation of recombinant Klebsiella oxytoca PDO-A1 was carried out.

[0099] 6.1. Seeds and fermentation medium

[0100] Refer to Example 5.

[0101] 6.2. Seed culture

[0102] Refer to Example 5.

[0103] 6.3. Fermentation

[0104] The Klebsiella oxytoca PDO-A1 seeds constructed in Example 2 were cultured overnight, inoculated at 2% (v / v) into a 250 ml conical flask, and the liquid volume was 50 ml of fermentation medium for shake flask fermentation. The fermentation time was 12 h and the culture temperature was 37° C. After sampling 1 mL, centrifugation was performed at 12000 rpm for 5 min, and filtered through a 0.22 μm aqueous phase filter membrane, and detected by high performance liquid chromatography.

[0105] According to the test results, after 36 hours of shake flask fermentation, the maximum OD 620 The maximum yield of glycerol in the fermentation broth increased to 0.24 g / L and the maximum yield of 1,3-propylene glycol increased to 0.18 g / L.

[0106] showed that disruption of the lactate pathway improved cell growth and enhanced the conversion of glycerol to 1,3-propanediol.

[0107] Example 7

[0108] In this example, shake flask fermentation of recombinant Klebsiella oxytoca PDO-A2 was carried out.

[0109] 7.1. Seeds and fermentation medium

[0110] Refer to Example 5.

[0111] 7.2. Seed culture

[0112] Refer to Example 5.

[0113] 7.3. Fermentation

[0114] The seeds of Klebsiella oxytoca PDO-A2 were cultured overnight, inoculated with 2% (v / v) and transferred to a 250 ml conical flask, filled with 50 ml fermentation medium for shake flask fermentation, fermentation time was 12 h, and the culture temperature was 37 ° C. After sampling 1 mL, centrifugation was performed at 12000 rpm for 5 min, filtered with a 0.22 μm aqueous filter membrane, and detected by high performance liquid chromatography.

[0115] According to the test results, after 36 hours of shake flask fermentation, the maximum OD 620 The maximum yield of glycerol in the fermentation broth was increased to 1.5 g / L, and the maximum yield of 1,3-propylene glycol was increased to 0.21 g / L.

[0116] showed that disruption of the ethanol pathway improved cell growth and enhanced the conversion of glycerol to 1,3-propanediol.

[0117] Example 8

[0118] In this example, shake flask fermentation of recombinant Klebsiella oxytoca PDO-A3 was carried out.

[0119] 8.1. Seeds and fermentation medium

[0120] Same as Example 5.

[0121] 8.2. Seed culture

[0122] Same as Example 5.

[0123] 8.3. Fermentation

[0124] The seeds of Klebsiella oxytoca PDO-A2 were cultured overnight, inoculated with 2% (v / v) and transferred to a 250 ml conical flask, filled with 50 ml fermentation medium for shake flask fermentation, fermentation time was 12 h, and the culture temperature was 37 ° C. After sampling 1 mL, centrifugation was performed at 12000 rpm for 5 min, filtered with a 0.22 μm aqueous filter membrane, and detected by high performance liquid chromatography.

[0125] According to the test results, after 36 hours of shake flask fermentation, the maximum OD 620 When the concentration was increased to 5.6, the maximum glycerol production in the fermentation broth was 1.8 g / L, and the maximum 1,3-propylene glycol production was 0.20 g / L.

[0126] It was shown that disruption of the acetate pathway improved cell growth and enhanced the conversion of glycerol to 1,3-propanediol.

[0127] In summary, the present invention develops a method for producing glycerol and / or 1,3-propanediol, designs a new transformation strategy, overexpresses glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, and realizes the synthesis of the intermediate glycerol from glucose. Glycerol is further synthesized into 1,3-propanediol through endogenous glycerol dehydratase and 1,3-propanediol reductase of the strain, and at the same time utilizes NADPH-dependent glycerol-3-phosphate dehydrogenase from archaea to control the consumption of intracellular NADH, so as to achieve high yield of glycerol and 1,3-propanediol.

[0128] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An engineered bacterium, characterized in that: The engineered bacteria overexpress glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, and the amino acid sequence of the glycerol-3-phosphate dehydrogenase includes: (1) the sequence shown in SEQ ID NO.1, or (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or, (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

2. The engineered bacteria according to claim 1, characterized in that The amino acid sequence of the glycerol-3-phosphatase includes the sequence shown in SEQ ID NO.

2.

3. The engineered bacteria according to claim 1 or 2, characterized in that: The starting strain of the engineered bacteria is selected from any one of Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Streptomyces, Methylobacterium, Salmonella, Bacillus, Pseudomonas, Klebsiella, Lactobacillus, Enterobacter, Citrobacter, Dark Bacillus, Silva or Clostridium.

4. The engineered bacteria according to any one of claims 1 to 3, characterized in that The engineered bacteria also lack any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase.

5. The engineered bacteria according to claim 4, characterized in that In the engineered bacteria, the gene encoding any one or a combination of at least two of lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase is deleted or the expression level is reduced.

6. A method for constructing an engineered bacterium according to any one of claims 1 to 5, characterized in that: The method comprises: The genes encoding glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase were overexpressed in the starting strain.

7. The method for constructing an engineering bacterium according to claim 6, characterized in that: The overexpression method comprises inserting the coding genes of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a starting strain.

8. The method for constructing an engineering bacterium according to claim 6 or 7, characterized in that: The method further comprises the step of modifying the starting strain to lack any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase; Preferably, the modified starting strain lacks any one or a combination of at least two of functional lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase, specifically comprises deleting or reducing the expression level of the coding gene of any one or a combination of at least two of lactate dehydrogenase, acetylphosphotransferase or aldehyde dehydrogenase.

9. Use of the engineered bacteria according to any one of claims 1 to 5 in the preparation of glycerol and / or 1,3-propylene glycol.

10. A method for producing glycerol and / or 1,3-propylene glycol, characterized in that: The method comprises: Cultivate the engineered bacteria according to any one of claims 1 to 5, collect the culture fluid, and purify the product.

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