Engineering Halomonas sp.

Abstract

The invention relates to the technical field of biological materials, in particular to engineering modified halomonas, a construction method of the engineering modified halomonas and a method for producing mannitol by using the engineering modified halomonas. In the engineering modified halomonas, a mannitol 2 dehydrogenase coding gene (mtlK) is over-expressed through a promoter porin 58. In the engineering modified halomonas provided by the embodiment of the invention, the mannitol 2 dehydrogenase coding gene (mtlK) is over-expressed through the promoter porin 58, so that the utilization rate of the engineering modified halomonas on a substrate (fructose) is increased, and the yield of mannitol is remarkably increased.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to an engineered Halomonas bacterium and a construction method thereof, and a method for producing mannitol using the engineered Halomonas bacterium. Background Art

[0002] Mannitol is a sugar alcohol compound with a molecular formula of C6H 14 O6, usually used as an additive in food, medicine and chemical products. It tastes slightly sweet, but not as sweet as ordinary sugar, so it is often used as a sweetener for low-sugar foods. Mannitol is also widely used in the medical field as a diuretic, kidney protector, or for the treatment of problems such as high intracranial pressure and intraocular pressure. The traditional production method of mannitol mainly relies on chemical synthesis. However, in recent years, biosynthetic mannitol has been considered a more environmentally friendly and sustainable production method. Biosynthetic mannitol usually uses microorganisms (such as Escherichia coli, yeast, Streptomyces, etc.) to convert sugars into mannitol through a fermentation process.

[0003] Although mannitol can be obtained through microbial fermentation, the yield of many biosynthetic pathways is still low, far lower than the efficiency of chemical synthesis methods. This means that the production cost is high, which limits large-scale application. In addition, microorganisms require strict environmental control (such as temperature, pH, oxygen supply, etc.) during the fermentation process, so fluctuations are prone to occur in large-scale production, resulting in unstable yield and quality.

[0004] Therefore, it is urgent to propose a method for efficiently producing mannitol. Summary of the invention

[0005] The present invention solves one of the problems of the related art from at least the following aspects.

[0006] The first aspect of the present invention provides an engineered Halomonas, wherein in the engineered Halomonas, a mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed by a promoter porin58, and the Halomonas is selected from the genus Halomonas.

[0007] In some embodiments, the nucleotide sequence of the mannitol 2 dehydrogenase encoding gene (mtlK) is shown as SEQ ID NO: 1.

[0008] In some embodiments, the nucleotide sequence of the promoter porin58 is as shown in SEQ ID NO:2.

[0009] In some embodiments, the mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed on the chromosome of the Halomonas via the promoter porin58.

[0010] In some embodiments, the Halomonas is selected from the group consisting of Halomonas bluephagenesis, Halomonas campaniensis, and Halomonas elongata.

[0011] In some embodiments, the Halomonas is selected from the group consisting of Halomonasbluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018, and Halomonas bluephagenesis WZY254.

[0012] In some embodiments, the engineered Halomonas is Halomonas bluephagenesis TD01-porin58-mtlK, Halomonas campaniensis LS21-porin58-mtlK, or Halomonas bluephagenesis WZY254-porin58-mtlK.

[0013] The second aspect of the present invention provides a method for constructing an engineered Halomonas according to any one of the first aspects, comprising: overexpressing a mannitol 2 dehydrogenase encoding gene (mtlK) through a promoter porin58.

[0014] In some embodiments, the overexpression of the mannitol 2 dehydrogenase encoding gene (mtlK) through the promoter porin58 includes: inserting the promoter porin58 and the mannitol 2 dehydrogenase encoding gene (mtlK) into a vector to obtain a recombinant plasmid; and transforming the recombinant plasmid into the Halomonas by conjugation.

[0015] In some embodiments, the vector is pSEVA341.

[0016] A third aspect of the present invention provides a method for producing mannitol, comprising: using the engineered Halomonas according to any one of the first aspects to ferment and produce mannitol.

[0017] In some embodiments, the carbon source of the fermentation medium of the engineered Halomonas comprises fructose, and the nitrogen source of the fermentation medium of the engineered Halomonas comprises urea.

[0018] In some embodiments, the initial fructose content in the fermentation medium is 10-30 g / L, more preferably 15-25 g / L.

[0019] Compared with the related art, the embodiments of the present application achieve at least the following beneficial effects:

[0020] In the engineered Halomonas provided in the examples of the present application, the mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed through the promoter porin58, so that the engineered Halomonas can utilize the substrate (fructose) and significantly increase the production of mannitol. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0022] In this application, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0023] The inventors of the present application unexpectedly discovered that by overexpressing the mannitol 2 dehydrogenase encoding gene (mtlK) in Halomonas via the promoter porin58, an engineered Halomonas capable of high mannitol production was obtained.

[0024] The first aspect of the present application provides an engineered Halomonas, in which a mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed by a promoter porin58, and the Halomonas is selected from the genus Halomonas.

[0025] In some embodiments, the nucleotide sequence of the mannitol 2 dehydrogenase encoding gene (mtlK) is shown as SEQ ID NO: 1.

[0026]

[0027] In some embodiments, the nucleotide sequence of the promoter porin58 is as shown in SEQ ID NO:2.

[0028] tcccagtatatatttgac(SEQ ID NO:2)

[0029] The fluorescence intensity (FI) of the promoter is 80291, which is the number of cells whose fluorescence intensity is greater than the standard fluorescence value of 1.0 when 100,000 cells containing the promoter-driven green fluorescent protein expression plasmid are screened by flow cytometry.

[0030] In some embodiments, the mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed on the chromosome of the Halomonas via the promoter porin58.

[0031] In some embodiments, the Halomonas is selected from the group consisting of Halomonas bluephagenesis, Halomonas campaniensis, and Halomonas elongata.

[0032] In some embodiments, the Halomonas is selected from the group consisting of Halomonasbluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018, and Halomonas bluephagenesis WZY254.

[0033] Bacteria used in the examples of this application:

[0034] Halomonas bluephagenesis TD01, the strain was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on November 19, 2010, with the deposit registration number of CGMCC No.4353, and the classification name is Halomonas sp.TD01 (also known as Halomonas bluephagenesis TD01; it is recorded in the patent application publication number CN102120973A, and the public can also obtain the strain from the applicant, which can only be used to repeat the experiment of the present invention.

[0035] Halomonas campaniensis LS21: It is a Gram-negative halophilic bacterium screened by this laboratory and has very good prospects for industrial production and application. The public can obtain this bacterium from the applicant and can only be used to repeat the experiments of the present invention. It is disclosed in Chinese patent CN115927137A and has a deposit number of CGMCC No.6593.

[0036] Halomonas bluephagenesis WZY254 strain (TDH4ΔphaP1,ΔlpxL,ΔlpxM,P Mmp1 -phaCAB was replaced with P porin -phaCAB) was prepared from the starting strain Halomonas bluephagenesis TDH4 by knocking out the phaP1 gene, lpxL gene, and lpxM gene, and using P porin -phaCAB replaces P Mmp1 -phaCAB, for details, refer to the literature: Wang, Ziyu et al. "Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas blue phagenesis grown in bioreactors." Microbial biotechnology.

[0037] The construction of Halomonas bluephagenesis TDH4 strain is described in the literature by Shen R, Yin J, Jian-WenYe et al. (Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis [J]. ACS Synthetic Biology, 7(8): 1897-1906). The public can also obtain the strain from the applicant for use only in repeating the experiments of the present invention.

[0038] The construction of Halomonas elongata OUT30018 strain is described in the literature by Huynh Cong Khanh et al. (Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata [J]. Appl Environ Microbiol, 90(9): e0119524.). The public can also obtain the strain from the applicant for use only in repeating the experiments of the present invention.

[0039] In some embodiments, the engineered Halomonas is Halomonas bluephagenesis TD01-porin58-mtlK, Halomonas campaniensis LS21-porin58-mtlK, or Halomonas bluephagenesis WZY254-porin58-mtlK.

[0040] It can be understood that Halomonas bluephagenesis TD01-porin58-mtlK is an engineered strain obtained by overexpressing mtlK through porin58 in Halomonasbluephagenesis TD01 as a starting strain; Halomonas campaniensis LS21-porin58-mtlK is an engineered strain obtained by overexpressing mtlK through porin58 in Halomonas campaniensis LS21 as a starting strain; Halomonasbluephagenesis WZY254-porin58-mtlK is an engineered strain obtained by overexpressing mtlK through porin58 in Halomonas bluephagenesis WZY254 as a starting strain.

[0041] The second aspect of the present application provides a method for constructing an engineered Halomonas as in any of the first aspect embodiments, comprising: overexpressing a mannitol 2 dehydrogenase encoding gene (mtlK) through a promoter porin58.

[0042] It is understandable that the gene encoding mannitol 2 dehydrogenase (mtlK) can be overexpressed in the starting strain (e.g., on its chromosome) through the promoter porin58 by conventional gene editing techniques in the art, including but not limited to CRISPR / Cas9 gene editing technology. CRISPR genome editing methods refer to the document CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018). The genome editing sites used in the embodiments of this application are referred to in the document CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0043] In some embodiments, the overexpression of the mannitol 2 dehydrogenase encoding gene (mtlK) through the promoter porin58 includes: inserting the promoter porin58 and the mannitol 2 dehydrogenase encoding gene (mtlK) into a vector to obtain a recombinant plasmid; and transforming the recombinant plasmid into the Halomonas by conjugation.

[0044] In some embodiments, the vector is pSEVA341.

[0045] The third aspect of the present application provides a method for producing mannitol, comprising: using the engineered Halomonas of any one of the first aspects to ferment and produce mannitol.

[0046] In some embodiments, the carbon source of the fermentation medium of the engineered Halomonas comprises fructose, and the nitrogen source of the fermentation medium of the engineered Halomonas comprises urea.

[0047] In some embodiments, the initial fructose content in the fermentation medium is 10-30 g / L (e.g., 15 g / L, 20 g / L, 25 g / L), more preferably 15-25 g / L (e.g., 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L).

[0048] The fermentation method adopted in the embodiment of the present application is a conventional fermentation method using urea as a nitrogen source. As an example, the specific formula of the MM culture medium used to cultivate halophilic microorganisms in the embodiment of the present application is as follows: 0.5 g / L urea; 0.2 g / L MgSO4; 1.5 g / L KH2PO4; and 0.05 g / L Fe(III)-NH4-Citrate (ammonium ferric (III) citrate), 0.02 g / L CaCl2·2H2O, 0.1 mg / L ZnSO4·7H2O, 0.03 mg / L MnCl2·4H2O, 0.3 mg / L H3BO3, 0.2 mg / L CoCl2·6H2O, 0.01 mg / LCuSO4·5H2O, 0.02 mg / L NiCl2·6H2O, 0.03 mg / L NaMoO4·2H2O.

[0049] In the embodiment of the present application, the aforementioned MM medium was used as the basal medium, and 20 g / L of glucose, 10 g / L of sodium chloride and 10 g / L of fructose were added to carry out the fermentation tank experiment. The fermentation tank used this time was a 7L fermentation tank, the fermentation liquid volume was 3L, the pH was set at 9.0, and the temperature was maintained at 37 ° C. A major advantage of halophilic fermentation is that it can be open fermented without additional sterilization. At the same time, it is not necessary to add various air filters and complex aseptic operations during the fermentation process to reduce the possibility of contamination, which greatly saves production costs and manpower and material resources. Conventional open fermentation relies on a high-salt and high-pH environment to inhibit the growth of other bacteria. Glucose 30 g / L was added to the culture medium as a carbon source, and 5-100 g / L sodium chloride was added to provide the osmotic pressure environment required for the growth of halophilic bacteria; and the pH was adjusted to 9.0.

[0050] Escherichia coli was grown in LB medium, and the medium composition contained: 10 g / L sodium chloride, 10 g / L peptone (OXID Company, UK, product catalog number LP0042), and 5 g / L yeast extract (OXID Company, UK, product catalog number LP0021).

[0051] Halomonas bluephagenesis TD01 was cultured in LB60 medium, and its components were the same as LB except that the concentration of sodium chloride was changed to 60 g / L. Halomonas bluephagenesis TD01, see Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01 (Tan et al, Bioresource Technology, 2011).

[0052] The following examples are used to further illustrate the advantages and features of the present method, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0053] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in the art (e.g., reference to "Molecular Cloning Experiment Guide" by J. Sambrook, etc., translated by Huang Peitang, etc., the third edition, Science Press) or the product instructions are followed. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0054] Unless otherwise specified, the quantitative analysis experiments in the following examples were performed three times and the results were averaged.

[0055] Example 1 Construction of mtlK overexpression plasmid

[0056] Construction of recombinant plasmid pKD+mtlK

[0057] The backbone of the pKD+mtlK plasmid is pSEVA341 (see the literature for plasmid: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41.D1 (2012): D666-D675.). The sequence inserted into the plasmid is SEQ ID NO: 3, which is arranged in the order of sgRNA expression module (nucleotides 1-152), upstream homology arm (nucleotides 153-1152), Pporin58 promoter module (nucleotides 1153-1170), mtlK gene (nucleotides 1171-2658), and downstream homology arm (nucleotides 2659-3496).

[0058]

[0059] Example 2 Construction of recombinant strains

[0060] The pQ08 plasmid used in the present application example (plasmid see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp [J]. Metabolic engineering, 2018, 47: 219-229.). The plasmid pKD-mtlK and the pQ08 plasmid were respectively transferred into Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018 and Halomonas bluephagenesis WZY254 by conjugation transformation. The porin58-mtlK gene was successfully inserted into the Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonaselongata OUT30018 and Halomonas bluephagenesis WZY254 genome by PCR and sequencing verification to obtain the corresponding recombinant strain. Then, the successfully edited strains were continuously and multiple times passaged in liquid culture medium and streaked on spectinomycin-resistant, chloramphenicol-resistant and non-resistant plates, respectively, and the CRISPR plasmid-lost strains Halomonasbluephagenesis TD01-porin58-mtlK, Halomonas campaniensis LS21-porin58-mtlK, Halomonas elongata OUT30018-porin58-mtlK and Halomonas bluephagenesis WZY254-porin58-mtlK were identified.

[0061] Example 3

[0062] Halomonas bluephagenesis TD01 and Halomonas bluephagenesis TD01-porin58-mtlK were used for small molecule fermentation to produce mannitol, and the accumulation of mannitol during the fermentation process was observed and analyzed. The results are shown in Table 1 below.

[0063] The fermentation medium adopts the above fermentation formula and process. The fermentation time is 96 hours and the fermentation temperature is 37 degrees Celsius.

[0064] Table 1

[0065] Fermentation strains Mannitol concentration Cell dry weight Halomonas bluephagenesis TD01 5.8g / L 20g / L Halomonas bluephagenesis TD01-porin58-mtlK 11.9g / L 29g / L

[0066] According to Table 1 above, by overexpressing mtlK through porin58 in Halomonas bluephagenesis TD01, a higher cell accumulation density can be achieved and a higher product yield can be achieved.

[0067] Example 4

[0068] Halomonas campaniensis LS21 and Halomonas campaniensis LS21-porin58-mtlK were used for small molecule fermentation to produce mannitol, and the accumulation of mannitol during the fermentation process was observed and analyzed. The results are shown in Table 2 below.

[0069] The fermentation medium adopts the above fermentation formula and process. The fermentation time is 96 hours and the fermentation temperature is 37 degrees Celsius.

[0070] Table 2

[0071]

[0072]

[0073] According to Table 2 above, by overexpressing mtlK through porin58 in Halomonas campaniensis LS21, a higher cell accumulation density can be achieved and a higher product yield can be achieved.

[0074] Example 5

[0075] Halomonas bluephagenesis WZY254 and Halomonas bluephagenesis WZY254-porin58-mtlK were used for small molecule fermentation to produce mannitol, and the accumulation of mannitol during the fermentation process was observed and analyzed. The results are shown in Table 3 below.

[0076] The fermentation medium adopts the above fermentation formula and process. The fermentation time is 96 hours and the fermentation temperature is 37 degrees Celsius.

[0077] Table 3

[0078] Fermentation strains Mannitol concentration Cell dry weight Halomonas bluephagenesis WZY254 6.0g / L 25g / L Halomonas bluephagenesis WZY254-porin58-mtlK 10.3g / L 31g / L

[0079] According to Table 3 above, by overexpressing mltK through porin58 in Halomonas bluephagenesis WZY254, a higher cell accumulation density can be achieved and a higher product yield can be achieved.

[0080] Example 6

[0081] Halomonas elongata OUT30018 and Halomonas elongata OUT30018-porin58-mtlK were used for small molecule fermentation to produce mannitol, and the accumulation of mannitol during the fermentation process was observed and analyzed. The results are shown in Table 4 below.

[0082] The fermentation medium adopts the above fermentation formula and process. The fermentation time is 96 hours and the fermentation temperature is 37 degrees Celsius.

[0083] Table 4

[0084] Fermentation strains Mannitol concentration Cell dry weight Halomonas elongata OUT30018 5.0g / L 22g / L Halomonas elongata OUT30018-porin58-mtlK 13.3g / L 33g / L

[0085] According to Table 4 above, by overexpressing mltK through porin58 in Halomonas elongata OUT30018, a higher cell accumulation density can be achieved and a higher product yield can be achieved.

[0086] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0087] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An engineered Halomonas, characterized in that: In the engineered Halomonas, a mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed by the promoter porin58, and the Halomonas is selected from the genus Halomonas.

2. The engineered Halomonas according to claim 1, characterized in that The nucleotide sequence of the mannitol 2 dehydrogenase encoding gene (mtlK) is shown in SEQ ID NO: 1; Optionally, the nucleotide sequence of the promoter porin58 is as shown in SEQ ID NO:

2.

3. The engineered Halomonas according to claim 1, characterized in that The mannitol 2 dehydrogenase encoding gene (mtlK) is overexpressed on the chromosome of the Halomonas via the promoter porin58.

4. The engineered Halomonas according to claim 1, characterized in that The Halomonas is selected from the group consisting of Halomonas bluephagenesis, Halomonas campaniensis and Halomonas elongata; Optionally, the Halomonas is selected from the group consisting of Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018 and Halomonasbluephagenesis WZY254.

5. The engineered Halomonas according to claim 1, characterized in that The engineered Halomonas is Halomonas bluephagenesis TD01-porin58-mtlK, Halomonas campaniensis LS21-porin58-mtlK or Halomonas bluephagenesis WZY254-porin58-mtlK.

6. A method for constructing an engineered Halomonas according to any one of claims 1 to 5, characterized in that: The method comprises: overexpressing a gene encoding mannitol 2 dehydrogenase (mtlK) via a promoter porin58.

7. The method according to claim 6, characterized in that The method of overexpressing the mannitol 2 dehydrogenase encoding gene (mtlK) through the promoter porin58 comprises: inserting the promoter porin58 and the mannitol 2 dehydrogenase encoding gene (mtlK) into a vector to obtain a recombinant plasmid; The recombinant plasmid is transformed into the Halomonas by conjugation.

8. The method according to claim 7, characterized in that The vector is pSEVA341.

9. A method for producing mannitol, characterized in that: The method comprises: using the engineered Halomonas according to any one of claims 1 to 5 to ferment and produce mannitol.

10. The method according to claim 9, characterized in that The carbon source of the fermentation medium of the engineered Halomonas comprises fructose, and the nitrogen source of the fermentation medium of the engineered Halomonas comprises urea; Preferably, the initial fructose content in the fermentation medium is 10-30 g / L, more preferably 15-25 g / L.

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