Production method of engineering modified halomonas and glutamic acid

Abstract

The invention relates to the technical field of biological materials, in particular to a production method of engineering modified halomonas and glutamic acid. The engineering modified halomonas does not express glutamate dehydrogenase B, and the engineering modified halomonas overexpresses glutamate dehydrogenase A. The invention further discloses a preparation method of the engineering modified halomonas. According to the engineering modified halomonas provided by the embodiment of the invention, higher cell accumulation density and higher glutamic acid yield are realized at the same time, and the advantage is more obvious especially under a high osmotic pressure fermentation condition.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an engineered halomonas bacterium and a method for producing glutamic acid. Background Art

[0002] As an important amino acid, glutamic acid has a variety of applications in industry, especially in the fields of chemical synthesis and biotechnology. For example, glutamic acid can be used as a raw material to synthesize other amino acids (such as proline and aspartic acid) or green materials polyhydroxyalkanoates, etc., or directly used as a nutritional supplement in the field of food and health products. Halomonas sp. is a type of moderate halophilic bacteria isolated from Xinjiang Aidin Salt Lake. Due to its tolerance to alkaline and high osmotic pressure environments and strong anti-pollution ability, it has been developed for industrial production of various metabolites. However, the yield of glutamic acid produced by this strain in the related art is only 5g / L, which is far below the requirements for industrial-grade glutamic acid production.

[0003] Therefore, there is an urgent need to provide an engineered Halomonas capable of efficiently producing glutamate. Summary of the invention

[0004] The present invention solves one of the problems of the related art from at least the following aspects.

[0005] The first aspect of the present invention provides an engineered Halomonas, wherein the engineered Halomonas does not express glutamate dehydrogenase B, the engineered Halomonas overexpresses glutamate dehydrogenase A, and the Halomonas is selected from the genus Halomonas.

[0006] In some embodiments, the Halomonas is selected from the group consisting of Halomonas bluephagenesis, Halomonas campaniensis, and Halomonas elongata.

[0007] In some embodiments, the Halomonas is selected from the group consisting of Halomonasbluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018, and Halomonas bluephagenesis WZY254.

[0008] In some embodiments, the amino acid sequence of the glutamate dehydrogenase B is shown in SEQ ID NO.2.

[0009] In some embodiments, the amino acid sequence of the glutamate dehydrogenase A is shown in SEQ ID NO.4.

[0010] In some embodiments, the nucleotide sequence of the gene gdhB encoding glutamate dehydrogenase B is shown as SEQ ID NO.1.

[0011] In some embodiments, the nucleotide sequence of the gene gdhA encoding glutamate dehydrogenase A is shown in SEQ ID NO.3.

[0012] The second aspect of the present invention provides a method for constructing an engineered Halomonas according to any one of the first aspects, comprising: knocking out glutamate dehydrogenase B in the starting strain; and overexpressing glutamate dehydrogenase A in the starting strain.

[0013] A third aspect of the present invention provides a method for producing glutamic acid, comprising fermenting the engineered Halomonas of any one of the first aspects.

[0014] In some embodiments, the culture medium used for the fermentation contains sodium chloride at a concentration of 20 to 80 g / L, preferably 50 to 70 g / L.

[0015] In some embodiments, the fermentation uses a culture medium comprising glucose at a concentration of 10 to 30 g / L and urea at a concentration of 4 to 10 g / L.

[0016] The fourth aspect of the present invention provides an embodiment of the use of the engineered Halomonas of any embodiment of the first aspect in the production of metabolites selected from the group consisting of: proteins, nucleic acids, acids, alcohols, ester products and / or other organic or inorganic compounds.

[0017] In some embodiments, the metabolite is selected from the group consisting of glutamic acid, nylon materials, N-vinyl pyrrolidone, and polyhydroxyalkanoates.

[0018] Compared with the related art, the embodiments of the present application achieve at least the following beneficial effects:

[0019] The engineered Halomonas provided in the examples of the present application simultaneously achieves a higher cell accumulation density and a higher glutamate yield, especially under high osmotic pressure fermentation conditions, and this advantage is more obvious. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[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] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0023] The term “include” or “comprising” described in the present invention is an open description, which contains the specified components or steps described and other specified components or steps that will not be substantially affected.

[0024] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0025] The first aspect of the present application provides an engineered Halomonas, wherein the engineered Halomonas does not express glutamate dehydrogenase B, the engineered Halomonas overexpresses glutamate dehydrogenase A, and the Halomonas is selected from the genus Halomonas.

[0026] In the examples of the present application, "the engineered bacteria does not express glutamate dehydrogenase B" means that in the engineered bacteria, the glutamate dehydrogenase B protein is fully or partially mutated (mutation includes insertion, deletion or replacement), that is, in the engineered bacteria, glutamate dehydrogenase B protein or its variant is not contained, or the glutamate dehydrogenase B protein or its variant contained therein is unable to convert glutamate into α-ketoglutarate.

[0027] In the embodiments of the present application, methods for achieving "the engineered bacteria not expressing glutamate dehydrogenase B" include but are not limited to CRISPR / Cas9 gene editing technology and zinc finger nuclease technology.

[0028] The engineered Halomonas provided in the examples of the present application simultaneously achieves a higher cell accumulation density and a higher glutamate yield, especially under high osmotic pressure fermentation conditions, and this advantage is more obvious.

[0029] In some embodiments, the Halomonas is selected from the group consisting of Halomonas bluephagenesis, Halomonas campaniensis, and Halomonas elongata.

[0030] In some embodiments, the Halomonas is selected from the group consisting of Halomonasbluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018, and Halomonas bluephagenesis WZY254.

[0031] In some embodiments, the amino acid sequence of the glutamate dehydrogenase B is shown in SEQ ID NO.2.

[0032] In some embodiments, the amino acid sequence of the glutamate dehydrogenase A is shown in SEQ ID NO.4.

[0033] In some embodiments, the nucleotide sequence of the gene gdhB encoding glutamate dehydrogenase B is shown as SEQ ID NO.1.

[0034] In some embodiments, the nucleotide sequence of the gene gdhA encoding glutamate dehydrogenase A is shown in SEQ ID NO.3.

[0035] The second aspect of the present application provides a method for constructing an engineered Halomonas according to any one of the first aspect embodiments, comprising: knocking out glutamate dehydrogenase B in the starting strain; and overexpressing glutamate dehydrogenase A in the starting strain.

[0036] A third aspect of the present application provides a method for producing glutamic acid, comprising fermenting the engineered Halomonas of any one of the first aspects.

[0037] A major advantage of using halophilic bacteria to ferment glutamic acid in the present embodiment is that, due to its high tolerance to high osmotic pressure and alkaline conditions, 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 bacterial 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.

[0038] In some embodiments, the culture medium used in the fermentation contains sodium chloride at a concentration of 20 to 80 g / L (e.g., 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L), preferably 50 to 70 g / L (55 g / L, 60 g / L, 65 g / L).

[0039] The applicant of the present invention unexpectedly discovered that the gdhB and gdhA genes have strong glutamate substrate binding activity under hypertonic conditions, especially under sodium chloride concentration conditions of 50 to 70 g / L. Under such conditions, fermentation of gdhB-knocked and gdhA-overexpressed Halomonas can simultaneously achieve significantly higher cell accumulation density and glutamate production.

[0040] In some embodiments, the culture medium used for the fermentation comprises glucose at a concentration of 10 to 30 g / L (e.g., 15 g / L, 20 g / L, 25 g / L) and 4 to 10 g / L (e.g., 5 g / L, 5.55 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.55 g / L, 8 g / L, 8.55 g / L, 9 g / L).

[0041] The fourth aspect of the present application provides an application of the engineered salt mononas of any embodiment of the first aspect in the production of metabolites selected from the group consisting of: proteins, nucleic acids, acids, alcohols, ester products and / or other organic or inorganic compounds.

[0042] In some embodiments, the metabolite is selected from the group consisting of glutamic acid, nylon materials, N-vinyl pyrrolidone, and polyhydroxyalkanoates.

[0043] The nucleotide sequence of the gene gdhB encoding glutamate dehydrogenase B is shown in SEQ ID NO.1:

[0044]

[0045] The amino acid sequence of glutamate dehydrogenase B is shown in SEQ ID NO.2:

[0046]

[0047] The nucleotide sequence of the gene gdhA encoding glutamate dehydrogenase A is shown in SEQ ID NO.3:

[0048]

[0049] The amino acid sequence of glutamate dehydrogenase A is shown in SEQ ID NO.4:

[0050] MQVYVLAVKYVIALASKSTFLTIKGCLFMPYVHDTLTRLAKSSPAQTEFYQATEEVLECLRPLFERAPHYHQHSIIERIVEPERQVMFRVSWVDDAGRVQVNKGYRVQFNSALGPYKGGL RFHPSVTSGTIKFLGFEQIFKNALTGLPIGGGKGGADFDPKGKSDNEIMRFCQAFMSELYRHIGPHTDVPAGDIGVGGREIGYLFGQYKRLTGRYEGVLTGKGLNWGGSLGRKEATGYGAV YFAENMLAAKDEALRGKTCLVSGAGNVAIYTIEKLYELGARPVTCSDSKGMIHDPSGIDLGLLKQLKEVQRVSLESYLHDHPEAHYIPARDYPQDGHAVWRIEGEAAFPCATQNELTEAD AKALLANGVTCVSEGANMPSTKEAVDVFLEAKIAYGPGKAANAGGVATSQLEMAQNSSMEQWPLEKVDQKLKQIMADIYRQCADTASEFGEPDNLVLGANIAGFRKVADAMIEQGVI(SEQ ID NO.4)

[0051] The present invention is further explained below by specific examples. It should be understood that these examples are merely used to describe and help understand the present invention in an exemplary manner, and are not intended to limit the present invention to these examples.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Strain source

[0054] The bacterium used in the embodiment is Halomonas bluephagenesis TD01, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on November 19, 2010, with a deposit registration number of CGMCC No. 4353, and is classified as Halomonas sp. TD01 (also known as Halomonas bluephagenesis TD01; which is recorded in patent application publication number CN102120973A; the public can obtain the bacterium from the applicant and can only be used to repeat the experiments of the present invention.

[0055] Halomonas bluephagenesis TD01 can also be found in Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01 (Tan et al, Bioresource Technology, 2011).

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] Plasmids :

[0061] pQ08 is described in the following document: "Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229". The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0062] pSEVA341 is described in the following document: "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 public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0063] Unless otherwise specified, the present invention adopts Gene Editing Technology All are CRISPR-Cas9 gene editing technologies, and the specific operation methods are recorded in the following document: "Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229". The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0064] Fermentation conditions

[0065] Amino acid fermentation process:

[0066] Fermentation medium: MM medium was used as the basal medium, and 20 g / L of glucose, sodium chloride (for specific contents, see the table of each embodiment) and 6 g / L of urea (i.e., the MM basal medium originally contained 0.5 g / L urea, and the urea content was increased to 6.5 g / L) were added to conduct a fermentation tank experiment. This time, a 7 L fermentation tank was used, the fermentation liquid volume was 3 L, the pH was set at 9.0, and the temperature was maintained at 37 ° C. The specific formula of MM culture medium is as follows: 0.5g / L urea; 1.2g / L MgSO4; 2.5g / L KH2PO4; and 0.05g / L Fe(III)-NH4-Citrate (ammonium ferric (III) citrate), 0.02g / L CaCl2·2H2O, 0.1mg / L ZnSO4·7H2O, 0.03mg / L MnCl2·4H2O, 0.3mg / L H3BO3, 0.2mg / L CoCl2·6H2O, 0.01mg / L CuSO4·5H2O, 0.02mg / L NiCl2·6H2O, 0.03mg / L NaMoO4·2H2O.

[0067] The growth medium of E. coli is LB60, and its formula is: 10g / L sodium chloride, 10g / L peptone (UK OXID Company, product catalog number LP0042), 5g / L yeast extract (UK OXID Company, product catalog number LP0021).

[0068] Growth medium of Halomonas bluephagenesis TD01: 60 g / L sodium chloride, 10 g / L peptone (OXID, UK, product catalog number LP0042), 5 g / L yeast extract (OXID, UK, product catalog number LP0021).

[0069] Example 1 Construction of recombinant strains

[0070] 1.1 Construction of plasmid pKDG for knocking out the gdhB gene

[0071] The backbone of the pKDG plasmid is pSEVA341, and the sequence inserted into the plasmid is SEQ ID NO.5, which is arranged in the order of sgRNA expression module (nucleotides 1-152), upstream homology arm (nucleotides 153-1152), and downstream homology arm (nucleotides 1153-2153).

[0072]

[0073] 1.2 Construction of plasmid pKDa for overexpression of gdhA gene

[0074] The backbone of the pKDa plasmid is pSEVA341, and the sequence inserted into the plasmid is SEQ ID NO.6, which is arranged in the order of sgRNA module (nucleotides 1-152), porin58 promoter module (nucleotides 153-170), and gdhA gene module (nucleotides 171-1607).

[0075]

[0076] 1.3 Construction of engineered bacteria for glutamate production

[0077] The gdhB gene was knocked out and the gdhA gene was overexpressed in the starting strains Halomonas bluephagenesis TD01, Halomonascampaniensis LS21, Halomonas elongata OUT30018 and Halomonas bluephagenesisWZY254, respectively, to obtain the strains: Halomonas bluephagenesis TD01ΔgdhB+gdhA, Halomonas campaniensis LS21ΔgdhB+gdhA, Halomonas elongata OUT30018ΔgdhB+gdhA and Halomonas bluephagenesis WZY254ΔgdhB+gdhA.

[0078] Knockout of gdhB gene: The plasmid pKDG and pQ08 plasmid prepared in step 1.1 above for knockout of gdhB gene were transferred into Halomonas by conjugation transformation. The successful knockout of gdhB gene was verified by PCR and sequencing. Then, the strain after successful editing was continuously and multiple times subcultured in liquid culture medium, and streaked on spectinomycin-resistant, chloramphenicol-resistant and non-resistant plates, respectively, to identify the strain with lost CRISPR plasmid, so as to facilitate culture test.

[0079] Overexpression of gdhA gene: The gdhA gene was overexpressed in the above strain using the plasmid pKDa for overexpression of gdhA gene prepared in step 1.2 above.

[0080] Example 2

[0081] The amino acid fermentation process was used to ferment Halomonas bluephagenesis TD01ΔgdhB+gdhA, and the accumulation of glutamate during the fermentation process was observed and analyzed. The fermentation medium and fermentation conditions were used, and the glutamate concentration in the fermentation broth was detected after 48 hours of fermentation. The results are shown in Table 1 below.

[0082] According to Table 1, knocking out gdhB and overexpressing gdhA in Halomonas bluephagenesis TD01 can simultaneously achieve higher cell accumulation density and higher product yield, especially under high osmotic pressure fermentation conditions.

[0083] Table 1

[0084]

[0085] Example 3

[0086] Halomonas campaniensis LS21ΔgdhB+gdhA was fermented using an amino acid fermentation process, and the accumulation of glutamate during the fermentation process was observed and analyzed. The fermentation medium and fermentation conditions were used, and the concentration of glutamate in the fermentation broth was detected after 48 hours of fermentation. The results are shown in Table 2 below.

[0087] Knocking out gdhB and simultaneously overexpressing gdhA in Halomonas campaniensis LS21 can simultaneously achieve higher cell accumulation density and higher product yield, especially under high osmotic pressure fermentation conditions.

[0088] Table 2

[0089]

[0090] Example 4

[0091] The amino acid fermentation process was used to ferment Halomonas bluephagenesis WZY254ΔgdhB+gdhA, and the accumulation of glutamate during the fermentation process was observed and analyzed. The fermentation medium and fermentation conditions were used, and the glutamate concentration in the fermentation broth was detected after 48 hours of fermentation. The results are shown in Table 3 below.

[0092] Knocking out gdhB and overexpressing gdhA in Halomonas bluephagenesis WZY254 can simultaneously achieve higher cell accumulation density and higher product yield, especially under high osmotic pressure fermentation conditions.

[0093] Table 3

[0094]

[0095] Example 5

[0096] The amino acid fermentation process was used to ferment Halomonas elongata OUT30018ΔgdhB+gdhA, and the accumulation of glutamate during the fermentation process was observed and analyzed. The fermentation medium and fermentation conditions were used, and the glutamate concentration in the fermentation broth was detected after 48 hours of fermentation. The results are shown in Table 3 below.

[0097] Knocking out gdhB and overexpressing gdhA in Halomonas elongata OUT30018 can simultaneously achieve higher cell accumulation density and higher product yield, especially under high osmotic pressure fermentation conditions.

[0098] Table 4

[0099]

[0100] Through the above description, it should be noted and understood that various modifications and improvements can be made to the present invention described in detail above without departing from the spirit and scope of the present invention required by the attached claims. Therefore, the scope of the technical solution claimed is not limited by any specific exemplary teaching given.

[0101] The applicant declares that the above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. An engineered Halomonas, characterized in that: The engineered Halomonas does not express glutamate dehydrogenase B, the engineered Halomonas overexpresses glutamate dehydrogenase A, and the Halomonas is selected from the genus Halomonas.

2. 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.

3. The engineered Halomonas according to claim 1 or 2, characterized in that The Halomonas is selected from the group consisting of Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas elongata OUT30018 and Halomonas bluephagenesis WZY254.

4. The engineered Halomonas according to any one of claims 1 to 3, characterized in that The amino acid sequence of the glutamate dehydrogenase B is shown in SEQ ID NO.2; Optionally, the amino acid sequence of the glutamate dehydrogenase A is shown in SEQ ID NO.4; Optionally, the nucleotide sequence of the glutamate dehydrogenase B encoding gene gdhB is as shown in SEQ ID NO.1; Optionally, the nucleotide sequence of the gene gdhA encoding glutamate dehydrogenase A is shown in SEQ ID NO.

3.

5. The method for constructing an engineered Halomonas according to any one of claims 1 to 4, characterized in that: include: Knock out glutamate dehydrogenase B in the starting strain; and overexpressing glutamate dehydrogenase A in the starting strain.

6. A method for producing glutamic acid, characterized in that: The method comprises fermenting the engineered Halomonas as described in any one of claims 1 to 4.

7. The method according to claim 6, characterized in that The culture medium used in the fermentation contains sodium chloride at a concentration of 20 to 80 g / L, preferably 50 to 70 g / L.

8. The method according to claim 6 or 7, wherein the culture medium used in the fermentation contains glucose at a concentration of 10 to 30 g / L and urea at a concentration of 4 to 10 g / L.

9. Use of the engineered Halomonas according to any one of claims 1 to 4 in the production of metabolites selected from the group consisting of proteins, nucleic acids, acids, alcohols, ester products and / or other organic or inorganic compounds.

10. The use according to claim 9, wherein the metabolite is selected from the group consisting of glutamic acid, nylon material, N-vinyl pyrrolidone and polyhydroxyalkanoate.

Images