Serinol-producing escherichia coli genetic engineering strain and application thereof

Abstract

The invention provides a serinol-producing escherichia coli genetic engineering strain and application thereof. The strain contains an expression plasmid vector or an expression plasmid vector combination, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, the first group of polynucleotides comprises at least one polynucleotide encoding phosphatase, and the second group of polynucleotides comprises at least one polynucleotide encoding phosphatase. The second group of polynucleotides comprises at least one polynucleotide encoding a dihydroxyacetone phosphate transaminase; the phosphatase is selected from at least one of histidine phosphatase, acid phosphatase and alkaline phosphatase, and the dihydroxyacetone phosphate transaminase is selected from at least one of CJ1437 and CJ1361. The strain realizes efficient fermentation production of serinol, and provides a new method for biosynthesis of serinol.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered bacteria technology, and in particular to genetically engineered strains of *Escherichia coli* producing serine alcohol and their applications. Background Technology

[0002] Serine alcohol, also known as 2-amino-1,3-propanediol, is an important chemical synthesis intermediate widely used in pharmaceuticals, chemicals, and other fields. One of its important uses is in the synthesis of the nonionic contrast agent iopamidol (pharmaceutical name: iodide), which serves as a diagnostic reagent for the hematologic, lymphatic, urinary, and nervous systems and is now widely used in developed countries. In addition, serine alcohol can also be used in the organic polymer materials industry.

[0003] Natural serine was initially discovered during the synthesis of long-spore glycoside toxin by sugarcane eye spot pathogens. Later, it was also found in the synthesis of rhizobium toxins by prokaryotes such as *Burkholderia granatum*, *Bradyrhizobium japonicum*, and *Bradyrhizobium elkanii*.

[0004] Currently, serine is mainly produced through chemical synthesis methods based on substances such as nitro-1,3-propanediol and dihydroxyacetone oxime. However, this method suffers from problems such as high pollution, high energy consumption, and difficulty in separation and purification. In contrast, the biological synthesis of serine through aminotransferases and other substances has attracted increasing attention due to its green, environmentally friendly, and low-energy consumption advantages.

[0005] Currently, there are few reports on the biosynthesis of serine. Although as early as 1975, different aldehydes were used to culture *Bacillus microflagellate*, *Candida albicans*, and *Coryneacterium glycinophilum* to produce serine and its derivatives. Among them, *Candida albicans*, using p-nitrobenzaldehyde or 3,4-dinitrobenzene as substrates, achieved the highest yield of serine derivatives, reaching 8 g / L. In 2019, Professor Feng Yan's research group at Shanghai Jiao Tong University achieved a fermenter serine yield of 14.6 g / L using recombinant *Escherichia coli*.

[0006] Microbial fermentation offers advantages such as mild reaction conditions, low raw material costs, a green, low-carbon, and environmentally friendly production process, and ease of large-scale production. *Escherichia coli*, with its clear genetic background, mature and well-developed gene modification methods and tools, abundant application examples in the fermentation industry, rapid growth rate, and mature fermentation regulation theory and practice, has become the preferred strain for green alternative production of chemical products. Therefore, the biomanufacturing of serine will become a future research hotspot with enormous economic application potential and market prospects. However, currently, there is no information released regarding the industrial implementation of serine production via bio-fermentation, making research on serine production via bio-fermentation highly promising. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a genetically engineered *Escherichia coli* strain that produces serine and its applications. It also provides a novel method for the biosynthesis of serine.

[0008] The first aspect of the present invention provides an expression plasmid vector or a combination of expression plasmid vectors, the vector or combination comprising: a first group of polynucleotides and a second group of polynucleotides, wherein the first group of polynucleotides comprises at least one polynucleotide encoding a phosphatase, and the second group of polynucleotides comprises at least one polynucleotide encoding dihydroxyacetone transaminase.

[0009] And the backbone plasmid, which can replicate autonomously in the host cell.

[0010] The phosphatase is selected from at least one of histamine phosphatase, acid phosphatase, and alkaline phosphatase, and the dihydroxyacetone phosphate transaminase is selected from at least one of CJ1437 or its mutant, CJ1361 or its mutant.

[0011] In one embodiment of the present invention, the histamine phosphatase is selected from histamine phosphatase hisB. In a specific embodiment of the present invention, the histamine phosphatase hisB is the amino acid sequence of protein accession number CAA31814, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by CAA31814.

[0012] In one embodiment of the present invention, the acid phosphatase is selected from acid phosphatase aphA. In a specific embodiment of the present invention, the acid phosphatase aphA is the amino acid sequence of protein accession number CAA60534, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by CAA60534.

[0013] In one embodiment of the present invention, the alkaline phosphatase is selected from alkaline phosphatase phoA. In a specific embodiment of the present invention, the alkaline phosphatase phoA is the amino acid sequence of protein accession number AAC73486, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC73486.

[0014] In one embodiment of the present invention, the amino acid sequence of CJ1437 or its mutant is as shown in SEQ ID NO:3 or has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3.

[0015] In one embodiment of the present invention, the amino acid sequence of CJ1361 or its mutant is as shown in SEQ ID NO:4 or has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4. In a specific embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of CJ1361 contains at least one of the following sites: E140, D270, S279, D156, I233, S248, D83, Q297, I287, N97 mutated to form a protein with dihydroxyacetone phosphate transaminase activity. In a further embodiment of the present invention, the amino acid sequence of CJ1361 contains at least one site substitution selected from the group consisting of: E140F, D270W, S279A, D156K, I233G, S248P, D83M, Q297P, I287L, N97M. Preferably, the mutation site of the amino acid sequence of CJ1361 is I233G.

[0016] In one embodiment of the present invention, the carrier or combination further comprises a third group of polynucleotides, the third group of polynucleotides encoding an enzyme that can act on an amino donor.

[0017] In one embodiment of the present invention, the amino donor is an amino acid, preferably glutamic acid. In one embodiment of the present invention, the enzyme that can act on the amino donor is a dehydrogenase, preferably glutamate dehydrogenase. In a specific embodiment of the present invention, the glutamate dehydrogenase is glutamate dehydrogenase gdhA. In a specific embodiment of the present invention, the glutamate dehydrogenase gdhA is the amino acid sequence of protein accession number AAC74831, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC74831.

[0018] In one embodiment of the present invention, the vector or combination comprises: a polynucleotide encoding histaminase (hisB) and a polynucleotide encoding dihydroxyacetone transaminase (CJ1361). In another embodiment of the present invention, the vector or combination comprises: a polynucleotide encoding histaminase (hisB), a polynucleotide encoding dihydroxyacetone transaminase (CJ1361), and a polynucleotide encoding glutamate dehydrogenase (gdhA).

[0019] In one embodiment of the present invention, the vector or combination comprises: a polynucleotide encoding hisamine phosphatase (hisB), a polynucleotide encoding dihydroxyacetone transaminase (CJ1437), and / or a polynucleotide encoding glutamate dehydrogenase (gdhA). In another embodiment of the present invention, the vector or combination comprises: a polynucleotide encoding hisamine phosphatase (AphA), a polynucleotide encoding dihydroxyacetone transaminase (CJ1437), and / or a polynucleotide encoding glutamate dehydrogenase (gdhA). In yet another embodiment of the present invention, the vector or combination comprises: a polynucleotide encoding hisamine phosphatase (PhoA), a polynucleotide encoding dihydroxyacetone transaminase (CJ1437), and / or a polynucleotide encoding glutamate dehydrogenase (gdhA).

[0020] In a specific embodiment of the present invention, the polynucleotides in the first, second and / or third groups of polynucleotides are homologous or heterologous to the host cell.

[0021] In a specific embodiment of the present invention, the polynucleotides in the first, second and / or third groups of polynucleotides contain codon optimizations for the host cell.

[0022] The nucleotide sequences, polynucleotides, and DNA molecules used in this invention are not limited to functional regions and may include at least one of expression repression regions, coding regions, leader sequences, exons, introns, and expression cassettes. Furthermore, the nucleotide sequences or polynucleotides may include double-stranded DNA or single-stranded DNA (i.e., the sense and antisense strands that make up double-stranded DNA) or RNA. Polynucleotides containing a specific polynucleotide sequence may include fragments and / or mutants of that specific polynucleotide sequence. A fragment of a polynucleotide refers to a portion of a polynucleotide that encodes a polypeptide that provides substantially the same function as the polypeptide encoded by the complete polynucleotide sequence. Examples of mutants of a specific polynucleotide sequence include naturally occurring allelic mutants, artificial mutants, and polynucleotide sequences obtained by deleting, substituting, adding, and / or inserting one or more nucleotides into said specific polynucleotide sequence. It should be understood that such fragments and / or mutants of a specific polynucleotide sequence encode a polypeptide that has substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.

[0023] In a specific embodiment of the present invention, the polynucleotides in the first, second and / or third groups of polynucleotides are operatively linked to promoters, which are homologous or heterologous to the host cell.

[0024] A second aspect of the invention provides a transformant that contains one or more of the above-described expression plasmid vectors or combinations thereof in a host cell.

[0025] In this invention, a transformant is a host cell that has been altered by introducing one or more expression plasmid vectors into a host cell, wherein the one or more expression plasmid vectors are the same or different. In some embodiments, the transformant is obtained by introducing the plasmid vector into a host cell that exhibits competence with the expression plasmid vector.

[0026] In a specific embodiment of the present invention, the transformant is a mutant host cell, and the expression plasmid vector or expression plasmid vector combination is integrated into the host cell chromosome.

[0027] In a specific embodiment of the present invention, the mutant host cell comprises a first group of polynucleotides, a second group of polynucleotides, and / or a third group of polynucleotides integrated into the host cell chromosome.

[0028] In this invention, the first group, the second group of polynucleotides and / or the third group of polynucleotides can be integrated into the host cell chromosome by methods such as plasmid transformation, phage-mediated transformation and genome editing.

[0029] In a further preferred embodiment of the present invention, the pathways for the generation of metabolic byproducts succinic acid, lactic acid, ethanol, acetaldehyde, acetic acid and formic acid in the transformed organism are inhibited or blocked.

[0030] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the genetically engineered strain of the transformant is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, and fumarate reductase flavin subunit frdA.

[0031] This invention employs gene knockout to inhibit, reduce, or eliminate the expression of the corresponding enzyme. Those skilled in the art will recognize that other methods for inhibiting enzyme expression or reducing / eliminating enzyme activity can also be applied to the construction of genetically engineered strains, such as promoter knockout or replacement, introduction of enzyme inactivation / reduction mutations, deletion (or partial deletion) of essential functional elements or regions for gene transcription / translation, introduction of mutations or nucleic acid sequences that accelerate mRNA degradation to guide enzyme synthesis, introduction of mutations or protein tags that accelerate enzyme degradation / decomposition / inactivation, inhibition of signal activation, RNA interference, gene silencing, CRISPRi, etc. In a specific embodiment of this invention, the expression level of the enzyme is reduced by more than 30% or the activity of the enzyme is reduced by more than 30%.

[0032] In specific embodiments of the present invention, the host strain of the transformant is selected from bacteria or fungi; optionally, the host strain is selected from wild or genetically modified Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the host strain is selected from wild or genetically modified Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; optionally, the host strain is selected from wild or genetically modified Escherichia coli. Escherichia coli cells can be Escherichia coli strains derived from Escherichia coli K12 (e.g., MG1655, W3110, DH10b, DH1, BW2952 and strains derived therefrom) or any Escherichia coli strain or strain derived therefrom of Escherichia coli B.

[0033] A backbone plasmid that can replicate autonomously in a host cell can be any plasmid that can replicate in a host cell. In one embodiment, the expression plasmid vector includes a backbone plasmid that can replicate in *E. coli*. Examples of backbone plasmids include, but are not limited to, backbone plasmids that can replicate in *E. coli* strains, such as pUC (e.g., pUC18 and pUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET, pSC101, and pZE plasmids, and plasmids derived therefrom.

[0034] In a third aspect, the present invention provides a composition comprising the above-described transformant or a culture thereof.

[0035] In a fourth aspect, the present invention provides a method for preparing serine, comprising culturing the above-mentioned transformant to obtain serine.

[0036] In one embodiment of the present invention, the culture medium for cultivation includes one or more of glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, vitamin B1, IPTG, and calcium carbonate.

[0037] In a fifth aspect, the present invention provides the use of the above-described expression plasmid vector or combination of expression plasmid vectors, transformants or their cultures, or compositions in the preparation of serine.

[0038] In a sixth aspect, the present invention provides a chassis strain, which is an Escherichia coli strain, wherein the pathways for the generation of metabolic byproducts succinic acid, lactic acid, ethanol, acetaldehyde, acetic acid and formic acid in the chassis strain are inhibited or blocked.

[0039] In a further preferred embodiment of the present invention, the expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, and fumarate reductase flavin subunit frdA.

[0040] In a seventh aspect, the present invention provides a dihydroxyacetone transaminase mutant, the amino acid sequence of which has at least 90% sequence identity with the parental dihydroxyacetone transaminase shown in SEQ ID NO:4, and comprises one or more of the following amino acid mutations: E140F, D270W, S279A, D156K, I233G, S248P, D83M, Q297P, I287L or N97M, the positions of which refer to the number in SEQ ID NO:4, and the mutant has dihydroxyacetone transaminase activity.

[0041] Preferably, it includes the I233G site.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1) This invention improves serine alcohol production by introducing phosphatases (histidine phosphatase, acid phosphatase, alkaline phosphatase) and dihydroxyacetone phosphate transaminase (CJ1437, CJ1361) into Escherichia coli; at the same time, it achieves efficient fermentation production of serine alcohol by knocking out or weakening genes related to metabolic byproduct synthesis pathways and mixed acid fermentation pathways.

[0044] 2) This invention improves serine production by introducing a mutation site into dihydroxyacetone phosphate transaminase, especially the I233G mutation in CJ1361, which increases serine production to 2.66 times that of the wild-type strain.

[0045] 3) Compared with traditional chemical synthesis methods, the engineered strain for producing serine provided by this invention has the advantages of high yield, non-toxicity, no pollution, mild conditions, and less environmental pollution. Because it uses food-grade strains for fermentation, it has broad application prospects in the food, pharmaceutical and other industries. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0047] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0048] Materials and Methods

[0049] LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0050] Fermentation medium: glucose 30g / L, yeast powder 5g / L, ammonium sulfate 8g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, potassium dihydrogen phosphate 0.8g / L, vitamin B1 0.005g / L, calcium carbonate 10g / L. The pH of the medium was adjusted to 7.3-7.4 with concentrated ammonia water and sterilized at 115℃ for 15min.

[0051] Shake-flask fermentation method: Single clones of the activated bacterial strain from LB agar plates (containing appropriate concentrations of antibiotics) at 37℃ are inoculated into LB liquid medium and cultured at 37℃ for 12-16 hours at 220 rpm. The overnight LB culture is inoculated at a 1% inoculum into a 250 ml shake flask containing 100 mg / L ampicillin and 25 ml of fermentation medium (containing 10 g / L CaCO3 as a pH stabilizer). After sealing with a breathable membrane, the flask is incubated at 30℃ and 220 rpm. When the OD600 reaches 0.6, IPTG (final concentration 0.3 mM) is added to induce plasmid expression. Fermentation is stopped after 40-48 hours, and samples are taken.

[0052] Concentration determination of serine: All standards used were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). 1 mL of fermentation broth was centrifuged at 12000 g for 5 min to remove bacterial cells. The filtrate was filtered through a 0.22 μm filter membrane. After appropriate dilution, the concentration of the above-mentioned product in the sample was determined by high-performance liquid chromatography (HPLC). The HPLC instrument was a Shimadzu LC-40D; the column was a Welch Ultimate AQ-C18, 4.6 × 250 mm; the column oven was set to 40 degrees Celsius; the detector was a UV detector (set to constant temperature at 40 degrees Celsius), with a detection wavelength of 338 nm; the mobile phase was sodium acetate aqueous solution and methanol, with gradient elution.

[0053] Information on the genes and enzymes involved in this invention is shown in Table 1.

[0054] Table 1. Types of enzymes involved in this invention.

[0055]

[0056]

[0057] In the context of this application, the enzymes mentioned include mutants that retain enzyme activity, said mutants having an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence represented by the protein accession numbers in Table 1. The nucleic acid sequence of wild-type CJ1437 is shown in SEQ ID NO:1; the nucleic acid sequence of wild-type CJ1361 is shown in SEQ ID NO:2.

[0058] The biomaterials constructed in this invention are shown in Table 2.

[0059] Table 2 Biomaterials involved in this invention

[0060]

[0061]

[0062] Example 1: Construction of chassis strain WD7

[0063] Using the E. coli Keio Knockout Collection 1Phages were created from the corresponding single-gene knockout strain (Horizon Discovery, CO, USA). The DNA deletion fragment carrying kanamycin (Kan) resistance was introduced into the starting strain *E. coli* W3110 using the P1 phage transfection method. The fragment was plated on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C to obtain a Kan-resistant deletion strain. Then, plasmid pCP20 was transformed into the aforementioned Kan-resistant transformants and plated on LB agar plates containing 100 mg / L ampicillin. After incubation at 30°C for 24 hours, PCR was used to identify the correct transformants with the Kan-resistant gene removed from the target gene, thus obtaining a non-resistant deletion strain. Finally, single colonies of the correctly removed Kan gene transformants were streaked onto LB agar plates and incubated at 37 or 42°C to obtain a non-resistant *E. coli* strain without the pCP20 plasmid. By repeating this process, a deletion strain WD7 (ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔfrdA) based on W3110 was constructed, in which the synthesis pathway of the metabolic byproduct acetic acid and the mixed acid fermentation pathway were knocked out to avoid the waste of carbon source.

[0064] Example 2: Construction of recombinant plasmids pAPD-1 to pAPD-7

[0065] Based on the protein sequences corresponding to wild-type CJ1437 (SEQ ID NO:1) and CJ1361 (SEQ ID NO:2), codon optimization was performed to synthesize relevant DNA sequences for E. coli; then, the genes hisB, gdhA, aphA, phoA, serC, and hisC were cloned from the E. coli MG1655 genome and assembled using Gibson assembly. 2 The DNA fragments obtained above were combined and ligated into plasmid pZElac in different ways. 3 After deactivating the lac promoter, the following recombinant plasmids were obtained (see the Materials and Methods section for details of the combination methods): pAPD-1 to pAPD-7. The above recombinant plasmids and the empty vector pZElac were then transformed into either WD7 or W3110 strains, respectively. See the Materials and Methods section for details of the specific plasmid and strain combinations.

[0066] Example 3: Shake-flask fermentation of strains APD00 to APD07

[0067] Using strain APD00 as a blank control, shake-flask fermentation was performed. The composition of the fermentation medium and fermentation conditions are detailed in the Materials and Methods section. Fermentation data after 40 hours are shown in Table 3.

[0068] Table 3. Data from serine shake-flask fermentation

[0069]

[0070]

[0071] The results show that CJ1361 and HisB are the optimal gene combination for serine production, and that simultaneous overexpression of glutamate dehydrogenase GdhA also promotes serine synthesis. Furthermore, transaminase CJ1437 and phosphatases AphA and PhoA can also be used in the serine biosynthesis pathway.

[0072] Example 4: Protein Engineering of CJ1361

[0073] Based on the protein sequence of CJ1361, a series of point mutations were constructed at different amino acid positions (see Table 4) and introduced into plasmid pAPD-7. Shake-flask fermentation was performed using pAPD-7 strain APD07 carrying wild-type CJ1361 as a control. The fermentation results are shown in Table 4. It can be seen that, except for the point mutation D291P, all other point mutations enhanced the serine production activity of CJ1361 to varying degrees. Among them, I233G showed the highest yield, which was 2.66 times that of the control strain.

[0074] Table 4. Data on serine shake-flask fermentation

[0075]

[0076] *Relative yield is the ratio of the experimental group to the control group.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0078] References

[0079] 1. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, KA; Tomita, M.; Wanner, BL; Mori, H., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006,2(1),2006.0008.

[0080] 2. Gibson, D.G.; Young, L.; Chuang, R.-Y.; Venter, J.C.; Hutchison, C.A.; Smith, H.O., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 2009, 6(5), 343 - 345.

[0081] 3. Zhang, K.; Li, H.; Cho, K.M.; Liao, J.C., Expanding metabolism for total biosynthesis of the nonnatural amino acid L - homoalanine. Proceedings of the National Academy of Sciences 2010, 107(14), 6234 - 6239.

[0082] Nucleic acid sequence:

[0083] SEQ ID NO:1 (CJ1437):

[0084]

[0085] SEQ ID NO:2(CJ1361):

[0086]

[0087] Amino acid sequence:

[0088] SEQ ID NO:3 (CJ1437):

[0089] MQANKNIQKLTPYLSIPHKIWNSSQSNILKLDWNEATIPPSPYVIESIKKFLVNGNLNWYPN

[0090] TKNLYLLDKIAEYTKQINSSFVELFEGSDSAHECIIDVFLDKCDKIGIVSPTYDNFRSRANGV

[0091] GIETISFTLDDNFNLDFDSLEYFIHEKRIKLLYLCNPNNPTGKSYNIQKIKSLIINNPNVMFIID

[0092] EAYYEFTSQSVCDLVEQCNNLIITRTFSKAFALASFRIGYIISHPENIESINKLRNPKSVPMLS

[0093] QIAANAALEDLQYMRDYVDEVSCARMEFVKFLNTLTTGGGGIFNDSVANFVLIQNENISLFVGFLEKEGIFIRNYSHLISKNCRISIGTRNQMSYVAEKIQEFAKKQGGFHLV*

[0094] SEQ ID NO:4 (CJ1361):

[0095] MLPNKNIQNLKPYMSIPHDIWNFKNYDNVLKLDWNEATINPSPKVFEHIYQFLNSGKLNW

[0096] YPNTKNIELLRALSLYTKQDSEDYIEIFGSSDAAHENIIDVFLEKNSIVCIISPTYDNFRARAN

[0097] GVGIKTINFMLDKNFELDFDELNNFLKQKKVDFLYICNPNNPTGVCYDNNKLEQLIINNPKI

[0098] MFLVDEAYYEFCKKSVQNLVKNCKNLIITRTFSKAFALASFRIGYVISHVDNIKSINKLRNS

[0099] KNISMLSQIAALAALKDVEYVDKFVEQVATSRKLFLKDIKAFDIRACEQTETNFVLLSMKNMQEVINYLKQNKIFIRNYNHIIPGYCRITIGTVEQMRYLLDKLKEFYER*.

Claims

1. An expression plasmid vector or a combination of expression plasmid vectors, characterized in that: The vector or combination comprises: a first group of polynucleotides and a second group of polynucleotides, wherein the first group of polynucleotides contains at least one polynucleotide encoding a phosphatase and the second group of polynucleotides contains at least one polynucleotide encoding dihydroxyacetone transaminase. And the backbone plasmid, which can replicate autonomously in the host cell. The phosphatase is selected from at least one of histamine phosphatase, acid phosphatase, and alkaline phosphatase, and the dihydroxyacetone phosphate transaminase is selected from at least one of CJ1437 and CJ1361.

2. The expression plasmid vector or combination of expression plasmid vectors as described in claim 1, characterized in that: The histidine phosphatase is selected from histidine phosphatase hisB. Preferably, histidine phosphatase hisB is the amino acid sequence of protein accession number CAA31814, or an amino acid sequence that has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by CAA31814. And / or the acid phosphatase is selected from acid phosphatase aphA. Preferably, the acid phosphatase aphA is the amino acid sequence of protein accession number CAA60534, or an amino acid sequence that has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by CAA60534. And / or the alkaline phosphatase is selected from alkaline phosphatase phoA. Preferably, the alkaline phosphatase phoA is the amino acid sequence of protein accession number AAC73486, or an amino acid sequence that has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC73486. And / or the amino acid sequence of CJ1437 as shown in SEQ ID NO:3 or an amino acid sequence having at least 96% or 97% or 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3; And / or the amino acid sequence of CJ1361 as shown in SEQ ID NO:4 or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4; preferably, an amino acid sequence with at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Compared to the amino acid sequence shown in IDNO.4, the amino acid sequence of CJ1361 contains at least one of the following sites: E140, D270, S279, D156, I233, S248, D83, Q297, I287, N97, which are amino acid mutations and have dihydroxyacetone phosphate transaminase activity; more preferably, the amino acid sequence of CJ1361 contains at least one site substitution selected from the group consisting of: E140F, D270W, S279A, D156K, I233G, S248P, D83M, Q297P, I287L, N97M; even more preferably, the mutation site of the amino acid sequence of CJ1361 is I233G.

3. The expression plasmid vector or combination of expression plasmid vectors as described in claim 1, characterized in that: The carrier or combination further comprises a third group of polynucleotides, which encodes an enzyme that can act on an amino donor; Preferably, the amino donor is an amino acid, more preferably glutamic acid; Preferably, the enzyme that can act on the amino donor is a dehydrogenase, more preferably glutamate dehydrogenase; Preferably, the glutamate dehydrogenase is glutamate dehydrogenase gdhA; more preferably, the glutamate dehydrogenase gdhA is the amino acid sequence of protein accession number AAC74831, or an amino acid sequence that has at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC74831. Preferably, the vector or combination comprises: a polynucleotide encoding histaminol phosphatase hisB and a polynucleotide encoding dihydroxyacetone phosphate transaminase CJ1361; Preferably, the vector or combination comprises: a polynucleotide encoding histaminol phosphatase hisB, a polynucleotide encoding dihydroxyacetone transaminase CJ1361, and a polynucleotide encoding glutamate dehydrogenase gdhA. Preferably, the vector or combination comprises: a polynucleotide encoding histaminol phosphatase hisB, a polynucleotide encoding dihydroxyacetone transaminase CJ1437, and / or a polynucleotide encoding glutamate dehydrogenase gdhA. Preferably, the vector or combination comprises: a polynucleotide encoding histamine phosphatase AphA, a polynucleotide encoding dihydroxyacetone transaminase CJ1437, and / or a polynucleotide encoding glutamate dehydrogenase gdhA. Preferably, the vector or combination comprises: a polynucleotide encoding histamine phosphatase PhoA, a polynucleotide encoding dihydroxyacetone transaminase CJ1437, and / or a polynucleotide encoding glutamate dehydrogenase gdhA.

4. An expression plasmid vector or combination of expression plasmid vectors as described in any one of claims 1-3, characterized in that: The polynucleotides in the first, second, and / or third groups of polynucleotides are homologous or heterologous to the host cell; Preferably, the polynucleotides in the first, second, and / or third groups of polynucleotides contain codons optimized for the host cell; Preferably, the polynucleotides in the first, second, and / or third groups of polynucleotides are operatively linked to promoters that are homologous or heterologous to the host cell.

5. A transformant, characterized in that: The transformant contains one or more expression plasmid vectors or combinations thereof as described in any one of claims 1-4; Preferably, the transformant is obtained by introducing one or more expression plasmid vectors into a host cell, wherein the one or more expression plasmid vectors are the same or different; Preferably, the transformant is obtained by introducing the plasmid vector into a host cell that exhibits a competent state for the expression plasmid vector through transformation; Preferably, the transformant is a mutant host cell, and the expression plasmid vector or combination of expression plasmid vectors is integrated into the host cell chromosome; Preferably, the mutant host cell contains a first group of polynucleotides, a second group of polynucleotides, and / or a third group of polynucleotides integrated into the host cell chromosome. Preferably, the first group, the second group of polynucleotides and / or the third group of polynucleotides are integrated into the host cell chromosome by means of plasmid transformation, phage-mediated transformation or genome editing; Preferably, the pathways for the formation of metabolic byproducts succinic acid, lactic acid, ethanol, acetaldehyde, acetic acid, and formic acid in the transformed organism are inhibited or blocked; Preferably, the expression of one, two or more of the following genes in the genetically engineered strain of the transformant is inhibited, weakened or eliminated: phosphoacetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, and fumarate reductase flavin subunit frdA. Preferably, the host strain of the transformant is selected from bacteria or fungi.

6. A composition, characterized in that: Includes the transformant or its culture as described in claim 5.

7. A method for preparing serinel, characterized in that: The process includes culturing the transformant according to claim 5 to obtain serinel; Preferably, the culture medium includes one or more of glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, vitamin B1, IPTG, and calcium carbonate.

8. The use of the expression plasmid vector or combination of expression plasmid vectors according to any one of claims 1-4, the transformant or culture thereof according to claim 5, or the composition according to claim 6 in the preparation of serine.

9. A chassis strain, which is an Escherichia coli strain, characterized in that: The pathways for the production of metabolic byproducts succinic acid, lactic acid, ethanol, acetaldehyde, acetic acid, and formic acid in the chassis strain were inhibited or blocked. Preferably, the expression of one, two or more of the following genes in the chassis strain is inhibited, weakened or eliminated: phosphorylated acetyltransferase pta, pyruvate oxidase poxB, alcohol dehydrogenase adhE, pyruvate-formate lyase pflB, pyruvate aldehyde synthase mgsA, D-lactate dehydrogenase ldhA, and fumarate reductase flavin subunit frdA.

10. A mutant of dihydroxyacetone phosphate transaminase, characterized in that: The mutant has at least 90% sequence identity with the parental dihydroxyacetone transaminase shown in SEQ ID NO:4 and contains one or more of the following amino acid mutations: E140F, D270W, S279A, D156K, I233G, S248P, D83M, Q297P, I287L or N97M, the positions of which refer to the number in SEQ ID NO:

4. The mutant has dihydroxyacetone transaminase activity. Preferably, it includes the I233G site.

Images