Recombinant escherichia coli strain and method for producing mannitol by fermentation of recombinant escherichia coli strain

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a genetic recombination escherichia coli strain and a method for producing mannitol through fermentation of the genetic recombination escherichia coli strain. The gene recombinant escherichia coli strain contains the following modifications: deletion, nonsense mutation, inactivation mutation or expression of inhibitory nucleic acid fragments of 6-phosphofructokinase 1pfkA gene and 6-phosphofructokinase 2pfkB gene. According to the strain and the production method, the mannitol can be produced by taking glucose as a substrate, and the conversion rate is very high.

Description

Technical Field

[0001] The present invention generally relates to the technical field of microbial fermentation, and in particular to an Escherichia coli strain and a method for producing mannitol by fermenting the same. Background Art

[0002] D-mannitol, as an isomer of sorbitol, is often used as an excipient in pharmaceutical preparations. It is non-hygroscopic, dries quickly, has good chemical stability, and has good granular properties. Mannitol can increase the maximum allowable temperature of the drying product in freeze-dried preparations. In addition, mannitol can also be used as an excipient to improve the recovery of powder cakes.

[0003] The mainstream production method of mannitol on the market currently uses food-grade crystalline glucose as raw material. After dissolving it in water, part of the glucose is converted into mannose and fructose through isomerization. After refining and removing impurities, it reacts with hydrogen under the action of a catalyst to reduce glucose, mannose and fructose to a mixture of mannitol and sorbitol. The catalyst is then removed through ion exchange, and the mixture is concentrated and cooled for crystallization. The mannitol and sorbitol are then separated using a centrifuge. After drying and screening, the mannitol is made into a finished product.

[0004] Biological fermentation has the advantages of high production efficiency and low cost, but the technical solutions for producing mannitol based on microbial fermentation technology in the prior art generally have the disadvantages of low conversion rate and unsatisfactory purity. For example, the Chinese patent application with publication number CN1064311A has a conversion rate of 45%, and the final concentration of mannitol in the fermentation broth is about 70g / L, which cannot meet the needs of large-scale industrial production. Summary of the invention

[0005] The present invention covers the following technical solutions:

[0006] Another object of the present invention is to provide a composition comprising a genetically recombinant Escherichia coli strain;

[0007] The genetically recombinant Escherichia coli strain contains the following modifications:

[0008] 6-phosphofructokinase 1pfkA gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; and

[0009] The 6-phosphofructokinase 2pfkB gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivating mutation, or has an expression-inhibiting nucleic acid fragment.

[0010] Optionally, the composition is in liquid, frozen or dry powder form.

[0011] Optionally, the composition is in the form of a frozen or dried powder, which further comprises a solid carrier.

[0012] Optionally, the solid carrier includes one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter press mud, sawdust, perlite, mica, silica, quartz powder, calcium-based bentonite, vermiculite, kaolin, light calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, white carbon black, fine sand and clay.

[0013] Optionally, the composition is liquid, and further comprises a liquid carrier.

[0014] Optionally, the liquid carrier is preferably an organic solvent and / or water.

[0015] Optionally, the composition comprises an auxiliary agent that can be used for bacterial agents.

[0016] Optionally, the auxiliary agent includes one or more of sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkylnaphthalene sulfonate polycondensate, nicotinic acid, alcohol, buffer salt, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extract, white carbon black, tea saponin, and skimmed milk;

[0017] Optionally, the number of viable Escherichia coli strains in the composition is 10 7～12 cfu·mL -1 or 10 7～ 12 cfu·g -1 .

[0018] The strain also includes the following modifications:

[0019] Pyridine nucleotide transhydrogenase sthA gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment;

[0020] Introducing and / or enhancing the expression of the pyridine nucleotide transhydrogenase α subunit pntA gene or its isozyme gene or its functional homolog gene; and

[0021] Introducing and / or enhancing the expression of the pyridine nucleotide transhydrogenase β subunit pntB gene or its isozyme gene or its functional homolog gene;

[0022] Optionally, the following modifications are also included:

[0023] The lauroyl acyltransferase LPXL gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivating mutation, or has an expression-inhibiting nucleic acid fragment;

[0024] Introducing and / or enhancing the expression of 6-phosphate glucose dehydrogenase zwf gene or its isozyme gene or its functional homolog gene;

[0025] Introducing and / or enhancing the expression of 6-phosphogluconate dehydrogenase gnd gene or its isozyme gene or its functional homolog gene; and

[0026] p119 promoter;

[0027] Optionally, the following modifications are also included:

[0028] The phosphogluconate dehydratase edd gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivating mutation, or has an expression-inhibiting nucleic acid fragment; and

[0029] KHG / KDPG aldolase eda gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment;

[0030] Optionally, the following modifications are also included:

[0031] Myristoyltransferase LPXM gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment;

[0032] Introducing and / or enhancing the expression of mannitol 1-phosphate-5-dehydrogenase M1PDH gene or its isozyme gene or its functional homolog gene; and

[0033] Introducing and / or enhancing the expression of mannitol-1-phosphatase M1PP gene or its isozyme gene or its functional homolog gene;

[0034] Optionally, the M1PDH gene is from at least one of Thermoanaerobacter sp. (TSM1PDH), actococcus lactis subsp. lactis bv. Diacetylactis (DM1PDH), eauveria bassiana (BBM1PDH), Oceanobacillus iheyensis (OIM1PDH), Bacillus sp. FW1 (BSM1PDH), Brochothrix thermosphacta (BTM1PDH), Aeromonas almonicida (ASM1PDH), Pectobacterium atrosepticum (PAM1PDH), and Escherichia coli (ECM1PDH);

[0035] Optionally, the M1PDH gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 1 to 9;

[0036] Optionally, the M1PP gene is from at least one of Pantoea stewartia (PSM1PP), Luteibacter sp. (LSM1PP), Pseudomonas graminis (PGM1PP), Muribaculaceae bacterium (MBM1PP), Eimeria tenella (ETM1PP), Salmonella diarizonae (SDM1PP), Photorhabdus cinerea (PCM1PP), Citrobacter pasteurii (CPM1PP), Corallincola holothuriorum (CHM1PP), and Escherichia coli (ECM1PPA, ECM1PPB);

[0037] Optionally, the M1PP gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 10 to 12;

[0038] Optionally, pntA and pntB comprise the sequences shown in Gene ID: 946628 and Gene ID: 946144, respectively;

[0039] Optionally, the zwf gene comprises the sequence shown in Gene ID: 946370;

[0040] Optionally, the gnd gene comprises the sequence shown in Gene ID:946554.

[0041] Still another object of the present invention is to provide a method for producing mannitol by fermentation, wherein a culture medium containing glucose as a main carbon substrate is fermented under aerobic conditions using the strain as described above, and the method includes then recovering the mannitol accumulated in the culture medium.

[0042] Optionally, the fermentation is stopped after mannitol is accumulated in the culture medium to a concentration of not less than 150 g / L.

[0043] Optionally, the strain is pre-cultured in an inorganic salt medium before fermentation.

[0044] Optionally, the inorganic salt culture medium uses glucose as the main carbon substrate and contains 5-15 g / L potassium dihydrogen phosphate, 4-8 g / L diammonium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 1.4-2.0 g / L citric acid monohydrate, 1-3 g / L yeast extract and a metal ion solution; the metal ion solution contains 600-1000 mg / L ethylenediaminetetraacetic acid, 100-300 mg / L cobalt chloride hexahydrate, 700-1300 mg / L manganese chloride tetrahydrate, 70-130 mg / L copper chloride dihydrate, 70-130 mg / L boric acid, 100-300 mg / L sodium molybdate dihydrate, 700-1300 mg / L zinc acetate dihydrate and 5-15 g / L ferric citrate.

[0045] Optionally, in step ii), when OD 600 =35-45 when induction begins.

[0046] Optionally, in step i) and / or step ii), the dissolved oxygen content during acclimatization culture is controlled at 35%±10%.

[0047] Optionally, the fermentation includes: adding arabinose to induce the strain, and then accumulating and producing mannitol.

[0048] Optionally, the strain is cultured to an OD of 600 =35-45 when induction begins.

[0049] Optionally, the fermentation medium for accumulating the production of mannitol adopts the inorganic salt medium as described above.

[0050] Optionally, the fermentation time for accumulating the production of mannitol is 48 to 60 hours.

[0051] Optionally, the dissolved oxygen content during the accumulative production of mannitol is controlled at 40±10%.

[0052] Optionally, the glucose content is controlled below 10 g / L during the accumulation and production of mannitol.

[0053] The present invention provides an Escherichia coli strain and a fermentation method for producing mannitol optimized therefor. The strain can produce mannitol with glucose as a substrate and has a very high conversion rate (in a preferred embodiment, it can reach more than 70%). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0055] Figure 1-1 This is a nucleic acid gel image of colony PCR. Lane M: 1kb plus DNA Marker; Lane 1: E. coli without knocking out the pfkA gene obtained a 2200bp band; Lane 2: E. coli with successfully knocking out the target gene pfkA gene obtained a 1200bp band; Lane 3: E. coli without knocking out the pfkB gene obtained a 2200bp band; Lane 4: E. coli with successfully knocking out the target gene pfkB gene obtained a 1200bp band; Lane 5: E. coli without knocking out the sthA gene obtained a 2601bp band; Lane 6: E. coli with successfully replacing the target gene sthA gene with the pntAB gene obtained a 4132bp band; Lane 7: E. coli without knocking out the LPXL gene obtained a 2121bp band; Lane 8: The target gene LPXL gene was successfully replaced with p119-zwf-g Lane 9: Escherichia coli with the nd gene not knocked out obtained a 4146bp band; Lane 9: Escherichia coli with the edd and eda genes not knocked out obtained a 3690bp band; Lane 10: Escherichia coli with the target genes edd and eda genes successfully knocked out obtained a 1200bp band; Lane 11: Escherichia coli with the lPXM gene not knocked out obtained a 2172bp band; Lane 12: Escherichia coli with the target gene lPXM gene successfully replaced with the p119-TSM1PDH-PSM1PP gene obtained a 3060bp band; Lane 13: Escherichia coli with the lPXM gene not knocked out obtained a 2172bp band, Lane 14: Escherichia coli with the target gene lPXM gene successfully replaced with the p119-ASM1PDH-PCM1PP gene obtained a 3120bp band.

[0056] Figure 1-2 This is the HPLC spectrum of D-mannitol standard.

[0057] Figure 1-3 HPLC spectrum of D-glucose standard.

[0058] Figure 1-4 The HPLC spectrum of the whole cell of the engineered bacterium ML06 catalytically converting glucose into D-mannitol according to an exemplary embodiment of the present disclosure.

[0059] Figure 2-1A This is the peak diagram of liquid phase detection of 1g / L glucose standard.

[0060] Figure 2-1B This is the peak diagram of liquid phase detection of 1g / L mannitol standard.

[0061] Figure 2-2 This is a comparison chart of OD600 of acclimation growth in LB / inorganic salt medium.

[0062] Figure 2-3 This is the growth curve of fermentation culture bacteria.

[0063] Figure 2-4A This is the peak diagram of liquid phase detection of the initial fermentation sample.

[0064] Figure 2-4B Liquid phase detection peak diagram of fermentation samples after optimizing dissolved oxygen conditions.

[0065] Figure 2-4C Liquid phase detection peak diagram of fermentation sample after optimizing sugar addition rate. DETAILED DESCRIPTION

[0066] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0067] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0068] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0069] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present invention, the technical solution undoubtedly includes technical solutions connected by "logical and", and undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0070] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0071] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0072] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0073] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0074] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0075] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0076] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0077] In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

[0078] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. For example, "optionally includes" means including or not including.

[0079] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features can also be incorporated into the present invention as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be modified adaptively according to the description of the present invention.

[0080] In the process of practical application, considering the possible need for transportation, it is necessary to expand the culture of E. coli strains to prepare them in the form of compositions (especially microbial agents) to expand their scope of application.

[0081] definition

[0082] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0083] Throughout this specification and the appended claims, the words "comprises" and "comprising" and variations thereof should be interpreted inclusively. That is, where the context permits, these words are intended to convey that other elements or integers not specifically listed may be included.

[0084] The articles "a / an" are used herein to refer to one / a or more than one / more than one (i.e., one / a or at least one / at least one) of the grammatical objects of the article. For example, "an element / an element" can mean one element / an element or more than one element / more than one element. When nouns (e.g., compounds, additives, etc.) are referred to in the singular, the plural is intended to be included. Thus, when referring to a particular part (e.g., "a gene"), this means "at least one" of the gene, e.g., "at least one gene", unless otherwise specified.

[0085] Unless explicitly indicated otherwise, the various embodiments of the invention described herein may be cross-combined.

[0086] The term "carbon source" refers to a source of carbon, preferably a compound or molecule comprising carbon. Preferably, the carbon source is a carbohydrate. Carbohydrate is understood herein as an organic compound consisting of carbon, oxygen and hydrogen. Suitably, the carbon source can be selected from the group consisting of: monosaccharides, disaccharides and / or polysaccharides, acids and acid salts. More preferably, the carbon source is a compound selected from the group consisting of: glucose and its various derivatives and salts, and various disaccharides, oligosaccharides, polysaccharides, etc. that can be decomposed and hydrolyzed into glucose.

[0087] The term "cell" refers to a eukaryotic organism or a prokaryotic organism, preferably existing as a single cell. In the present invention, the cell can be a recombinant Escherichia coli. That is, the recombinant cell is selected from a cell population of the genus consisting of Escherichia coli.

[0088] As used herein, the term "recombination" / "engineered" (e.g., referring to "recombinant E. coli," "recombinant cells," "recombinant microorganisms," and / or "recombinant strains") may refer to cells, microorganisms, or strains containing nucleic acids as a result of one or more genetic modifications. In short, cells, microorganisms, or strains contain different combinations of nucleic acids from one or more of their parents (any of which). In order to construct recombinant cells, microorganisms, or strains, one or more recombinant DNA techniques and / or one or more additional mutagenesis techniques may be used. For example, recombinant E. coli and / or recombinant E. coli cells may include nucleic acids that are not present in corresponding wild-type E. coli and / or cells, which have been introduced into the E. coli or E. coli cells (i.e., transgenic E. coli and / or cells) using recombinant DNA techniques, or which are not present in the wild-type E. coli and / or cells are the result of one or more mutations (e.g., using recombinant DNA techniques or another mutagenesis technique such as UV irradiation) in the nucleic acid sequence (such as a gene encoding a wild-type polypeptide) present in the wild-type E. coli and / or E. coli cells. Furthermore, the term "recombinant" may appropriately refer to a cell, microorganism or strain from which a nucleic acid sequence has been removed, for example using recombinant DNA techniques.

[0089] In this article, the recombinant E. coli containing or having a certain activity is understood to mean that the recombinant E. coli may contain one or more nucleic acid sequences encoding a protein having such activity. Therefore, the recombinant E. coli is allowed to functionally express such a protein or enzyme.

[0090] The term "functionally expressed" means that there is functional transcription of the relevant nucleic acid sequence, allowing the nucleic acid sequence to actually be transcribed, for example resulting in the synthesis of a protein.

[0091] As used herein, the term "transgenic" (e.g., referring to "transgenic E. coli" and / or "transgenic cells") refers to E. coli and / or cells, respectively, that contain a nucleic acid that is not naturally present in the E. coli and / or cells and that has been introduced into the E. coli and / or cells using, for example, recombinant DNA technology, such as recombinant yeast and / or cells.

[0092] As used herein for a protein or polypeptide, the term "mutation" means that at least one amino acid has been replaced, inserted into or deleted from an amino acid sequence by a different amino acid compared to a wild-type or naturally occurring protein or polypeptide sequence. Amino acid substitution, insertion or deletion can be achieved, for example, via mutagenesis of nucleic acids encoding these amino acids. Mutagenesis is a method well known in the art, and includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis, as described in Sambrook et al., Molecular Cloning-A Laboratory Manual, 2nd edition, Vol. 1-3 (1989), published by Cold Spring Harbor Publishing.

[0093] As used herein with respect to a gene, the term "mutation" means that at least one nucleotide in the nucleic acid sequence of a gene or its regulatory sequence has been replaced, inserted into, or deleted from a nucleic acid sequence by a different nucleotide compared to a wild-type or naturally occurring nucleic acid sequence. The replacement, insertion, or deletion of an amino acid can be achieved, for example, via mutagenesis, resulting in, for example, the transcription of a protein sequence with a qualitatively or quantitatively altered function or the knockout of the gene. In the context of the present invention, "altered gene" has the same meaning as a mutant gene.

[0094] As used herein, the term "gene" may refer to a nucleic acid sequence that can be transcribed into mRNA and then translated into a protein. A gene encoding a certain protein refers to one or more nucleic acid sequences encoding the protein.

[0095] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomer unit in a deoxyribonucleotide or ribonucleotide polymer (i.e., polynucleotide) in a single-stranded or double-stranded form, and unless otherwise limited, known analogs with the essential properties of natural nucleotides are encompassed because they hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). For example, a certain enzyme defined by a nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence hybridized with a reference nucleotide sequence encoding the enzyme. A polynucleotide can be the full length or subsequence of a natural or heterologous structure or regulatory gene. Unless otherwise indicated, the term includes reference to a specified sequence and its complementary sequence. Therefore, a DNA or RNA with a modified backbone for stability or other reasons is a term "polynucleotide" as intended herein. In addition, a DNA or RNA containing a rare base (such as inosine) or a modified base (such as a tritylated base) (just to name two examples) is a term polynucleotide as used herein. It will be understood that a variety of modifications have been made to DNA and RNA for many useful purposes known to those skilled in the art. The term polynucleotide as used herein includes such chemically, enzymatically, or metabolically modified forms of the polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells (especially including simple and complex cells).

[0096] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein. An example of a nucleic acid sequence is a DNA sequence.

[0097] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, such as those exhibited by an amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. An essential property of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein has specific reactivity with antibodies elicited by proteins consisting of the same but entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0098] The term "enzyme" refers to a protein with catalytic function in this article. In the case of a certain biological reaction of protein catalysis, the terms "protein" and "enzyme" can be used interchangeably in this article. When enzymes are mentioned with reference to enzymes (EC), enzymes are such categories, wherein enzymes are classified or can be classified according to the enzyme nomenclature provided by the International Union of Biochemistry and Molecular Biology Nomenclature Committee (the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, NC-IUBMB), which can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . It is intended to include other suitable enzymes that have not yet (yet) been classified in a given category but can be so classified.

[0099] If a protein or nucleic acid sequence (such as a gene) is referred to herein by reference to an accession number, unless otherwise specified, that number is specifically used to refer to the protein or nucleic acid sequence (gene) having a sequence that can be found via www.ncbi.nlm.nih.gov / (available as of October 1, 2020).

[0100] Each nucleic acid sequence encoding a polypeptide herein also includes any conservatively modified variants thereof. This includes, by reference to the genetic code, that it describes every possible silent variation of nucleic acid. The term "conservatively modified variant" is applicable to both amino acids and nucleic acid sequences. With regard to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding identical amino acid sequences or conservatively modified amino acid sequence variants due to the degeneracy of the genetic code. The term "degeneracy of the genetic code" refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode amino acid alanine. Therefore, at each position where a codon specifies alanine, a codon can be changed to any described corresponding codon without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent a conservatively modified variation.

[0101] As used herein, the term "functional homolog" (or simply "homolog") of a polypeptide and / or amino acid sequence or a gene having a specific sequence (e.g., "SEQ ID NO: X") refers to a polypeptide and / or amino acid sequence comprising the specific sequence, or refers to a nucleic acid sequence comprising a polypeptide and / or amino acid sequence encoding the specific sequence, provided that one or more amino acids are mutated, substituted, deleted, added and / or inserted, and the polypeptide has (qualitatively) the same enzymatic function for substrate conversion.

[0102] As used herein, the term "functional homologue" (or simply "homologue") of a polynucleotide and / or nucleic acid sequence having a specific sequence (e.g., "SEQ ID NO: X") refers to a polynucleotide and / or nucleic acid sequence comprising the specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added and / or inserted, and the polynucleotide encodes a polypeptide sequence having (qualitatively) the same enzymatic function for substrate conversion. With respect to nucleic acid sequences, the term functional homologue is intended to include nucleic acid sequences that differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0103] Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing sequences. Typically, sequence identity or similarity is compared over the entire length of the compared sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by matching between strings of such sequences.

[0104] When a certain level of similarity is exhibited, amino acid or nucleotide sequences are said to be homologous. Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by a "percent identity" or "percent similarity", which is high or low, respectively. Although controversial, to indicate "percent identity" or "percent similarity", "level of homology" or "percent homology" are often used interchangeably. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available for aligning two sequences and determining homology between two sequences (Kruskal et al., "An overview of sequence comparison: Time warps, string edits, and macromolecules", (1983), Society for Industrial and Applied Mathematics (SIAM), Vol. 25, No. 2, pp. 201-237 and the handbook edited by D. Sankoff and JB Kruskal (eds.), "Time warps, string edits and macromolecules: the theory and practice of sequence comparison", (1983), pp. 1-44, published by Addison-Wesley Publishing Company, Massachusetts USA).

[0105] The percent identity between two amino acid sequences can be determined by aligning two sequences using the Needleman and Wunsch algorithm. (Needleman et al. "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins" (1970) J. Mol. Biol. 48, pp. 443-453). This algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or higher, see Rice et al., "EMBOSS: The European Molecular Biology Open Software Suite", (2000), Trends in Genetics Vol. 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ) is used. For protein sequences, EBLOSUM62 is used as the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices may be specified. Optional parameters for amino acid sequence alignments are a gap opening penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences does not change significantly when different algorithms are used.

[0106] Homology or identity is the percentage of identical matches between two complete sequences over the total alignment area including any gaps or extensions. Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions showing identical amino acids in the two sequences in the alignment divided by the total length of the alignment including gaps. Identity as defined herein can be obtained from NEEDLE and is labeled "identity" in the output of the program.

[0107] The homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions showing identical amino acids in the two sequences in the alignment divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is labeled "longest-identity" in the output of the program.

[0108] Variants of the nucleotide or amino acid sequences disclosed herein may also be defined as nucleotide or amino acid sequences having one or more mutations, substitutions, insertions and / or deletions compared to the nucleotide or amino acid sequences specifically disclosed herein (eg, in the sequence listing).

[0109] Optionally, when determining the degree of amino acid similarity, the skilled person may also consider so-called "conservative" amino acid substitutions, which will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine and isoleucine; a group of amino acids with aliphatic-hydroxy side chains are serine and threonine; a group of amino acids with amide-containing side chains are asparagine and glutamine; a group of amino acids with aromatic side chains are phenylalanine, tyrosine and tryptophan; a group of amino acids with basic side chains are lysine, arginine and histidine; and a group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. In one embodiment, the conservative amino acid substitution group is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. The substitution variants of the amino acid sequences disclosed herein are variants in which at least one residue in the disclosed sequence has been removed and a different residue is inserted in its place. Preferably, the amino acid changes are conservative. In one embodiment, conservative substitutions of each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp;

[0110] Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg;

[0111] Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0112] The nucleotide sequences of the present invention can also be defined by their ability to hybridize with parts of the specific nucleotide sequences disclosed herein, respectively, under moderate hybridization conditions or preferably under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow a nucleic acid sequence of at least about 25 nucleotides, preferably about 50, 75 or 100 nucleotides, most preferably about 200 or more nucleotides to hybridize at a temperature of about 65°C in a solution containing about 1M salt (preferably 6xSSC or any other solution with comparable ionic strength), and washed at 65°C in a solution containing about 0.1M or less salt (preferably 0.2x SSC or any other solution with comparable ionic strength). Preferably, hybridization is performed overnight, i.e., for at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with about 90% or higher sequence identity. Moderate conditions are defined herein as conditions that allow a nucleic acid sequence of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at a temperature of about 45°C in a solution containing about 1M salt (preferably 6x SSC or any other solution with comparable ionic strength), and to wash at room temperature in a solution containing about 1M salt (preferably 6x SSC or any other solution with comparable ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions so as to specifically identify sequences with identities varying between 50% and 90%.

[0113] "Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), followed by translation into protein.

[0114] "Overexpression" refers to the expression of a gene (corresponding to a nucleic acid sequence) by a recombinant cell exceeding its expression in a corresponding wild-type cell. Such overexpression can be arranged, for example, by increasing the transcription frequency of one or more nucleic acid sequences, for example by operably linking the nucleic acid sequence to a promoter functional in the recombinant cell; and / or by increasing the copy number of a nucleic acid sequence.

[0115] The term "upregulation" and its variants refer to a process by which a cell increases the amount of a cellular component, such as an RNA or protein. Such upregulation may be in response to or caused by a genetic modification.

[0116] The term "pathway" or "metabolic pathway" is understood herein as a series of chemical reactions that build and break down molecules in a cell.

[0117] The nucleic acid sequence (ie, polynucleotide) or protein (ie, polypeptide) may be native or heterologous to the genome of the host cell.

[0118] "Native", "homologous" or "endogenous" with respect to a host cell means that the nucleic acid sequence does exist naturally in the genome of the host cell, or the protein is naturally produced by the cell. The terms "native", "homologous" and "endogenous" are used interchangeably herein.

[0119] As used herein, "heterologous" or "exogenous" may refer to a nucleic acid sequence or protein. For example, with respect to a host cell, "heterologous" may refer to a polynucleotide that is not naturally present in the genome of the host cell in this manner, or a polypeptide or protein that is not naturally produced by the cell in this manner. A heterologous nucleic acid sequence is a nucleic acid derived from an alien species, or if from the same species, substantially modified in composition and / or genomic loci relative to its native form by deliberate human intervention. For example, a promoter operably linked to a natural structural gene is from a species different from the species from which the structural gene is derived, or if from the same species, one or both are substantially modified relative to their original form. A heterologous protein may be derived from an alien species, or if from the same species, substantially modified relative to its original form by deliberate human intervention. That is, heterologous protein expression relates to the expression of a protein that is not naturally expressed in this manner in a host cell. The term "heterologous expression" refers to the expression of heterologous nucleic acids in a host cell. The expression of heterologous proteins in eukaryotic host cell systems (such as Escherichia coli) is well known to those skilled in the art. Polynucleotides containing nucleic acid sequences encoding genes for certain proteins or enzymes having specific activities can be expressed in such eukaryotic systems. In some embodiments, transformed / transfected cells can be used as expression systems for expressing enzymes. The expression of heterologous proteins in Escherichia coli is well known. Published by Cold Spring Harbor Laboratory is a recognized work describing a variety of methods for expressing proteins in Escherichia coli.

[0120] As used herein, a "promoter" is a DNA sequence that directs the transcription of a (structural) gene or other (partial) nucleic acid sequence. Suitably, the promoter is located in the 5' region of the gene, near the transcription start site of the (structural) gene. The promoter sequence may be constitutive, inducible or repressible. In one embodiment, no (external) inducer is required.

[0121] As used herein, the term "vector" includes reference to autosomal expression vectors and integration vectors for integration into a chromosome.

[0122] The term "expression vector" refers to a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest, which segment is under the control of (i.e., operably linked to) another nucleic acid segment that provides for its transcription. Such another segment may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0123] "Plasmid" refers to an autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microorganism and is usually circular in nature.

[0124] "Host cell" is understood herein to be a cell (such as an E. coli cell) that is transformed with one or more nucleic acid sequences encoding one or more heterologous proteins to construct a transformed cell (also referred to as a recombinant cell). For example, a transformed cell can contain a vector and can support replication and / or expression of the vector.

[0125] As used herein, "conversion" refers to the insertion of exogenous polynucleotides into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-engagement or electroporation. Exogenous polynucleotides can be maintained as a non-integrated vector (e.g., a plasmid), or alternatively can be integrated into the host cell genome. As used herein, "conversion" refers to the insertion of exogenous polynucleotides (i.e., exogenous nucleic acid sequences) into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-engagement or electroporation. Exogenous polynucleotides can be maintained as a non-integrated vector (e.g., a plasmid), or alternatively can be integrated into the host cell genome.

[0126] The present disclosure is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present disclosure, and are not intended to limit the scope of the present disclosure. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0127] In the following examples, Escherichia coli MG1655 (CGSC#: 6300) was purchased from the Escherichia coli Genetic Collection Center of Yale University, USA.

[0128] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the 6-phosphofructokinase 1 (pfkA) is NP_418351.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948412 (02-May-2024).

[0129] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the 6-phosphofructokinase 2 (pfkB) is NP_416237.3 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946230 (02-May-2024).

[0130] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the pyridine nucleotide transhydrogenase (sthA) is NP_418397.2 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 948461 (26-Jun-2024).

[0131] In the present invention, the NCBI reference sequence number (NCBIReference Sequence) of the pyridine nucleotide transhydrogenase alpha subunit (pntA) is NP_416120.1 (09-MAR-2022), and the NCBI reference sequence number (NCBIReference Sequence) of its encoding gene is Gene ID: 946628 (26-Jun-2024).

[0132] In the present invention, the NCBI reference sequence number (NCBIReference Sequence) of the pyridine nucleotide transhydrogenase β subunit (pntB) is NP_416119.1 (09-MAR-2022), and the NCBI reference sequence number (NCBIReference Sequence) of its encoding gene is Gene ID: 946144 (26-Jun-2024).

[0133] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the myristoyltransferase (LPXM) is NP_416369.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 945143 (6-Jun-2024).

[0134] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the lauroyl acyltransferase (LPXL) is NP_415572.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of the gene encoding the lauroyl acyltransferase (LPXL) is Gene ID: 946216 (2-May-2024).

[0135] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the 6-phosphate glucose dehydrogenase (zwf) is NP_416366.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946370 (02-May-2024).

[0136] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the 6-phosphogluconate dehydrogenase (gnd) is NP_416533.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946554 (23-Nov-2023)

[0137] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the phosphogluconate dehydratase (edd) is NP_416365.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946362 (02-May-2024).

[0138] In the present invention, the NCBI Reference Sequence Number (NCBI Reference Sequence) of the KHG / KDPG aldolase (eda) is NP_416364.1 (09-MAR-2022), and the NCBI Reference Sequence Number (NCBI Reference Sequence) of its encoding gene is Gene ID: 946367 (26-Jun-2024).

[0139] In the present invention, 9 mannitol 1-phosphate-5-dehydrogenase M1PDHs from different sources are introduced exogenously, and the sequences are all synthesized by Qingke Biological Company. Mannitol 1-phosphate-5-dehydrogenase M1PDHs are derived from Thermoanaerobacter sp. (TSM1PDH), actococcus lactis subsp. lactis bv. Diacetylactis (DM1PDH), eauveriabassiana (BBM1PDH), Oceanobacillus iheyensis (OIM1PDH), Bacillus sp. FW1 (BSM1PDH), Brochothrix thermosphacta (BTM1PDH), Aeromonas almonicida (ASM1PDH), Pectobacterium atrosepticum (PAM1PDH), Escherichia coli (strain K12) (ECM1PDH). The nucleotide and protein sequences are shown in Sequence 1 to Sequence 9, respectively.

[0140] Furthermore, in the present invention, 11 different sources of mannitol-1-phosphatase M1PP are introduced exogenously, and the sequences are all synthesized by Qingke Biotechnology Co., Ltd. Mannitol-1-phosphatase M1PP is derived from Pantoea stewartia (PSM1PP), Luteibacter sp. (LSM1PP), Pseudomonas graminis (PGM1PP), Muribaculaceaebacterium (MBM1PP), Eimeria tenella (ETM1PP), Salmonella diarizonae (SDM1PP), Photorhabdus cinerea (PCM1PP), Citrobacter pasteurii (CPM1PP), Corallincolaholothuriorum (CHM1PP), Escherichia coli (strain K12) (ECM1PPA, ECM1PPB). The nucleotide and protein sequences are shown in SEQ ID NOs. 10 to 21, respectively. Among them, M1PDH from different sources and M1PP from different sources can be freely combined.

[0141] It will be appreciated by those skilled in the art that the genes disclosed herein or their functional homologues comprise nucleic acid sequences having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the exemplified sequences, or amino acid sequences having one or more mutations, substitutions, insertions and / or deletions when compared to the amino acid sequences encoded therein.

[0142] Preferably, compared to the amino acid sequence encoded by a gene, the amino acid sequence encoded by any functional homolog of the gene has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid mutations, substitutions, insertions and / or deletions.

[0143] By way of example and not limitation, a functional homolog of the TSM1 PDH gene shown in SEQ ID NO.1 comprises a nucleic acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with any of those nucleic acid sequences, or a functional homolog thereof comprises a nucleic acid sequence having one or more mutations, substitutions, insertions and / or deletions when compared to any of those nucleic acid sequences; preferably, the nucleic acid sequence of any such functional homolog has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid mutations, substitutions, insertions and / or deletions compared to such nucleic acid sequences. More preferably, the functional homolog of phosphoglucomutase comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the polypeptide shown by SEQ ID NO.8.

[0144] Those skilled in the art will appreciate that other sequences include the above situations.

[0145] The composition of the present invention (preferably, when used as a starter culture) can be a pure culture or a mixed culture. Therefore, the present invention defines a pure culture as a culture in which all or substantially all of the culture consists of the same E. coli strain of the present invention. In an alternative form, a mixed culture is defined as a culture comprising several microorganisms, in particular several bacterial strains, including the E. coli strain of the present invention.

[0146] The composition can be prepared in the form of liquid, frozen or dry powder; or can be expressed in the form of preparations commonly used in the industry, such as granules, suspensions, wettable powders, emulsions or solutions.

[0147] Any carriers can be used, whether they are solid or liquid, as long as they are commonly used in the field of biological fermentation and are biologically inert. It is not limited to any specific carrier.

[0148] In some specific embodiments, when the composition is in the form of frozen or dried powder, it includes a solid carrier; examples of solid carriers include mineral powders such as clay, talc, montmorillonite, bentonite, medical stone, calcite, zeolite, calcium carbonate, diatomaceous earth and white carbon. On the other hand, typical liquid carriers include organic solvents and / or water, organic solvents such as decane and dodecane, vegetable oils, mineral oils, etc. Any one or more.

[0149] The composition contains an auxiliary agent that can be used for bacterial agents; the auxiliary agent preferably includes one or more of sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkylnaphthalene sulfonate condensate, nicotinic acid, alcohol, buffer salt, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extract, white carbon black, tea saponin, and skimmed milk.

[0150] The total content of these additives can be 0wt% to 80wt%, for example 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt%.

[0151] The content of the carrier may be a value obtained by subtracting the content of the effective ingredient and the auxiliary agent from 100 wt %.

[0152] In some specific embodiments, the number of viable Escherichia coli strains in the composition is 10 7 ～12 cfu·mL -1 or 10 7～12 cfu·g -1 .

[0153] The present invention also provides a method for producing mannitol by fermentation, wherein a culture medium containing glucose as a major carbon substrate is fermented under aerobic conditions using the strain described above, and wherein the mannitol accumulated in the culture medium is then recovered.

[0154] Glucose can account for at least 50wt% of the carbon substrate in the culture medium, and of course it can also be at least 60wt%, at least 70wt%, at least 80wt%, at least 90wt%, at least 95wt%. Other additional carbon substrates used as supplements can be sorbitol, sucrose, maltose, fructose, dextrin, molasses, starch, organic acids / alcohols / hydrocarbons, etc., or some complex carbon sources such as fatty carbon sources (such as soybean oil, rapeseed oil, lard, cottonseed oil, corn oil, linseed oil, olive oil and other oils). In some embodiments, the carbon substrate does not contain any one or more of sorbitol, sucrose, maltose, fructose, dextrin, molasses, starch, organic acids / alcohols / hydrocarbons, and fatty carbon sources; in some embodiments, glucose is used as the sole carbon source.

[0155] In some embodiments, the fermentation is stopped after mannitol is accumulated in the culture medium to a concentration of not less than 150 g / L.

[0156] In some embodiments, the strain is first cultured in an inorganic salt medium before fermentation. More preferably, the strain is cultured and activated before the culture is cultured in an inorganic salt medium.

[0157] Inoculating the activated strain into an inorganic salt culture medium can enable it to adapt to nutrient-deficient conditions in advance and shorten the lag period in accumulating and producing mannitol.

[0158] In some embodiments, the inorganic salt culture medium uses glucose as the main carbon substrate and contains 5-15 g / L potassium dihydrogen phosphate, 4-8 g / L diammonium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 1.4-2.0 g / L citric acid monohydrate, 1-3 g / L yeast extract and a metal ion solution; the metal ion solution contains 600-1000 mg / L ethylenediaminetetraacetic acid, 100-300 mg / L cobalt chloride hexahydrate, 700-1300 mg / L manganese chloride tetrahydrate, 70-130 mg / L copper chloride dihydrate, 70-130 mg / L boric acid, 100-300 mg / L sodium molybdate dihydrate, 700-1300 mg / L zinc acetate dihydrate and 5-15 g / L ferric citrate. Specifically, for example, it contains 10g / L potassium dihydrogen phosphate, 6g / L diammonium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 1.7g / L citric acid monohydrate, 2g / L yeast extract and a metal ion solution; the metal ion solution contains 800mg / L ethylenediaminetetraacetic acid, 200mg / L cobalt chloride hexahydrate, 1000mg / L manganese chloride tetrahydrate, 100mg / L cupric chloride dihydrate, 100mg / L boric acid, 200mg / L sodium molybdate dihydrate, 1000mg / L zinc acetate dihydrate and 10g / L ferric citrate.

[0159] In some embodiments, during acclimatization culture, when the bottom carbon is exhausted and the dissolved oxygen rebounds, feeding is started at a rate of 2g / L / h to 6g / L / h (e.g., 3g / L / h, 4g / L / h, 5g / L / h). The content of the feeding may include glucose, magnesium sulfate heptahydrate and a metal ion solution. Taking a 5L fermentation tank as an example, the fermentation feed medium includes 600g / L glucose, 2g / L magnesium sulfate heptahydrate and 10mL / L 100× metal ion solution.

[0160] In some embodiments, the dissolved oxygen content during acclimation culture is controlled at 35%±10%.

[0161] According to the general understanding of those skilled in the art, it is usually difficult to strictly maintain the values ​​of the fermentation system within a constant range during the fermentation process. For example, the dissolved oxygen content may decrease near the end of the fermentation, which will not have a significant impact on the fermentation process. Therefore, for example, the "control" in the dissolved oxygen content control at 35% ± 10% means that 35% ± 10% is used as the target value during the fermentation process, and the dissolved oxygen content is maintained within this range for most of the time during the fermentation process. Other parameters (such as glucose) are also understood in this way.

[0162] In some embodiments, arabinose is added to induce the strain, and then the strain accumulates and produces mannitol.

[0163] In some embodiments, the strain is cultured to an OD of 600=35-45 (e.g. 37, 40, 43) when induction begins.

[0164] In some embodiments, the concentration of arabinose used for induction is 0.01 g / L or more, such as 0.02 g / L, 0.05 g / L, 0.1 g / L, etc.

[0165] In some embodiments, the fermentation medium for accumulating the production of mannitol uses an inorganic salt medium as defined above.

[0166] In some embodiments, the fermentation time for accumulating and producing mannitol is 48 to 60 hours.

[0167] In some embodiments, the dissolved oxygen content during the accumulative production of mannitol is controlled at 40±10%.

[0168] In some embodiments, the glucose content is controlled below 10 g / L during the accumulation and production of mannitol.

[0169] In some specific embodiments, the fermentation process is as follows Figure 2-3 The exemplary method of differentiation includes:

[0170] i) The strain has been acclimated and cultured on an inorganic salt medium in advance;

[0171] ii) adding arabinose to induce the strain;

[0172] iii) Accumulate production of mannitol.

[0173] A complete fermentation cycle takes about 96-120 hours, and step iii) takes about 48-60 hours.

[0174] Of course, in a continuous culture process, steps i) and ii) may be omitted.

[0175] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0176] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0177] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting 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 do not constitute a limitation of the present invention in any way.

[0178] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0179] Unless otherwise specified herein, the nouns or terms used herein should be understood according to the common knowledge and usage of ordinary technicians in the field. Unless otherwise specified, the specific operating methods (including: preparation process, experimental steps, detection means, etc.) used in this application adopt conventional biochemical experiments, cell biology experiments, molecular biology experiments, gene editing (e.g., recombinant DNA technology), zoology experiments and conventional techniques in related fields in the field of this technology. These techniques have been well described in the existing literature, for example, see Sam Brook et al., Molecular Cloning: a Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Publishing, updated from time to time; Kursad Turksen et al., Embryonic Stem Cell Protocols, 3rd edition, Springer Press, 2016; P. Nagarajan et al., Essentials of Laboratory Animal Science: Principles and Practices, Springer Press, 2021; and Jann Hau et al., Handbook of Laboratory Animal Science: Essential Principles and Practices, 4th edition, CRC Press, 2021. The primer sequences in the following examples are as follows:

[0180] Table 1 Primer sequences

[0181]

[0182]

[0183]

[0184] Example 1-1. Construction of recombinant Escherichia coli ML05

[0185] In this example, a basic bacterium ML05 was prepared that can use glucose, glycerol, sucrose or fructose syrup as a carbon source for bacterial growth and can convert D-glucose into D-mannitol. The preparation method of the strain is as follows, and the primers used in the preparation process are shown in Table 1.

[0186] 1. Construction of strain ML01

[0187] The strain ML01 was constructed by knocking out 6-phosphofructokinase 1 (pfkA) and 6-phosphofructokinase 2 (pfkB) of Escherichia coli K12 using CRISPR technology (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81: 2506-2514., which is incorporated herein by reference in its entirety), thereby obtaining the mutant ML01 of Escherichia coli K12.

[0188] The specific construction steps of the mutant ML01 of Escherichia coli K12 are as follows:

[0189] 1. Preparation of electrocompetent cells: The pCas plasmid (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia cooligenome via the CRISPRCas9 system. Appl Environ Microbiol 2015, 81: 2506-2514.) was transformed into Escherichia coli K12 (MG1655) by chemical transformation, and positive clones were screened at 30°C on LB plates containing kanamycin (kanamycin concentration was 50 μg / ml). Positive clones were inoculated in LB liquid culture medium containing 2 g / L arabinose and cultured at 30°C until OD600 was approximately 0.6, and then electrocompetent cells were prepared.

[0190] 2. Construction of pTarget plasmid: Select the knockout site N20 using the website https: / / crispy.secondarymetabolites.org, and design primers to construct pTarget plasmid. Using pTargetF (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81: 2506-22514.) as a template, use primers pTarget-pfkA-F and pTarget-pfkA-R, pTarget-pfkB-F and pTarget-pfkB-R to perform PCR amplification, and amplify fragments of about 2100 bp in size. After digestion with DpnI methylase for about 3 hours, the competent Escherichia coli DH5α was directly transformed by chemical transformation. Positive clones were screened on LB plates containing streptomycin (streptomycin concentration was 50 μg / mL) and sequenced and verified with primer pTarget-cexu-F. After correct sequencing, they were named pTarget-pfkA and pTarget-pfkB, respectively.

[0191] 3. Amplification of the target fragment: PCR amplification was performed with primer pairs pfkA-up500-F and pfkA-up500-R, and pfkA-down500-F and pfkA-down500-R, respectively, to obtain fragments of approximately 500 bp and 500 bp, respectively. Using the mixture of the two fragments as a template, overlap PCR amplification was performed with primer pairs pfkA-up500-F and pfkA-down500-R, and the target fragment pfkA of approximately 1000 bp was recovered. The pfkA fragment contains the upstream homology arm of the pfkA gene and the downstream homology arm of the pfkA gene in sequence. PCR amplification was performed with primer pairs pfkB-up500-F and pfkB-up500-R, and pfkB-down500-F and pfkB-down500-R, respectively, to obtain fragments of approximately 500 bp and 500 bp, respectively. Using the mixture of the two fragments as template, primers pfkB-up500-F and pfkB-down500-R were used for overlap PCR amplification, and the target fragment pfkB with a size of about 1000 bp was recovered. The pfkB fragment contains the upstream homology arm of the pfkB gene and the downstream homology arm of the pfkB gene in sequence.

[0192] 4. Electroporation: Mix 400ng pTarget-pfkA plasmid, 800ng targeting fragment pfkA and 100μl electroporation competent cells prepared in step 1, place in a 2mm electroporation cup, electroporate at 2.55kV, add 1ml LB liquid medium to revive at 30℃, then spread on LB plate containing kanamycin and streptomycin (kanamycin concentration is 50ug / ml, streptomycin concentration is 50μg / ml), and culture at 30℃. Primer pair pfkA-up600-F and pfkA-down600-R are designed for gene sequences other than homology arms. Colony PCR is used to verify and screen positive transformants. Colony PCR results are shown in Figure 1-1 A. By Figure 1-1 A shows that lane 1 is a 2200 bp band obtained from E. coli without knocking out the pfkA gene, and lane 2 is a 1200 bp band obtained from E. coli with successfully knocking out the target gene pfkA gene. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0193] 5. Elimination of pTarget plasmid: The positive clones verified by sequencing were inoculated into LB liquid medium containing 0.1mM IPTG and kanamycin and cultured overnight at 30°C to eliminate the pTarget plasmid. The strains after overnight culture were streaked on LB solid plates containing kanamycin and cultured overnight at 30°C to obtain the E. coli mutant MG1655△pfkA containing the pCas plasmid.

[0194] 6. Pick a single clone from the plate in step 5, prepare electrocompetent cells, mix with pTarget-pfkB plasmid and pfkB targeting fragment, repeat steps 4-5, and perform colony PCR with primers pfkB-up600-F and pfkB-down600-R to verify and screen positive transformants. The colony PCR results are shown in Figure 1-1 B. By Figure 1-1 Lane 3 shows a 2200bp band obtained from E. coli without knocking out the pfkB gene, and lane 4 shows a 1200bp band obtained from E. coli with successfully knocking out the target gene pfkB. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. The E. coli mutant MG1655△pfkA△pfkB containing the pCas plasmid was obtained.

[0195] 7. Elimination of pCas plasmid: The E. coli mutant MG1655△pfkA△pfkB containing the pCas plasmid verified by sequencing was inoculated into LB liquid culture medium and cultured overnight at 42°C to eliminate the pCas plasmid. The strain after overnight culture was streaked on an LB solid plate and cultured overnight at 42°C to obtain the E. coli mutant MG1655△pfkA△pfkB, referred to as ML01.

[0196] 2. Construction of strain ML02

[0197] ML02 is a product that uses CRISPER technology to replace the sthA gene of Escherichia coli K12 with

[0198] The recombinant bacteria are obtained by fragment pntAB of gene pntA and gene pntB, and other genes remain unchanged.

[0199] The preparation method of recombinant bacteria ML02 is basically the same as the preparation method of recombinant bacteria ML01 in the above one, except that MG1655 electroporation competent cells are replaced by ML01 electroporation competent cells, pTarget-pfkA is replaced by pTarget-sthA, and the targeting fragment pfkA is replaced by sthA.

[0200] 1. Construction of pTarget plasmid: Use the website https: / / crispy.secondarymetabolites.org to select the knockout site N20, design primers to construct pTarget plasmid. Using pTargetF as a template, use primers to PCR amplify pTarget-sthA-F and pTarget-sthA-R to amplify a fragment of about 2100 bp. After digestion with DpnI methylase for about 3 hours, directly use chemical transformation to transform Escherichia coli DH5α competent cells, screen positive clones on LB plates containing streptomycin (streptomycin concentration is 50 μg / mL), and sequence verification with primer pTarget-cexu-F. After sequencing is correct, it is named pTarget-sthA.

[0201] 2. Amplification of the target fragment: PCR amplification was performed with primer pairs sthA-up500-F and sthA-up500-R, pntAB-F and pntAB-R, sthA-down500-F and sthA-down500-R, respectively, to obtain fragments of approximately 500 bp, 3000 bp and 500 bp, respectively. Using the mixture of the three fragments as a template, overlap PCR amplification was performed with primer pairs sthA-up500-F and sthA-down500-R, and a target fragment sthA::pntAB of approximately 4000 bp was recovered. The sthA::pntAB fragment contains the upstream homology arm of the sthA gene-pntAB replacement fragment-downstream homology arm of the sthA gene in sequence.

[0202] The primer pair sthA-up600-F and sthA-down600-R was introduced for positive clone screening and used for colony PCR verification. The results of colony PCR are shown in Figure 1-1 C. By Figure 1-1 As can be seen from the C, lane 5 is a 2601bp band obtained from E. coli without knocking out the sthA gene, and lane 6 is a 4132bp band obtained from E. coli with the target gene sthA gene successfully replaced with the pntAB gene. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. After the pTarget plasmid and pCas plasmid were eliminated in sequence, the E. coli mutant MG1655△pfkA△pfkB△sthA::pntAB, referred to as ML02, was obtained.

[0203] 3. Construction of strain ML03

[0204] ML03 is a recombinant bacterium that uses CRISPER technology to replace the LPXL gene of Escherichia coli K12 with a fragment p119-zwf-gnd containing the promoter p119 and the genes zwf and gnd, while keeping other genes unchanged, in order to strengthen the ppp pathway.

[0205] The preparation method of recombinant bacteria ML03 is basically the same as the preparation method of recombinant bacteria ML01 in the above one, except that MG1655 electroporation competent cells are replaced by ML02 electroporation competent cells, pTarget-pfkA is replaced by pTarget-LPXL, and the targeting fragment pfkA is replaced by p119-zwf-gnd.

[0206] 1. Construction of pTarget plasmid: Use the website https: / / crispy.secondarymetabolites.org to select the knockout site N20, design primers to construct pTarget plasmid. Using pTargetF as a template, use primers to perform PCR amplification on pTarget-LPXL-F and pTarget-LPXL-R, and amplify a fragment of about 2100 bp. After digestion with DpnI methylase for about 3 hours, directly use chemical transformation to transform Escherichia coli DH5α competent cells, screen positive clones on LB plates containing streptomycin (streptomycin concentration is 50 μg / mL), and sequence verification with primer pTarget-cexu-F. After sequencing is correct, it is named pTarget-LPXL.

[0207] 2. Amplification of target fragments: PCR amplification was performed with primer pairs LPXL-up500-F and LPXL-up500-R, P119-zwf-F and P119-zwf-R, gnd-F and gnd-R, LPXL-down500-F and LPXL-down500-R, respectively, to obtain fragments of approximately 500 bp, 1500 bp, 1500 bp and 500 bp, respectively. Using the mixture of the four fragments as a template, overlap PCR amplification was performed with primer pairs LPXL-up500-F and LPXL-down500-R, and a target fragment of approximately 4000 bp, LPXL::p119-zwf-gnd, was recovered. The LPXL::p119-zwf-gnd fragment contains the upstream homology arm of the LPXL gene, the P119 promoter, the RBS, the zwf-gnd, and the downstream homology arm of the sthA gene.

[0208] The primer pair LPXL-up600-F and LPXL-down600-R were introduced for positive clone screening and used for colony PCR verification. The results of colony PCR are shown in Figure 1-1 D. By Figure 1-1 D shows that lane 7 is a 2121bp band obtained from E. coli without knocking out the LPXL gene, and lane 8 is a 4146bp band obtained from E. coli with the target gene LPXL gene successfully replaced with the p119-zwf-gnd gene. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. After the pTarget plasmid and pCas plasmid were eliminated in sequence, the E. coli mutant MG1655△pfkA△pfkB△sthA::pntAB△LPXL::p119-zwf-gnd, referred to as ML03, was obtained.

[0209] IV. Construction of strain ML04

[0210] ML04 uses CRISPER technology to knock out the edd and eda genes of Escherichia coli K12, while keeping other genes unchanged, in order to block the ED pathway.

[0211] The preparation method of recombinant bacteria ML04 is basically the same as the preparation method of recombinant bacteria ML01 in the above one, except that MG1655 electroporation competent cells are replaced by ML03 electroporation competent cells, pTarget-pfkA is replaced by pTarget-edd and pTarget-eda, and the targeting fragment pfkA is replaced by edd-eda.

[0212] 1. Construction of pTarget plasmid: Select the knockout site N20 using the website https: / / crispy.secondarymetabolites.org, design primers to construct pTarget plasmid. Using pTargetF as a template, use primers pTarget-edd-F and pTarget-edd-R, pTarget-eda-F and pTarget-eda-R to perform PCR amplification, and amplify a fragment of about 2100 bp. After digestion with DpnI methylase for about 3 hours, directly use chemical transformation to transform Escherichia coli DH5α competent cells, screen positive clones on LB plates containing streptomycin (streptomycin concentration is 50 μg / mL), and sequence verification with primer pTarget-cexu-F. After sequencing is correct, they are named pTarget-edd and pTarget-eda.

[0213] 2. Amplification of the target fragment: PCR amplification was performed with primer pairs edda-up500-F and edda-up500-R, edda-down500-F and edda-down500-R, respectively, to obtain fragments of approximately 500 bp and 500 bp in size, respectively. Using the mixture of the two fragments as a template, overlap PCR amplification was performed with primer pairs edda-up500-F and edda-down500-R, and the target fragment edda of approximately 1000 bp in size was recovered. The edda fragment contains the upstream homology arm of the edda gene and the downstream homology arm of the edda gene in sequence.

[0214] The primer pair edda-up600-F and edda-down600-R was introduced for positive clone screening and used for colony PCR verification. The results of colony PCR are shown in Figure 1-1 E. By Figure 1-1 Lane 9 shows a 3690bp band obtained from E. coli without knocking out the edd and eda genes, and lane 10 shows a 1200bp band obtained from E. coli with successfully knocking out the target genes edd and eda genes. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. The E. coli mutant MG1655△pfkA△pfkB△sthA::pntAB△LPXL::p119-zwf-gnd△edd△eda, referred to as ML04, was obtained after the pTarget plasmid and pCas plasmid were eliminated in sequence.

[0215] 5. Construction of strain ML05

[0216] ML05 is a product that uses CRISPER technology to replace the gene lPXM of Escherichia coli K12 with

[0217] Promoter p119 and the fragment p119-TSM1PDH-PSM1PP of the mannitol 1-phosphate-5-dehydrogenase gene TSM1PDH from Thermoanaerobacter sp. and the mannitol-1-phosphatase gene PSM1PP from Pantoea stewartii, while other genes remain unchanged, to obtain recombinant bacteria to enhance mannitol synthesis.

[0218] The preparation method of recombinant bacteria ML05 is basically the same as the preparation method of recombinant bacteria ML01 in the above one, except that MG1655 electroporation competent cells are replaced by ML04 electroporation competent cells, pTarget-pfkA is replaced by pTarget-lPXM, and the targeting fragment pfkA is replaced by p119-TSM1PDH-PSM1PP.

[0219] 1. Construction of pTarget plasmid: Use the website https: / / crispy.secondarymetabolites.org to select the knockout site N20, design primers to construct pTarget plasmid. Using pTargetF as a template, use primers to perform PCR amplification on pTarget-lPXM-F and pTarget-lPXM-R, respectively, to amplify a fragment of about 2100 bp. After digestion with DpnI methylase for about 3 hours, directly use chemical transformation to transform Escherichia coli DH5α competent cells, screen positive clones on LB plates containing streptomycin (streptomycin concentration is 50 μg / mL), and sequence verification with primer pTarget-cexu-F. After sequencing is correct, they are named pTarget-lPXM.

[0220] 2. Acquisition of Targeting Clips:

[0221] PCR amplification was performed with primer pairs lPXM-up500-F and lPXM-up500-R, lPXM-down500-F and lPXM-down500-R, respectively, to obtain fragments of approximately 500 bp and 500 bp, respectively. Sequence 1TSM1PDH and sequence 10PSM1PP were used as templates, and primer pairs P119-TSM1PDH-F and P119-TSM1PDH-R, PSM1PP-F and PSM1PP-R were used for PCR amplification, respectively, to obtain fragments of approximately 1200 bp and 660 bp, respectively. Using the mixture of four fragments as template, overlap PCR amplification was performed with primers lPXM-up500-F and lPXM-down500-R, and the target fragment p119-TSM1PDH-PSM1PP with a size of about 3000 bp was recovered. This fragment contains the upstream homologous arm of lPXM gene-p119 promoter-RBS-TSM1PDH-RBS-PSM1PP-downstream homologous arm of lPXM gene in sequence.

[0222] The primer pair lPXM-up600-F and lPXM-down600-R were introduced for positive clone screening and used for colony PCR verification. The results of colony PCR are shown in Figure 1-1 F. By Figure 1-1 It can be seen that lane 11 is a 2172bp band obtained by E. coli without knocking out the lPXM gene, and lane 12 is a 3060bp band obtained by E. coli that successfully replaced the target gene lPXM gene with the p119-TSM1PDH-PSM1PP gene. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. After the pTarget plasmid and pCas plasmid were eliminated in sequence, the E. coli mutant MG1655△pfkA△pfkB△sthA::pntAB△LPXL::p119-zwf-gnd△edd△eda△lPXM::p119-TSM1PDH-PSM1PP, referred to as ML05, was obtained. Among them, TSM1PDH and PSM1PP can be replaced by M1PDH and M1PP from other sources.

[0223] Example 1-2. Construction of recombinant Escherichia coli ML06

[0224] In this example, a basic bacterium ML06 was prepared that can use glucose, glycerol, sucrose or fructose syrup as a carbon source for bacterial growth and can convert D-glucose into D-mannitol. The preparation method of the strain is as follows, and the primers used in the preparation process are shown in Table 1.

[0225] ML06 is a recombinant bacterium that uses CRISPER technology to replace the gene lPXM of Escherichia coli K12 with a fragment p119-ASM1PDH-PCM1PP containing the promoter p119 and the mannitol 1-phosphate-5-dehydrogenase gene ASM1PDH from Aeromonas salmonicida and the mannitol-1-phosphatase gene PCM1PP from Photorhabdus scinerea, while keeping other genes unchanged, to enhance mannitol synthesis.

[0226] The preparation method of the recombinant bacterium ML06 is basically the same as the preparation method of the recombinant bacterium ML05 in the above Example 1, except that the targeting fragment p119-TSM1PDH-PSM1PP is replaced by p119-ASM1PDH-PCM1PP.

[0227] Acquisition of target shooting clips:

[0228] PCR amplification was performed with primer pairs lPXM-up500-F and lPXM-up500-R, lPXM-down500-F2 and lPXM-down500-R, respectively, to obtain fragments of approximately 500 bp and 500 bp, respectively. Sequence 7 (ASM1PDH) and sequence 16 (PCM1PP) were used as templates, and primer pairs P119-ASM1PDH-F and P119-ASM1PDH-R, PCM1PP-F and PCM1PP-R were used for PCR amplification, respectively, to obtain fragments of approximately 1200 bp and 660 bp, respectively. Using the mixture of the four fragments as a template, overlap PCR amplification was performed with primers lPXM-up500-F and lPXM-down500-R, and the target fragment p119-ASM1PDH-PCM1PP with a size of about 3000 bp was recovered. This fragment contains the upstream homologous arm of the lPXM gene-p119 promoter-RBS-ASM1PDH-RBS-PCM1PP-downstream homologous arm of the lPXM gene in sequence.

[0229] The primer pair lPXM-up600-F and lPXM-down600-R were introduced for positive clone screening and used for colony PCR verification. The results of colony PCR are shown in Figure 1-1 G. By Figure 1-1The G of lane 13 shows a 2172bp band obtained from E. coli without knocking out the lPXM gene, and lane 14 shows a 3120bp band obtained from E. coli with the target gene lPXM gene successfully replaced with the p119-ASM1PDH-PCM1PP gene. The PCR product was sequenced and verified as the expected product by Suzhou Jinweizhi Biotechnology Co., Ltd. After the pTarget plasmid and pCas plasmid were eliminated in sequence, the E. coli mutant MG1655△pfkA△pfkB△sthA::pntAB△LPXL::p119-zwf-gnd△edd△eda△lPXM::p119-ASM1PDH-PCM1PP was obtained.

[0230] Abbreviated as ML06.

[0231] The genotypes of the Escherichia coli mutants in the embodiment are shown in Table 2:

[0232] Table 2 Genotypes of Escherichia coli mutants

[0233]

[0234]

[0235] Example 1-3. Application of recombinant Escherichia coli ML05 in the preparation of D-mannitol using glucose as a substrate

[0236] 1. Glycerol Growth Medium Composition

[0237] Growth medium components and final concentrations are as follows:

[0238] 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 3% (g / 100mL) glycerol, 0.5% (g / 100mL) yeast powder, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μMZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, and the rest is water.

[0239] 2. Transformation medium composition

[0240] 25mM Na2HPO4, 25mM KH2PO4, 50mM MgCl2, 20% (g / 100mL) D-glucose, and the balance is water.

[0241] 3. Bacterial culture and enzyme induction

[0242] The wild-type strain MG1655, recombinant strain ML05 and control strains ML01, ML02, ML03, and ML04 cultured overnight were inoculated at 1% in a shake flask containing 200 ml of glycerol growth medium, cultured at 37°C for 16 h, and the cells were collected by centrifugation at 8000g for 10 min.

[0243] 4. Whole-cell catalysis of D-mannitol

[0244] The collected bacteria were resuspended in a beaker containing 10 ml of transformation medium. The reaction process was monitored regularly using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to maintain 7.0. After reacting at 37°C for 24 hours, the reaction product was centrifuged at 8000 g for 10 minutes, the supernatant was filtered through a 0.22 μm filter membrane, and the D-mannitol content in the supernatant was detected by HPLC. HPLC used a Hi-Plex Ca chromatographic column (300 mm × 7.7 mm, 8 μm); the mobile phase was ultrapure water, the flow rate was 0.5 mL / min; the column temperature was 78°C; the detector was a differential refractive index detector; the temperature was 40°C.

[0245] D-mannitol standard was purchased from Shanghai McLean Biotechnology (Cat. No.: M813424) with a retention time of 16.838 min.

[0246] The conversion rate was calculated as the ratio of D-mannitol production in the whole-cell catalytic stage (step 4) to D-glucose consumption*100%.

[0247] The results are shown in Table 3. The D-mannitol production of the wild-type strain MG1655 was 0, and the average D-mannitol production of the engineered strain ML05 was 98.7 g / L, with a conversion rate of 80.5%.

[0248] Table 3 D-mannitol production and conversion rate of various engineered bacteria using glucose as carbon source

[0249]

[0250]

[0251] ML0* represents ML02 / ML03 / ML04 / ML05

[0252] Example 1-4. Application of recombinant Escherichia coli ML06 in the preparation of D-mannitol using fructose syrup as substrate

[0253] 1. Glucose growth medium composition

[0254] Growth medium components and final concentrations are as follows:

[0255] 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 2% (g / 100mL) glucose, 0.5% (g / 100mL) yeast powder, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μMZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, and the rest is water.

[0256] 2. Transformation medium composition

[0257] 25mM Na2HPO4, 25mM KH2PO4, 50mM MgCl2, 20% (g / 100mL) fructose syrup, and the balance is water.

[0258] 3. Bacterial culture and enzyme induction

[0259] The wild-type strain MG1655, recombinant strain ML06 and control strains ML01, ML02, ML03, ML04 and ML05 cultured overnight were inoculated into a shake flask containing glucose growth medium at a 1% inoculation rate, cultured at 37°C for 16 h, and the cells were collected by centrifugation at 8000g for 10 min.

[0260] 4. Whole-cell catalysis of D-mannitol

[0261] The collected bacteria were resuspended in a beaker containing 10 ml of transformation medium. The reaction process was monitored regularly using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to maintain 7.0. After reacting at 37°C for 24 hours, the reaction product was centrifuged at 8000 g for 10 minutes, the supernatant was filtered through a 0.22 μm filter membrane, and the D-mannitol content in the supernatant was detected by HPLC. HPLC used a Hi-Plex Ca chromatographic column (300 mm × 7.7 mm, 8 μm); the mobile phase was ultrapure water, the flow rate was 0.5 mL / min; the column temperature was 78°C; the detector was a differential refractive index detector; the temperature was 40°C.

[0262] The results are shown in Table 4. The D-mannose production of the wild-type strain MG1655 was 0, and the average D-mannose production of the engineered strain ML06 was 132.8 g / L, with a conversion rate of 88.2%.

[0263] Table 4 D-mannitol production and conversion rate of various engineered bacteria using fructose syrup as carbon source

[0264]

[0265] ML0* represents ML02 / ML03 / ML04 / ML05 / ML06

[0266] In addition, the purity of D-mannitol in the reaction solution is relatively high, and other sugars are not obviously detected under the same HPLC conditions, which is conducive to simplifying the downstream separation and purification process.

[0267] The bacterial cells used in the following examples are all ML06.

[0268] Sample testing:

[0269] After conversion, the sample was centrifuged to obtain the supernatant, which was diluted and filtered before being analyzed by high performance liquid chromatography (HPLC, Agilent 1260). For the analysis of glucose and mannitol, an Aminex HPX-87H column and a differential refractive index detector (RID) were used. The mobile phase was a 6mM H2SO4 solution. The injection volume was 10μL, the flow rate was 0.5mL / min, and the column temperature was 55°C. The reference cell temperature of the RID detector was 40°C and the time was 20min. The standard curve was obtained by linear regression of standards of different concentrations, and then the concentration of the compound in the sample was determined. The peak diagram of the liquid phase detection of glucose and mannitol standards is shown in the figure. Figure 2-1A and Figure 2-1B shown. Different from the above-mentioned embodiment in which the bacteria are cultured in a triangular culture bottle of a smaller volume (under ideal conditions), the following embodiment cultured the bacteria in a triangular culture bottle of a smaller volume. The strain was cultured in a fermenter in the production environment, and the final yield, maximum yield and final product yield under different dissolved oxygen conditions in the actual scale-up production environment were measured. Final yield and hourly production efficiency .

[0270] Example 2-1 Optimization of early fermentation bacterial culture

[0271] 1. Bacteria activation

[0272] Use cotton wool soaked in 75% alcohol to wipe the purchased ampoule tube, then use the flame of an alcohol lamp to moderately heat the top of the ampoule tube, then use a pipette to take a small amount of sterile water, drip the sterile water onto the top of the heated ampoule tube to crack the glass, and finally use tweezers to knock off the top of the cracked ampoule tube. Use a pipette to draw 0.3-0.5mL of appropriate liquid culture medium (LB liquid culture medium), drip it into the ampoule tube, shake it gently to dissolve the bacteria into a suspended state, then take 0.1-0.2mL of the bacterial suspension and spread it on the LB solid plate culture medium, and add the remaining bacterial liquid to 3-5mL of liquid LB culture medium and culture at 37°C.

[0273] LB liquid medium includes: 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride (W / V); sterilized at 121°C for 30 min. LB solid medium includes: 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride; 15 g / L agarose; sterilized at 121°C for 30 min.

[0274] After 12-16 hours of incubation, take the plate out of 37°C, take a small amount with an inoculation loop, use the streak method or spread to revive, and place it in a 37°C constant temperature incubator for incubation. Repeat this operation 2-3 times to ensure bacterial viability.

[0275] 2. Acclimation of inorganic salt culture medium

[0276] The activated strain is inoculated into an inorganic salt culture medium to adapt it to the nutrient-deficient conditions in advance and shorten the lag period in the fermentation tank.

[0277] The specific components of the inorganic salt medium include 20g / L glucose, 10g / L potassium dihydrogen phosphate, 6g / L diammonium hydrogen phosphate, 2g / L magnesium sulfate heptahydrate, 1.7g / L citric acid monohydrate, 2g / L yeast extract, and sterilized at 115°C for 20min. The additional metal ion solution is added at a ratio of 10mL per liter of fermentation inorganic salt medium, and the specific formula concentration is: 800mg / L ethylenediaminetetraacetic acid, 200mg / L cobalt chloride hexahydrate, 1000mg / L manganese chloride tetrahydrate, 100mg / L copper chloride dihydrate, 100mg / L boric acid, 200mg / L sodium molybdate dihydrate, 1000mg / L zinc acetate dihydrate, 10g / L ferric citrate, and sterilized at 121°C for 30min.

[0278] Take the activated bacterial solution in liquid LB in step 1 and transfer it to the above inorganic salt medium at a rate of 1% and wait until it grows to OD 600 Repeat this process until the growth cycle can be shortened to 12-16 hours and the cells can grow stably. Figure 2-2 The domesticated strains were added with 50% glycerol and stored at -80°C for long-term storage. The glycerol tube strains can be directly used for activation in later use.

[0279] Example 2-2 Effect of fermentation factors on mannitol yield and bacterial biomass in fermentation broth

[0280] 1. High-density fermentation of Escherichia coli to produce mannitol

[0281] Take out the freeze-dried tube of engineered bacteria ML06 from the -80℃ refrigerator, streak it on a 2% LB solid plate for activation, repeat the operation, transfer the obtained monoclone to a 10ml test tube, and culture it in a shaker at 37℃ and 220rpm until 2-3OD, take a small amount of bacterial liquid for PCR identification to ensure that the strain chassis and plasmid are correct, and then transfer it to the first-level LB seed liquid; LB liquid culture medium includes: 10g / L tryptone; 5g / L yeast extract; 10g / L sodium chloride (W / V); sterilize at 121℃ for 30min. LB solid culture medium includes: 10g / L tryptone; 5g / L yeast extract; 10g / L sodium chloride; 15g / L agarose; sterilize at 121℃ for 30min.

[0282] The first-level LB seed liquid is transferred to 100ml LB liquid medium at an inoculation amount of 1%-2%, and cultured in a shaking table at 37°C and 220rpm to 1-2OD to obtain the second-level LB seed liquid; the second-level LB seed liquid is transferred to 100ml inorganic salt liquid medium at an inoculation amount of 2%-5%, and cultured in a shaking table at 37°C and 220rpm to 1.5-2.5OD to obtain the fermentation inorganic salt seed liquid. The inorganic salt seed liquid is transferred to the fermentation inorganic salt seed liquid and cultured in an inorganic salt medium in a 5L fermentation tank at an inoculation amount of 5%-10%. The specific components of the fermentation inorganic salt medium include 20g / L glucose, 10g / L potassium dihydrogen phosphate, 6g / L diammonium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 1.7g / L citric acid monohydrate, 2g / L yeast extract, and sterilized at 115°C for 20min. Additional metal ion solution: 800 mg / L EDTA, 200 mg / L cobalt chloride hexahydrate, 1000 mg / L manganese chloride tetrahydrate, 100 mg / L copper chloride dihydrate, 100 mg / L boric acid, 200 mg / L sodium molybdate dihydrate, 1000 mg / L zinc acetate dihydrate, 10 g / L ferric citrate, sterilized at 121°C for 30 min. Add 10 mL per L of fermentation inorganic salt medium.

[0283] 2. Optimize culture conditions

[0284] The specific culture conditions for fermentation are as follows: the reaction temperature is 37.5°C, the pH is adjusted to 7.5 with ammonia water in advance, the initial rotation speed is 500rpm, and the dissolved oxygen is linked to the rotation speed and controlled at 35%±10%. When the bottom carbon is exhausted and the dissolved oxygen rebounds, feed is started at a rate of 4g / L / h. The fermentation feed medium includes 600g / L glucose, 2g / L magnesium sulfate heptahydrate and 10mL / L 100× metal ion solution. When OD 600 =40, induction began, the inducer was 0.02g / L arabinose, and feeding was dynamically regulated after induction. The entire fermentation time was maintained at 96-120h, and the yield could reach at least 150g / L and the conversion rate was above 70%. The fermentation curve is shown in Figure 2-3 shown.

[0285] Example 2-3 Improving the purity of mannitol in fermentation broth by optimizing fermentation conditions

[0286] According to the initial culture conditions, the purity of mannitol in fermentation is maintained at about 50%. Figure 2-4A to Figure 2-4C As shown, it contains salt peaks and some by-products, and the purity is improved by optimizing the fermentation conditions.

[0287] 1. Change dissolved oxygen in the late fermentation period

[0288] According to the existing fermentation conditions, the dissolved oxygen was adjusted after 60 hours and set to 0±10%, 10±10%, 20±10%, 30±10%, 40±10%, 50±10%, and 60±10% for testing to compare the yield, conversion rate, and purity.

[0289] Table 5 Comparison of data under different dissolved oxygen conditions

[0290]

[0291]

[0292] 2. Change the sugar replenishment rate in the late fermentation period

[0293] On the existing basis, 40%±10% is selected as the dissolved oxygen control condition, and the sugar replenishment rate after 60 hours is finely controlled. The residual sugar is tested every 2-3 hours and controlled below 10g / L. If the accumulation is high, the sugar is stopped for 1 hour to improve the yield, conversion rate and purity.

[0294] The embodiment provided by the present invention improves the vitality of seeds by optimizing the fermentation process in the seed culture stage, thereby effectively improving the production intensity of mannitol without additionally increasing the cost of raw materials. By optimizing the fermentation mode, the production intensity and output of mannitol are improved, the production cost of biological preparation of mannitol is reduced, and it is beneficial to the biological industrial production of mannitol.

[0295] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A composition comprising a genetically recombinant Escherichia coli strain; The genetically recombinant Escherichia coli strain contains the following modifications: 6-phosphofructokinase 1pfkA gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; and 6-phosphofructokinase 2pfkB gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; Preferably, the composition is in liquid, frozen or dry powder form; Preferably, the composition is in the form of a frozen or dried powder, which further includes a solid carrier; the solid carrier preferably includes one or more of clay, talc, montmorillonite, bentonite, medical stone, calcite, zeolite, calcium carbonate, diatomaceous earth and white carbon; preferably, the composition is a liquid, which further includes a liquid carrier, and the liquid carrier is preferably an organic solvent and / or water. The composition contains an auxiliary agent that can be used for bacterial agents; the auxiliary agent preferably includes one or more of sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerol, sodium lignin sulfonate, sodium alkylnaphthalene sulfonate polycondensate, nicotinic acid, alcohol, buffer salt, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extract, white carbon black, tea saponin, and skim milk; The number of live bacteria of the Escherichia coli strain in the composition is 10 7～12 cfu·mL -1 or 10 7～12 cfu·g -1 .

2. The composition according to claim 1, wherein the strain further comprises the following modifications: Pyridine nucleotide transhydrogenase sthA gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; Introducing and / or enhancing the expression of the pyridine nucleotide transhydrogenase α subunit pntA gene or its isozyme gene or its functional homolog gene; and Introducing and / or enhancing the expression of the pyridine nucleotide transhydrogenase β subunit pntB gene or its isozyme gene or its functional homolog gene; Optionally, the following modifications are also included: The lauroyl acyltransferase LPXL gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivating mutation, or has an expression-inhibiting nucleic acid fragment; Introducing and / or enhancing the expression of 6-phosphate glucose dehydrogenase zwf gene or its isozyme gene or its functional homolog gene; Introducing and / or enhancing the expression of 6-phosphogluconate dehydrogenase gnd gene or its isozyme gene or its functional homolog gene; and p119 promoter; Optionally, the following modifications are also included: The phosphogluconate dehydratase edd gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivating mutation, or has an expression-inhibiting nucleic acid fragment; and KHG / KDPG aldolase eda gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; Optionally, the following modifications are also included: Myristoyltransferase LPXM gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment; Introducing and / or enhancing the expression of mannitol 1-phosphate-5-dehydrogenase M1PDH gene or its isozyme gene or its functional homolog gene; and Introducing and / or enhancing the expression of mannitol-1-phosphatase M1PP gene or its isozyme gene or its functional homolog gene; Optionally, wherein The M1PDH gene is from at least one of Thermoanaerobacter sp. (TSM1PDH), actococcus lactis subsp. lactis bv. Diacetylactis (DM1PDH), eauveria bassiana (BBM1PDH), Oceanobacillus iheyensis (OIM1PDH), Bacillus sp. FW1 (BSM1PDH), Brochothrix thermosphacta (BTM1PDH), Aeromonas almonicida (ASM1PDH), Pectobacterium atrosepticum (PAM1PDH), and Escherichia coli (ECM1PDH); Optionally, the M1PDH gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 1 to 9; Optionally, the M1PP gene is from at least one of Pantoea stewartia (PSM1PP), Luteibacter sp. (LSM1PP), Pseudomonas graminis (PGM1PP), Muribaculaceae bacterium (MBM1PP), Eimeria tenella (ETM1PP), Salmonella diarizonae (SDM1PP), Photorhabdus cinerea (PCM1PP), Citrobacter pasteurii (CPM1PP), Corallincola holothuriorum (CHM1PP), and Escherichia coli (ECM1PPA, ECM1PPB); Optionally, the M1PP gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 10 to 12; Optionally, pntA and pntB comprise the sequences shown in Gene ID: 946628 and Gene ID: 946144, respectively; Optionally, the zwf gene comprises the sequence shown in Gene ID: 946370; Optionally, the gnd gene comprises the sequence shown in Gene ID:946554.

3. A method for producing mannitol by fermentation, wherein a culture medium containing glucose as a main carbon substrate is fermented under aerobic conditions using the strain of claim 1, and wherein the mannitol accumulated in the culture medium is then recovered.

4. The method according to claim 3, wherein the fermentation is stopped after mannitol is accumulated in the culture medium to a concentration of not less than 150 g / L.

5. The method according to claim 3, wherein the strain is first acclimated and cultured in an inorganic salt medium before fermentation.

6. The method according to claim 5, wherein the inorganic salt culture medium uses glucose as a main carbon substrate and contains 5-15 g / L potassium dihydrogen phosphate, 4-8 g / L diammonium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 1.4-2.0 g / L citric acid monohydrate, 1-3 g / L yeast extract and a metal ion solution; the metal ion solution contains 600-1000 mg / L ethylenediaminetetraacetic acid, 100-300 mg / L cobalt chloride hexahydrate, 700-1300 mg / L manganese chloride tetrahydrate, 70-130 mg / L cupric chloride dihydrate, 70-130 mg / L boric acid, 100-300 mg / L sodium molybdate dihydrate, 700-1300 mg / L zinc acetate dihydrate and 5-15 g / L ferric citrate.

7. According to the method of claim 5 or 6, the dissolved oxygen content during acclimatization culture is controlled at 35%±10%.

8. The method according to claim 3, wherein the fermentation comprises: After adding arabinose to induce the strain, it accumulates and produces mannitol; Preferably, the strain is cultured to OD 600 =35-45 when induction begins; Preferably, the fermentation medium for accumulating and producing mannitol adopts the inorganic salt medium defined in claim 6; more preferably, the fermentation time for accumulating and producing mannitol is 48 to 60 hours.

9. The method according to claim 8, wherein the dissolved oxygen content during the accumulative production of mannitol is controlled at 40±10%.

10. The method according to claim 3 or 8, wherein the glucose content is controlled below 10 g / L during the accumulation and production of mannitol.

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